JP2021514637A - How to Amplify Nucleic Acids with Improved Specificity - Google Patents

Abstract

The present invention relates to a method of amplifying a nucleic acid having improved specificity using specific oligonucleotide primers and controller nucleotides. In the first amplification, the controller oligonucleotide is amplified in a sequence-specific manner. Allows chain opening of products. The present invention further relates to corresponding kits for carrying out the methods according to the invention. [Selection diagram] Fig. 1

Description

The present invention relates to a method for amplifying a nucleic acid with improved specificity.

This application claims the priority of the following applications: European Patent Application, EP18158722.1 (February 26, 2018), EP18195312.6 (September 18, 2018), EP18159337.7 (February 2018) 28th); German Patent Application 10 2018 001586.7 (February 28, 2018). These and all other patent documents referred to herein are deemed to be incorporated herein by reference (incorporated as a reference).

Nucleic acid chain synthesis plays a central role in today's biotechnology. Methods such as PCR have made significant advances in both research prospects and applied industrial fields, such as the diagnostic and food industries. The combination of PCR with technologies such as sequencing, real-time detection, microarray technology, and microfluidic management has contributed to technological development and has partially overcome the shortcomings of the original PCR. Other amplification methods such as isothermal amplification techniques have also been developed. Their use was specifically aimed at POCT (Point of Care Testing). Despite major advances in this area, PCR plays a central role, thus determining individual technical barriers to its application.

During the amplification procedure, PCR does not control the amplified sequence segments between the primers. The focus of PCR optimization is primer binding. At the start of PCR and in the process, synthesis of major products and by-products is continuously initiated, for example, by non-specific binding or extension of primers. When a reverse synthetic reaction occurs, the non-specifically elongated primers are read as templates, usually forming a complete primer binding site. Thus, incomplete sequence information is transferred from one synthesis cycle to the next synthesis cycle, which as a whole leads to an exponential increase in by-products as well as early formation.

Such side reactions result in an exponential increase in fragments that interfere with the main reaction (amplification of the target sequence) and can result in interference in the next step of the analysis, respectively. Such by-products usually contain the left and right primer sequences, so their amplification can occur in parallel with the main reaction. However, instead of the target sequence, such a by-product comprises another nucleic acid target sequence.

Primarily, the specificity of PCR amplification is achieved by optimizing the binding of primers to the target sequence. For example, additional oligonucleotides can be used that can partially bind to the primer and thus be competitively involved in primer binding to other nucleic acid strands.

Such probes generally bind to the sequence segment of the primer and do not occupy the single-stranded sequence segment, so that the primer with said segment can bind to the target nucleic acid and initiate a synthetic reaction. Primer template mismatches are competitively replaced by such oligonucleotides, improving initiation specificity. Such additional oligonucleotides do not interact with the nucleic acid strand amplified at the site between both primers. However, the molar excess of primers causes non-specific interactions between the primers and the template during amplification, resulting in the synthesis of complementary strands of by-products, resulting in the formation of fully functional primer binding sites. The presence of such a fully functional primer binding site in the by-product leads to a reduced competitive effect of such additional oligonucleotides on primer binding. Therefore, controlling the specificity of the primer that binds to the template with such an oligonucleotide only makes the initiation of side reactions less likely, and once the by-product is formed, it has little effect on its exponential amplification. Can't give.

The PCR side reactions increase with the number of synthetic cycles performed, often up to 40 PCR cycles. After PCR amplification, other methods are involved, such as detection, library generation, cloning, sequencing. The method can only tolerate differences in by-products or sequences produced to some extent during PCR amplification. In general, the specificity of such methods is also affected by the problem of side reactions during PCR.

Improving the synthetic specificity of amplification methods with the appearance of by-products and reduced co-amplification contributes to improved diagnostic methods.

An object of the present invention is to provide methods and means for improving the specificity of enzymatic amplification of nucleic acid chains.

A further object of the present invention is to provide a combination of amplification methods and PCR amplification, in which signal acquisition (monitoring or detection, respectively) or amplification of a target sequence is first performed with a controller oligonucleotide and a corresponding specific primer set. It is performed by controlled amplification using, and is continued by PCR, especially using PCR-specific primers.

A further object of the present invention is to reduce the number of PCR synthesis cycles required to obtain a sufficient amount of PCR product.

A further object of the present invention is to provide new enzymatic methods and components for nucleic acid chain synthesis and nucleic acid chain amplification, providing continuous (online) signal acquisition and signal acquisition (monitoring / detection). ) Is mediated or supported by a sequence-specific probe oligonucleotide.

The object is solved by the method and kit according to the independent claims. Further advantageous embodiments of the present invention are listed in the dependent claims and the following description.

FIG. 1 graphically shows the components of a particular embodiment of the amplification method according to the invention in a heterogeneous form. A) First amplification system; B) Second amplification system. FIG. 2 graphically shows the components of a particular embodiment of the first amplification system. FIG. 3 shows the temperature profile of a particular embodiment of the method according to the invention in a heterogeneous form. FIG. 4 shows the temperature profile of a particular embodiment of the method according to the invention in a heterogeneous form. FIG. 5 shows the components of a particular embodiment of the amplification method according to the invention in uniform form. A) First amplification system; B) Second amplification system. 6-11 show temperature profiles of embodiments of the method according to the invention. 6-11 show temperature profiles of embodiments of the method according to the invention. 6-11 show temperature profiles of embodiments of the method according to the invention. 6-11 show temperature profiles of embodiments of the method according to the invention. 6-11 show temperature profiles of embodiments of the method according to the invention. 6-11 show temperature profiles of embodiments of the method according to the invention. FIG. 12 shows (A) the structure of the first oligonucleotide primer, (B) complementary binding of the first primer to the amplified nucleic acid strand, and (C) the first primer to the amplified nucleic acid strand. The complementary binding of and the extension of the primer are shown graphically. FIG. 13 shows (A) the relationship between individual components during the synthesis of the first primer extension product, (B) the synthesis of the first primer extension product (1 = nucleic acid amplified in the first amplification, 2 = The controller oligonucleotide, 3 = first oligonucleotide primer, 4 = extension product of the first oligonucleotide primer) is shown graphically. FIG. 14 schematically shows the synthesis of the second primer extension product. As shown in FIG. 13, see marks 1-4; 5 = second oligonucleotide primer; 6 = extension product of the second oligonucleotide primer. FIG. 15 schematically shows the synthesis of the third and fourth primer extension products; P3.1-Ext part1 = extension product (intermediate product) of P3.1 synthesized in P2.1-Ext as a template. P4.1-Ext part1 = extension product (intermediate product) of P4.1 synthesized by p1.1-Ext as a template, P3.1-Ext = P4.1-Ext-part1 or P4.1-Ext as a template P3.1 extension product synthesized using P4.1-Ext = P4.1 extension product synthesized using P3.1-Ext-part1 as a template or P3.1-Ext. FIG. 16 graphically illustrates the synthesis of a partial third primer extension product (P3.1-Ext part1); 7 = P3 complementary to the second primer extension product (P2.1-Ext). Segment of .1; 8 = segment of P3.1 that is not complementary to the extension product (P2.1-Ext) of the second primer, P3.1-Ext-part1 = using P2.1-Ext as a template The extension product of the third primer (P3.1) synthesized in the above. FIG. 17 graphically illustrates the synthesis of the complete fourth primer extension product; P2.1-Ext = segments 5 and 6; P3.1-Ext-part1 = synthesized in P2.1-Ext as a template. Extension product of P3.1; P4.1 = 4th primer; 9 = segment of P4.1 capable of complementarily binding to P3.1-Ext-part1; 10 = complementary to P3.1-Ext-part1 Unbound P4.1 segment; P4.1-Ext = extension of P4.1 synthesized in P3.1-Ext-part1 as a template. FIG. 18 schematically shows the synthesis (A) of the intermediate product (P3.1-Ext-part1) of the third primer extension product using the second primer extension product (P2.1-Ext). (B) Complete third primer extension product (P3.1-Ext) using P4.1-Ext as a template; P4.1-Ext = P4.1 synthesized on P3.1-Ext part1 as a template. Extension product of P3.1-Ext = Extension product of P3.1 synthesized by P4.1-Ext as a template. Figures 19-26 graphically show the relationship between the individual components of the first primer extension product and the third PCR primer during synthesis; 1 = extension of P2.1 in the first amplification; 2 = controller. Oligonucleotide; 3 = 1st oligonucleotide primer; 4 = primer 3.1. Figures 19-26 graphically show the relationship between the individual components of the first primer extension product and the third PCR primer during synthesis; 1 = extension of P2.1 in the first amplification; 2 = controller. Oligonucleotide; 3 = 1st oligonucleotide primer; 4 = primer 3.1. Figures 19-26 graphically show the relationship between the individual components of the first primer extension product and the third PCR primer during synthesis; 1 = extension of P2.1 in the first amplification; 2 = controller. Oligonucleotide; 3 = 1st oligonucleotide primer; 4 = primer 3.1. Figures 19-26 graphically show the relationship between the individual components of the first primer extension product and the third PCR primer during synthesis; 1 = extension of P2.1 in the first amplification; 2 = controller. Oligonucleotide; 3 = 1st oligonucleotide primer; 4 = primer 3.1. Figures 19-26 graphically show the relationship between the individual components of the first primer extension product and the third PCR primer during synthesis; 1 = extension of P2.1 in the first amplification; 2 = controller. Oligonucleotide; 3 = 1st oligonucleotide primer; 4 = primer 3.1. Figures 19-26 graphically show the relationship between the individual components of the first primer extension product and the third PCR primer during synthesis; 1 = extension of P2.1 in the first amplification; 2 = controller. Oligonucleotide; 3 = 1st oligonucleotide primer; 4 = primer 3.1. Figures 19-26 graphically show the relationship between the individual components of the first primer extension product and the third PCR primer during synthesis; 1 = extension of P2.1 in the first amplification; 2 = controller. Oligonucleotide; 3 = 1st oligonucleotide primer; 4 = primer 3.1. Figures 19-26 graphically show the relationship between the individual components of the first primer extension product and the third PCR primer during synthesis; 1 = extension of P2.1 in the first amplification; 2 = controller. Oligonucleotide; 3 = 1st oligonucleotide primer; 4 = primer 3.1. 27-31 schematically show the components of different embodiments of the first amplification system; (A) components of the first amplification system; (B) components of the second amplification system. FIGS. 27-29: The fourth primer is the same as the second primer, and the third primer is different from the first primer. The binding can be shifted as compared to the first oligonucleotide primer. 27-31 schematically show the components of different embodiments of the first amplification system; (A) components of the first amplification system; (B) components of the second amplification system. FIGS. 27-29: The fourth primer is the same as the second primer, and the third primer is different from the first primer. The binding can be shifted as compared to the first oligonucleotide primer. 27-31 schematically show the components of different embodiments of the first amplification system; (A) components of the first amplification system; (B) components of the second amplification system. FIGS. 27-29: The fourth primer is the same as the second primer, and the third primer is different from the first primer. The binding can be shifted as compared to the first oligonucleotide primer. In FIG. 30, the fourth primer is different from the second oligonucleotide primer. The 3'end of the fourth primer can be shifted relative to the 3'end of the second oligonucleotide primer. The fourth primer contains a copyable sequence segment that is not complementary to the target sequence or does not bind to P1.1-Ext. In FIG. 31, the third oligonucleotide primer contains a copyable sequence segment that is not complementary to the target sequence or does not bind to P2.1-Ext. FIG. 32 schematically shows (A) the first oligonucleotide primer and (B) the amplified nucleic acids (P1.1-Ext and P2.1-Ext). FIG. 33 graphically illustrates chain substitution with a controller oligonucleotide. FIG. 34 schematically illustrates the interaction between the components in the reaction of the method according to the invention, with the second primer extension product from a complex having a duplex containing the first primer extension product and a controller oligonucleotide. It is a visualization of the intermediate steps to be separated. FIG. 35 schematically shows amplification by simultaneous synthesis of double strands and separation of the synthesized amplified fragments. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. FIGS. 36-49 show the experimental results of the sample. Figures 50 and 51 schematically show the preparation of a particular type of starting nucleic acid strand from genomic DNA. Figures 50 and 51 schematically show the preparation of a particular type of starting nucleic acid strand from genomic DNA. 52-54 schematically show a particular embodiment of amplification according to the present invention. 52-54 schematically show a particular embodiment of amplification according to the present invention. 52-54 schematically show a particular embodiment of amplification according to the present invention. FIGS. 55-57 graphically show a particular embodiment of topography of the components of the first amplification system and the second amplification system. FIGS. 55-57 graphically show a particular embodiment of topography of the components of the first amplification system and the second amplification system. FIGS. 55-57 graphically show a particular embodiment of topography of the components of the first amplification system and the second amplification system. Figures 58-59 graphically show specific embodiments of possible localized regions of probe oligonucleotides. Figures 58-59 graphically show specific embodiments of possible localized regions of probe oligonucleotides. FIG. 60 shows the localization of probe segments that can predominantly bind to the third (and optionally the first) primer extension products (P3.1-Ext and P1.1-Ext) in a complementary manner. Specific embodiments are shown graphically. FIG. 61 identifies the localization of probe segments that can predominantly bind complementarily to fourth (and optionally second) primer extension products (P4.1-Ext and P2.1-Ext). The embodiment of the above is shown graphically. Figures 62-67 graphically show specific embodiments of the probe. Figures 62-67 graphically show specific embodiments of the probe. Figures 62-67 graphically show specific embodiments of the probe. Figures 62-67 graphically show specific embodiments of the probe. Figures 62-67 graphically show specific embodiments of the probe. Figures 62-67 graphically show specific embodiments of the probe. Figures 68-71 show the starting nucleic acid 1.1, the amplified fragment 1.1 (P1.1-Ext and P2.1-Ext), and the second amplified fragment 2.1 (P3.1-Ext and P4.1). Specific embodiments of localization of target sequences 1 to 3 in −Ext) are shown graphically. Figures 68-71 show the starting nucleic acid 1.1, the amplified fragment 1.1 (P1.1-Ext and P2.1-Ext), and the second amplified fragment 2.1 (P3.1-Ext and P4.1). Specific embodiments of localization of target sequences 1 to 3 in −Ext) are shown graphically. Figures 68-71 show the starting nucleic acid 1.1, the amplified fragment 1.1 (P1.1-Ext and P2.1-Ext), and the second amplified fragment 2.1 (P3.1-Ext and P4.1). Specific embodiments of localization of target sequences 1 to 3 in −Ext) are shown graphically. Figures 68-71 show the starting nucleic acid 1.1, the amplified fragment 1.1 (P1.1-Ext and P2.1-Ext), and the second amplified fragment 2.1 (P3.1-Ext and P4.1). Specific embodiments of localization of target sequences 1 to 3 in −Ext) are shown graphically.

Description of the Invention A first aspect of the invention relates to a method for amplifying nucleic acids, which method comprises the following steps:
a) The first amplification step, which includes the next step:
-The step of hybridizing the first oligonucleotide primer (P1.1) to the amplified nucleic acid containing the target sequence, the first oligonucleotide primer (P1.1) contains the following regions:
A first region (P1.) In which the region of the nucleic acid to be amplified contains at least the 5'end of the target sequence or is located in the 5'direction of the target sequence and can be sequence-specifically bound to the region of the nucleic acid to be amplified. 1.1);
The second region can be attached by a controller oligonucleotide and remains essentially uncopied by the polymerase used under selected reaction conditions, adjacent to the 5'end of the first region, or a linker. Second region (P1.1.2) connected via
-Obtain a first primer extension product (P1.1-Ext) containing a synthetic region that is essentially complementary to the amplified nucleic acid or target sequence in addition to the first oligonucleotide primer (P1.1). It is a step of extension of the first oligonucleotide primer (P1.1) by the first polymerase for the purpose, and the first primer extension product and the amplified nucleic acid are present as double strands;
-A step of binding the controller oligonucleotide (C1.1) to the first primer extension product (P1.1-Ext), the controller oligonucleotide (C1.1) contains the following regions;
The first region (C1.1.1), which can bind to the second region (overhang) of the first primer extension product (P1.1-Ext),
A second region (C1.1.2) that is essentially complementary to the first region of the first oligonucleotide primer (P1.1), and a first primer extension product (P1.1-). A third region (C1.1.3) that is essentially complementary to at least a portion of the synthetic region of Ext); and where the controller oligonucleotide (C1.1) is the first oligonucleotide primer (P1). The first controller oligonucleotide (C1.1), which does not function as a template for primer extension in 1), replaces the amplified nucleic acid (region complementary to the first and second regions). It binds to the first and second regions of the primer extension product (P1.1-Ext) of 1.
-The step of hybridizing the second oligonucleotide primer (P2.1) to the first primer extension product, where the second oligonucleotide primer (P2.1) is at least complementary to the 5'end of the target sequence. Includes a region (P2.1.1) capable of sequence-specific binding to the synthetic region of the first primer extension product (P1.1-Ext), which is, or is located in the 3'direction thereof;
-A second primer extension product (P2.1-Ext) containing a synthetic region that is essentially identical to the nucleic acid or target sequence to be amplified in addition to the second oligonucleotide primer (P.2.1). It is a step of extension of the second oligonucleotide primer (P2.1) by the first polymerase to obtain, the first primer extension product (P1.1-Ext) and the second primer extension product (P2.1). ) Form the first double-stranded amplification product;
b) The second amplification step, which has the following steps:
-A step of hybridizing a third oligonucleotide primer (P3.1; P3.2) to a second primer extension product (P1.1-Ext) of the first amplification product, which is a third oligonucleotide primer. (P3.1; P3.2) can bind specifically to the region of the second primer extension product (P1.1-Ext) and contains at least the 5'end of the target sequence, or in the 5'direction thereof. It has a first region (P3.1.1) in which it is located;
-A third oligonucleotide containing a third oligonucleotide primer (P3.1; P3.2) plus a second primer extension product (P2.1-Ext) or a synthetic region that is essentially complementary to the target sequence. It is a step of extension of the third oligonucleotide primer (P3.1; P3.2) by the second polymerase to obtain the primer extension product (P3.1 / 2-Ext) of the second primer extension. The product (P2.1) and the third primer extension product (P3.1) are present as double strands;
-A step of hybridizing a fourth oligonucleotide primer (P4.1; P4.2) to a first primer extension product (P1.1-Ext), which is a step of hybridizing a fourth oligonucleotide primer (P4.1; P4.1; P4.2) binds specifically to the synthetic region of the first primer extension product (P1.1-Ext), which is at least complementary to the 5'end of the target sequence or located in the 3'direction thereof. Has a first region (P4.1.1) capable of;
-In addition to the fourth oligonucleotide primer, a fourth primer extension that contains a synthetic region that is essentially complementary to the first primer extension product (P1.1-Ext) or is identical to the target sequence. A step of extension of a fourth oligonucleotide primer (P4.1; P4.2) by a second polymerase to obtain a product (P4.1 / 2-Ext), which is a step of extension of the first primer extension product (P4.1; P4.2). P1.1-Ext) and the fourth primer extension product (P4.1 / 2-Ext) are present as double strands;
-Double strands from the first primer extension product (P1.1-Ext) and the fourth primer extension product (P4.1 / 2-Ext), and the second primer extension product (P2.1) and the second Step of double-stranded separation from primer extension product (P3.1) of 3;
-Hybrid the third oligonucleotide primer (P3.2) to the fourth primer extension product (P4.2-Ext) and extend the third oligonucleotide primer (P3.2) with the second polymerase. Step; and − Hybridize the 4th oligonucleotide primer to the 3rd primer extension product (P3.2-Ext) and use the 2nd polymerase to obtain the 4th oligonucleotide primer (P4.1; P4.2). Steps to extend.

Alternatively, aspects of the invention can also be configured as a method of amplifying nucleic acid (FIGS. 1, 55-56), including a first target sequence M1 [and a sequence M1'complementary to M1]. In the sample containing the nucleic acid polymer of the above, in the 5'-3'direction, M contains the sequence segments M1.5, M1.4, M1.3, M1.2, and M1.1 in succession immediately after. The sample is contacted with the following components in the first amplification step:
i. Suitable cofactors such as the first template-dependent nucleic acid polymerase, especially DNA polymerase, and substrates for template-dependent nucleic acid polymerases (particularly ribonucleoside triphosphate or deoxyribonucleoside triphosphate) and Mg salts.
ii. The first (right) oligonucleotide primer P1.1, which contains the sequence segments P1.1.2 and P1.1.1 in the 5'-3'direction in the immediate vicinity, is P1.1. It has a sequence [hybridizing] complementary to M1.1 [which can bind essentially sequence-specifically], and the sequence segment P1.1.2 is M1 [or 3'with respect to sequence M1 of M1.1. Immediately after sequence] cannot be attached, and here P1.1 (especially in segment P1.1.2) serves as a template for P1.1.2 to activate the first template-dependent nucleic acid polymerase. Includes modified nucleotide building blocks to prevent; and iii. To (essentially) identical to M1.5 [and to the reverse complementary sequence of M1.5 on the opposite strand of M1 or an extension of P1.1, P1.1-Ext (called P1.1E1). Can bind sequence-specifically] Second (left) oligonucleotide primer P2.1;
iv. In the 5'-3'direction, the sequence segments C1.2.3, C1.2.2, and C1.2.1 are the controller oligonucleotides C1.2 containing the most recent sequence, and C1.2.3 is a controller oligonucleotide C1.2. Identical to segment M1.2 of M1, which is located in the 5'direction of M1.1 [and first read at the start of polymerase of P1.1, thus C1.2.3 is primer P1.1. Can bind to Polymerase of −Ext], C1.2.2 is complementary to P1.1.1 (and is identical to M1.1) and C1.2.1 is complementary to P1.1.2. Being targeted
And here, C1.2 contains a nucleotide building block modified with C1.2.1 so that C1.2.1 cannot function as a template for activating a template-dependent nucleic acid polymerase.
And the sample is contacted with the following components in the second amplification step:
v. It is (reverse) complementary to M1.1 of M1 [can complementarily bind to P2.1-Ext] and contains a sequence segment 3.1.1 at the 3'end [or essentially 3.1. Consists of 1], 3rd (right) oligonucleotide primer P3.1,
vi. Identical and / or essentially identical to M1.5 of M1 [can complementarily bind to P1.1-Ext], containing sequence segment 4.1.1 at the 3'end [or essentially 4. Consisting of 1.1] Fourth (left) oligonucleotide primer P4.1,
vii. A second template-dependent nucleic acid polymerase, in particular a DNA polymerase, and optionally a substrate for the template-dependent nucleic acid polymerase (particularly ribonucleoside triphosphate or deoxyribonucleoside triphosphate) and suitable cofactors.

In the first amplification step, the first primer extension product P1.1-Ext is obtained, which sequences in the 5'-3'direction in addition to the sequence regions P1.1.2 and P1.1.1. Containing a synthetic region containing segments P1.1E4, P1.1E3, P1.1E2, and P1.1E1 in the 5'-3'direction, P1.1E4 essentially complements the sequence section M1.2 of target sequence M1. P1.1E3 is essentially complementary to M1.3, P1.1E2 is essentially complementary to M1.4, and P1.1E1 is essentially complementary to M1.5. is there. Similarly, in addition to the sequence region P2.1, a second primer extension product P2.1-Ext containing the synthetic region P2.1-Ext was obtained, which is the region following M1.5 in 3'. It is essentially the same as the target sequence M1 of. The reaction conditions and / or length or melting temperature of P1.1.2 and optionally M1.4, M1.3, M1.2 and M1.1 of the first amplification step are M1 and, or P2.1-. Ext and P1.1-Ext can form a double strand, P1.1-Ext can form a double strand with C1.2, and P1.1-Ext can form a double strand with C1.2. It is selected to take precedence over the formation of the double strand of P1.1-Ext and P2.1-Ext.

In a more general form, the invention can also be configured as a method for amplifying nucleic acids, with a sample containing a first nucleic acid polymer containing a first target sequence M1 in a first amplification step. Contact with the following ingredients:
a. First template-dependent nucleic acid polymerases, especially the first DNA polymerase, and substrates for template-dependent nucleic acid polymerases (particularly ribonucleoside triphosphate or deoxyribonucleoside triphosphate) and suitable cofactors.
b. The first (right) oligonucleotide primer P1.1 containing the sequence segments P1.1.2 and P1.1.1 in the 5'-3'direction in the immediate vicinity, P1.1.1. At least the 3'segment of is (essentially) sequence-specific and can bind to the segment of M1, the sequence segment P1.1.2 cannot bind to M1, and here P1.1 (particularly the segment P1.1. (2) includes a modified nucleotide building block such that P1.1.2 cannot function as a template for activating the first template-dependent nucleic acid polymerase; and c. It is (essentially) identical to the sequence segment of M1 located in the 5'direction of the binding site of P1.1, or is an inverse complementary sequence on the opposite strand of M1 or an extension of P1.1 (as P1.1E1). A second (left) oligonucleotide primer P2.1 containing at least one 3'segment capable of binding (called P1.1-Ext);
d. Controller oligonucleotide C1.2 containing the sequence segments C1.2.3, C1.2.2 and C1.2.1 in the 5'-3'direction in the most recent sequence:
i. A third region of the controller oligonucleotide (C1.2.3) that is essentially complementary to at least a portion of the synthetic region of the extension product (P1.1-Ext) of the first primer formed during the amplification reaction. Thus, in other words, the third region is identical to the segment of M1 in the 5'direction of the binding site of the first primer with respect to the polarity of the strand to be read [and first at the start of the polymerase of P1.1. Read, therefore, the third region of the controller can bind to at least a portion of the oligonucleotide [P1.1-Ext] bound to the polymerization product of the first primer.
ii. A second of controller oligonucleotides that is essentially complementary (and at least partially identical to the binding site of the first primer on target sequence M1) to the first region of the first oligonucleotide primer (P1.1). Region (C1.2.2) and iii. The first region of the controller oligonucleotide that can bind to the sequence segment P1.1.2 of the first primer or the 5'end region of the extension product (P1.1-Ext) of the first primer formed during the amplification reaction. (C1.2.1), and where C1.2 is at least C1.2.1 so that C1.2.1 cannot function as a template for activating template-dependent nucleic acid polymerases. Includes C1.2.3 modified nucleotide building blocks;
Under conditions that allow the first amplification, the sample is incubated with the above components and the primer extension product of the first oligonucleotide primer (P1.1-Ext) and the primer extension product of the second oligonucleotide primer. An amplification product containing (P2.1-Ext) is formed, which contains the target sequence M or at least a portion thereof and the corresponding complementary sequence; and the sample is subsequently or simultaneously subjected to a second amplification. Contact with the following ingredients in the step:
e. It is (reverse) complementary to the amplification product formed in the first amplification step, in other words, it is sequence-specific and 3'of the extension product of the second primer formed in the first amplification reaction. Third (right) oligonucleotide primer P3.1, which can bind to the terminal region and contains the sequence segment 3.1.1 at the 3'end [or consists essentially of 3.1.1],
f. It can (reversely) complementarily bind to the amplification product formed in the first amplification step, in other words, it is specific to the 3'end region of the extension product of the first primer formed in the first amplification reaction. Fourth (left) oligonucleotide primer P4.1, which contains the sequence segment 4.1.1 at the 3'end and [or essentially consists of 4.1.1] capable of binding to.
g. A second template-dependent nucleic acid polymerase, in particular a DNA polymerase, and optionally a substrate for the template-dependent nucleic acid polymerase (particularly ribonucleoside triphosphate or deoxyribonucleoside triphosphate) and suitable cofactors.

The sample was incubated with the above components under conditions that allowed a second amplification, and the primer extension product of the third oligonucleotide primer (P3.1-Ext) and the primer extension product of the fourth oligonucleotide primer (P3.1-Ext). An amplification product containing P4.1-Ext) is formed, which comprises the target sequence M or at least a portion thereof and the corresponding complementary sequence.

However, the present invention is not necessarily limited to the topologies shown in FIGS. 50-56.

A further aspect of the invention relates to a kit containing the following ingredients:
a. The first (right) oligonucleotide primer P1.1 containing two sequence segments P1.1.2 and P1.1.1 in the 5'-3'direction in the immediate vicinity.
i. P1.1.1 is an arbitrary sequence segment that complementarily [hybridizes] to the amplified sequence [M1.1], and ii. The sequence segment P1.1.2 cannot bind to the amplified sequence [or the sequence immediately following 3'with respect to the amplified sequence]; and iii. P1.1.2 contains a nucleotide building block modified such that P1.1.2 cannot function as a template for the activity of a template-dependent nucleic acid polymerase; and b. A second (left) oligonucleotide primer P2.1 that can bind to the sequence on the opposite strand of the sequence to which the first oligonucleotide primer is bound in a sequence-specific manner;
c. A controller oligonucleotide C1.2 containing the sequence segments C1.2.3, C1.2.2 and C1.2.1 in the immediate vicinity in the 5'-3'direction.
i. C1.2.3 is identical to segment M1.2 of the amplified sequence [M1] and is located at 5'of the binding site of [M1.1] P1.1.1 [and of P1.1]. First read at the start of the polymerase, therefore C1.2.3 can bind to the polymerization product P1.1-Ext of the oligonucleotide primer],.
ii. C1.2.2 is complementary to P1.1.1 (and identical to M1.1), and iii. C1.2.1 is complementary to p1.1.2 and
And C1.2 contains a nucleotide building block modified with C1.2.1 so that C1.2.1 cannot function as a template for activating a template-dependent nucleic acid polymerase;
d. Suitable cofactors such as the first template-dependent nucleic acid polymerase, especially DNA polymerase, and optionally the substrate of the template-dependent nucleic acid polymerase (particularly ribonucleoside triphosphate or deoxyribonucleoside triphosphate) and Mg salt;
e. Unlike the first oligonucleotide primer P1.1, it does not specifically have section P1.1.2 and is (reverse) complementary to [section M1.1] of the target sequence [M1] at the 3'end. Third (right) oligonucleotide primer P3.1, which contains the sequence segment 3.1.1 [or consists essentially of 3.1.1];
f. It contains a sequence segment 4.1.1 at the 3'end [or essentially consists of 4.1.1] capable of sequence-specific binding to the sequence opposite to the sequence to which the third oligonucleotide primer is attached. Fourth (left) oligonucleotide primer P4.1;
g. A second template-dependent nucleic acid polymerase, in particular a DNA polymerase, and optionally a substrate for the template-dependent nucleic acid polymerase (particularly ribonucleoside triphosphate or deoxyribonucleoside triphosphate) and suitable cofactors.

A further aspect of the present invention relates to a method for amplifying nucleic acid, which is a sample containing nucleic acid containing the first target sequence M1 in the 5'-3'direction with sequence segments M1.5, M1.4, The sample containing M1.3, M1.2, and M1.1 in succession immediately after is contacted with the following components in the first amplification step;
-The first template-dependent nucleic acid polymerase, especially the DNA polymerase, and the substrate for the template-dependent nucleic acid polymerase,
The first (right) oligonucleotide primer P1.1, which contains the sequence segments P1.1.2 and P1.1.1 in the most recent sequence in the −5'-3'direction, is P1.1.1. It has a sequence that [hybridizes] complementary to M1.1, and the sequence segment P1.1.2 cannot bind to M1, and where P1.1 (especially at site P1.1.2) Includes a modified nucleotide building block such that P1.1.2 cannot function as a template for activating the first template-dependent nucleic acid polymerase; and-is (essentially) identical to M1.5, the second. 2 (left) oligonucleotide primer P2.1,
A controller oligonucleotide C1.2 in which the sequence segments C1.2.3, C1.2.2, and C1.2.1 are consecutively contained immediately after the sequence segment C1.2.3, C1.2.2, and C1.2.1 in the −5'-3'direction, and C1.2.3. Is the same as the segment M1.2 of M1 located in the 5'direction of M1.1, C1.2.2 is complementary to P1.1.1 (and is the same as M1.1), and And C1.2.1 are complementary to P1.1.2,
And here, C1.2 comprises a nucleotide building block modified with C1.2.1 so that C1.2.1 cannot function as a template for activating a template-dependent nucleic acid polymerase.
Then, the sample is further contacted with the first probe oligonucleotide, and the first probe oligonucleotide is:
a. Containing a sequence segment, the sequence segment is i. The sequence is identical to the sequence of M1 located in the sequence segments M1.2, M1.3, and M1.4; or ii. Complementary to the M1 sequence located in sequence segments M1.3 and M1.4,
b. It binds to a fluorescent dye, and the fluorescent dye is
i. Form a donor quencher pair or FRET pair with a second probe oligonucleotide linked to a first probe oligonucleotide; or ii. It forms a donor quencher pair or FRET pair with a fluorescent dye linked to a second probe oligonucleotide that can bind in close proximity to the binding site of the first probe oligonucleotide.

Further, aspects of the invention can be configured as methods involving the following steps:
A) A step of providing a nucleic acid fragment (hereinafter, also referred to as a starting nucleic acid chain) containing (first target sequence).
B) A first amplification system (amplification) containing a first oligonucleotide primer, a second oligonucleotide primer, a controller oligonucleotide, a first DNA polymerase, and a substrate (dNTP) for the first polymerase, a suitable buffer solution. Steps to provide the system).
C) A step of providing a second amplification system containing a third oligonucleotide primer, a fourth oligonucleotide primer, a second thermostable polymerase and a substrate for the second polymerase (dNTP), and a suitable buffer solution. ..
Optionally, a detection system is further provided as long as the object of the present invention also includes the detection of nucleic acid fragments amplified by the first and / or second amplification system, especially using a probe.
D) The first template to obtain a first amplified fragment 1.1 containing a first target sequence and containing a first primer extension product and a second primer extension product capable of forming complementary duplexes. In the step of performing the first amplification using the starting amplification chain as the strand, the first primer extension product is the first with a polymerase using the starting nucleic acid chain as a template and / or the second primer extension product. The first primer extension results from the template-dependent extension of the first primer, and the second primer extension product results from the template-dependent extension of the second primer by a polymerase that uses the first primer extension product as a template. And the second primer extension products act as templates for their respective primer extensions, and both complementary strands of the first amplification product 1.1 are the respective primer extension products from which new primers have been synthesized. Coordination of controller oligonucleotides can at least partially convert to a single-stranded form so that it can bind to complementary segments.
The reaction conditions used for the first amplification are at least one temperature step that allows hybridization of both primers in the first amplification system and template-dependent primer extension, and the first primer extension product is the controller oligonucleotide. Includes at least one temperature step that is separated from the second primer extension product by the involvement of. The reaction conditions used do not allow spontaneous separation of the first primer extension product from the second primer extension product in the absence of the controller oligonucleotide. The first amplification is carried out until the desired amount of the first amplification product 1.1 is synthesized.
E) First amplification as the first template strand to obtain a second amplification fragment 2.1 containing a third primer extension product and a fourth primer extension product capable of forming complementary duplexes. In the step of performing the second amplification using at least one of the two strands of fragment 1.1 and using the second amplification system, both strands of the growth fragment 2.1 are primer extension. Hybridization of the two primers of the second amplification system and binding to the complementary region in at least one temperature step, depending on the reaction conditions used in the second amplification, which can act as templates for each other in. Allows template-dependent primer extension of the primers of the second amplification system and allows double-stranded separation in at least one additional temperature step, including a third primer extension product and a fourth primer extension product. The third and fourth primer extension products are converted to single strands for reprimer binding and extension. The second amplification is carried out until the desired amount of the second amplification product 2.1 is synthesized.

Specifically, the properties of the components of each amplification system, the properties of the nucleic acid fragment containing the first target sequence, and the properties of the reaction conditions used determine the process of the two amplification reactions.

Generally, the first amplification is done with the involvement of controller oligonucleotides. Separation of the two synthetic primer extension products depends in part on the sequence of at least the first primer extension product and its complementarity to the sequence of the controller oligonucleotide.

During the second amplification, the second amplified fragment is essentially amplified without the involvement of the controller oligonucleotide.

Both amplification reaction processes can form both the amplification products of interest (amplification fragment 1.1 and amplification fragment 2.1) and their intermediates (intermediate products).

The composition of the intermediate is essentially dependent on the components of the first and second amplification systems provided.

Detailed Description of the Invention The object of the invention mentioned at the beginning is particularly solved by a combination of two consecutive amplifications, with PCR amplification being the final step.

Another object of the invention is solved by using the probe system described below in certain embodiments and embodiments.

In the following, some molecular processes and their resulting products, and possible intermediate steps thereof, will be described in an exemplary and schematic manner. According to aspect 1 of the present invention, a method of amplification is provided. This method involves the following steps:
1) First amplification step including the next step 1. A) A step of hybridizing the first oligonucleotide primer to the 3'segment of a nucleic acid fragment that functions as a template (template chain, starting nucleic acid chain) of the first nucleic acid to be amplified, including the target sequence. Oligonucleotide Primers contain the following regions:
-A first region that can sequence-specifically bind to the 3'segment of the template strand of the first nucleic acid to be amplified and is complementary to at least a portion of the target sequence;
-The 5'end of the first region, where the second region can be bound by the controller oligonucleotide and remains essentially uncopied by the polymerase used for the first amplification under selected reaction conditions. Second region adjacent to or connected via a linker;
1. 1. B) In addition to the first oligonucleotide primer, a first template-dependent to obtain a first primer extension product containing a region synthesized by the polymerase that is essentially complementary to the template strand of the target sequence. A step of extension of the first oligonucleotide primer when hybridized to a complementary segment of the first nucleic acid amplified by sex polymerase, which is a template chain of the first primer extension product and the first nucleic acid to be amplified. Exists essentially as a double strand under the reaction conditions used in the first amplification;
1. 1. C) The step of binding the controller oligonucleotide to the first primer extension product, the controller oligonucleotide contains the following regions;
A first region capable of binding to a second region of a first oligonucleotide primer and / or a first primer extension product;
At least in the second region, which is essentially complementary or completely complementary to the first region of the first oligonucleotide primer and / or the first primer extension product, and in the region of the primer extension product synthesized by the polymerase. A third region that is partially complementary or essentially complementary;
Here, the controller oligonucleotide does not function as a template for primer extension of the first oligonucleotide primer, and the controller oligonucleotide is identical or essentially identical to one strand of the template strand (target sequence). The sequence segment is included, and the controller oligonucleotide binds to the first region of the first primer extension product, and
The controller oligonucleotide at least partially binds to the second region of the first primer extension product and the synthetic region of the first primer extension product, thereby complementing the template strand of the first nucleic acid to be amplified. Substitute the target portion; where the polymerase synthesis region of the first primer extension product, which comprises a segment of the first primer extension product complementary to the second oligonucleotide primer, becomes single-stranded under reaction conditions. Become;
1. 1. D) The step of hybridizing the second oligonucleotide primer to the single-stranded complementary segment of the first primer extension product, the second oligonucleotide primer being sequenced in the synthetic region of the first primer extension product. Contains regions that can specifically hybridize and contains at least a portion of the target sequence;
1. 1. E) In addition to the second oligonucleotide primer, the first primer extension product is used as a template to obtain a second primer extension product containing a polymerase synthesis region containing at least a portion of the first target sequence. , The step of extension of the second oligonucleotide primer by the first polymerase, in which the first primer extension product and the second primer extension product are the first double strand under the reaction conditions of the first amplification. Form amplification products, and 1. F) A step of repeating the first amplification step, including a plurality of iterations, as needed.
2) Second amplification step including the next step (FIG. 15):
2. A) A step of hybridizing a third oligonucleotide primer to a second primer extension product of the first amplification product, wherein the third oligonucleotide primer is essentially in the segment of the second primer extension product. Contains a first region that can bind sequence-specifically;
2. B) In addition to the third oligonucleotide primer, a third primer extension containing a region synthesized by a polymerase containing a second oligonucleotide primer or a sequence segment that is essentially complementary to a portion of the amplified nucleic acid or target sequence. A step of extension of a third oligonucleotide primer by a second polymerase using a second primer extension product as a template to obtain a product (P3.1-Ext part1), wherein the second oligonucleotide primer and the second The 3-primer extension product (P3.1-Ext part1) is double-stranded;
2. C) A step of hybridizing the fourth oligonucleotide primer to the first primer extension product of the first amplification product, wherein the fourth oligonucleotide primer is essentially in the polymerase synthesis region of the first primer extension product. Contains a first region that can bind sequence-specifically;
2. D) In addition to the 4th oligonucleotide primer, a 4th primer extension product (P4) that is essentially complementary to the 1st oligonucleotide primer and contains a region of the target sequence that is synthesized by the polymerase. A step of extension of a fourth oligonucleotide primer by a second polymerase using the first primer extension product as a template to obtain 1-Ext part1), the first primer extension product and the fourth primer. The extension product (P4.1-Ext part1) exists as a double strand;
2. E) The double strand was separated from the first primer extension product and the fourth primer extension product (P4.1-Ext part1), and the second primer extension product and the third primer extension product (P3.1-Ext) were separated. Step to separate double strand from part1);
2. F) The step of hybridizing the third oligonucleotide primer to the fourth primer extension product (obtained in step 2.D, P4.1-Ext part1), wherein the third oligonucleotide primer is essentially. Contains a first region that is sequence-specific and capable of binding to the fourth primer extension product segment (P4.1-Ext part1);
2. G) A third oligo by a second polymerase using a fourth primer extension product (P4.1-Ext part1) as a template to obtain a complete third primer extension product (P3.1-Ext). In the step of nucleotide primer extension, the third primer extension product (P3.1-Ext) and the fourth primer extension product (P4.1-Ext part1) exist as double strands;
2. H) The step of hybridizing the fourth oligonucleotide primer to the third primer extension product (obtained in step 2B, P3.1-Ext part1), wherein the fourth oligonucleotide primer is the third primer. Includes a first region that can essentially bind sequence-specifically to the polymerase synthesis region of the elongation product;
2. I) A fourth primer extension product (P3.1-Ext part1) by a second polymerase using a third primer extension product (P3.1-Ext part1) as a template to obtain a complete fourth primer extension product (P4.1-Ext). In the step of oligonucleotide primer extension, a third primer extension product (P3.1-Ext part1) and a fourth primer extension product (P4.1-Ext) are present as double strands;
2. J) Separation of the duplex by the third primer extension product (P3.1-Ext part1) and the complete fourth primer extension product (P4.1-Ext), and the fourth primer extension product (P4. Separation of double strands with 1-Ext part1) and complete third primer extension product (P3.1-Ext);
2. K) The step of hybridizing the third oligonucleotide primer to the complete fourth primer extension product (obtained in step 2I, P4.1-Ext), wherein the third oligonucleotide primer is essentially. Contains a first region that is sequence-specific and capable of binding to the fourth primer extension product segment (P4.1-Ext);
2. L) A third with a second polymerase using the complete fourth primer extension product (P4.1-Ext) as a template to obtain the complete third primer extension product (P3.1-Ext). In the step of oligonucleotide primer extension, the complete third primer extension product (P3.1-Ext) and the complete fourth primer extension product (P4.1-Ext) are present as double strands. ;
2. M) The step of hybridizing the fourth oligonucleotide primer to the complete third primer extension product (obtained in step 2G, P3.1-Ext), wherein the fourth oligonucleotide primer is the complete third primer. Includes a first region that is sequence-specific and essentially capable of binding to the polymerase synthesis region of the primer extension of 3;
2. N) A fourth with a second polymerase using the complete third primer extension product (P3.1-Ext) as a template to obtain the complete fourth primer extension product (P4.1-Ext). In the step of oligonucleotide primer extension, the third primer extension product (P3.1-Ext) and the complete fourth primer extension product (P4.1-Ext) are present as double strands;
2. O) Step 2. Consisting of a complete third primer extension product (P3.1-Ext) and a complete fourth primer extension product (P4.1-Ext). L and 2. The step of separating the double strand formed by N;
2. P) Optionally, repeat the second amplification step to complete the third primer extension product (P3.1-Ext) and the complete fourth primer extension product (P4.1-Ext). Steps to increase and repeat arbitrarily several times.

In particular, aspects and embodiments relating to the detection of the resulting amplification product with a probe may be aimed at adding a detection system to the reaction mixture.

Alternatively, said aspect of the invention can also be configured as a method of amplifying nucleic acid (FIGS. 1, 5):
1) Performing a first amplification to propagate the growth of the first amplification fragment 1.1 containing the target sequence, the first amplification comprising the following steps:
a) The step of hybridizing the first oligonucleotide primer to the 3'segment of the chain of the amplified nucleic acid chain, wherein the amplified nucleic acid chain comprises the target sequence.
The first oligonucleotide primer contains the following regions:
-The first primer region of the 3'segment of the first oligonucleotide primer that can be sequence-specifically bound to the strand of the amplified nucleic acid chain-Binding the controller oligonucleotide and substituting the strand with the controller oligonucleotide (step c) ), A second region attached to the 5'end of the first primer region of the first oligonucleotide primer, either directly or via a linker, comprising a polynucleotide tail suitable for supporting the polymerase. The polynucleotide tail remains essentially uncopied under selected reaction conditions.
b) The step of extension of the first oligonucleotide primer by a polymerase to form a first primer extension product containing a sequence complementary to the target sequence (a) of the amplified nucleic acid chain,
c) In the step of attaching the controller oligonucleotide to the polynucleotide tail of the second region of the first extended oligonucleotide primer, the controller oligonucleotide contains the following regions:
A first single-stranded region that can bind to the polynucleotide tail of the second region of the first oligonucleotide primer,
A second single-stranded region that is essentially complementary to and can bind to the first region of the first oligonucleotide primer,
A third single-stranded region that is essentially complementary to at least one segment of the polymerase synthetic extension of the first primer extension,
Here, the controller oligonucleotide does not function as a template for primer extension of the first oligonucleotide primer.
d) The step of binding the controller oligonucleotide to the first primer region of the first extended oligonucleotide primer and substituting the complementary strand for the first primer region of the nucleic acid strand amplified thereby.
e) The step of binding the controller oligonucleotide to the complementary segment of the extension product of the first extension oligonucleotide primer, thereby substituting the strand of the amplified nucleic acid chain complementary to the extension product. The 3'segment of the primer extension product of 1 becomes a single strand,
f) In the step of hybridizing the second oligonucleotide primer to the first primer extension product, the 3'segment of the second oligonucleotide primer contains a sequence capable of hybridizing to the first primer extension product. , And g) A step of extending a second oligonucleotide primer with a polymerase to form a second primer extension product, the extension comprising the first primer region of the first oligonucleotide primer. The first primer region is copied by the polymerase and the polynucleotide tail of the second region remains uncopied.
(H) A step of repeating steps (a) to (g) until a desired level of amplification is achieved.
2) Using the first amplified fragment 1.1 obtained in the first amplification reaction as a template, and using the third primer, the fourth primer, the second polymerase, and an appropriate cofactor, the target sequence or target A step of performing a second amplification leading to an increase in the second amplification fragment 2.1 containing at least a segment of the sequence (or a complementary sequence thereof), wherein the third oligonucleotide primer is a second. It can bind to the primer extension product and / or the fourth primer extension product in an essentially sequence-specific manner and can be extended by the second polymerase.
The fourth oligonucleotide primer can bind to the first primer extension product and / or the third primer extension product in an essentially sequence-specific manner and can be extended by the second polymerase.
And the primer extension products obtained from the third and fourth primers can function as a template for exponential amplification of the second amplification fragment 2.1.

Further, in the method according to any of the above-described embodiments, the condition that the second amplification is PCR (polymerase chain reaction, also referred to as PCR amplification) and enables the amplification of the second amplification fragment 2.1 containing the target sequence. It can be characterized by being done below.

Furthermore, the method according to any one of the aforementioned embodiments may be characterized in that at least the second amplification is PCR amplification performed in the presence of a detection system containing probe oligonucleotides.

Further, in the method according to any one of the above embodiments, the second amplification is PCR amplification, at least one step in which the primers are hybridized and / or extended by the polymerase, and one primer extension product obtained. It can be characterized by being performed under conditions involving at least one step of conversion (modification) to chain form.

Furthermore, the method according to any one of the above embodiments may be characterized in that the second amplification is PCR amplification and is carried out until a desired amount of amplification fragment 2.1 is synthesized.

Further, in the method according to any one of the above-described embodiments, the third oligonucleotide primer of the second amplification is the 3'segment of the second primer extension product and / or the 3'segment of the fourth primer extension product. It can be characterized in that it can bind to and can be extended by a polymerase.

Further, in the method according to any one of the above-described embodiments, the fourth oligonucleotide primer of the second amplification is the 3'segment of the first primer extension product and / or the 3'segment of the third primer extension product. It can be characterized in that it can bind to and can be extended by a polymerase.

In addition, the method according to any one of the aforementioned embodiments allows the third oligonucleotide primer of the second amplification to bind to the second primer extension product and / or the fourth primer extension product in its 3'segment. , Can be characterized in that it can be extended by a polymerase.

In addition, the method according to any one of the aforementioned embodiments allows the fourth oligonucleotide primer of the second amplification to bind to the first primer extension product and / or the third primer extension product in its 3'segment. , Can be characterized in that it can be extended by a polymerase.

Further, in the method according to any one of the above-described embodiments, the extension product of the third oligonucleotide primer and / or the extension product of the fourth oligonucleotide primer of the second amplification is at least one segment of the target sequence or It may be characterized by containing its complementary sequence.

In the method according to any one of the above embodiments, the third single-stranded region of the controller oligonucleotide is a segment of the extension product of the first primer extension product synthesized by the polymerase (immediately after the first primer region). It is characterized by being essentially complementary to (adjacent).

Further, in the method according to any one of the above-described embodiments, the substitution of the amplified nucleic acid strand complementary to the first primer extension product is carried out, and the complementary strand of the amplified nucleic acid is the first primer extension. It can be characterized by being carried out until it leaves the product.

In addition, the method according to any one of the aforementioned embodiments is modified in that step (f) of the method comprises hybridization of the second oligonucleotide primer to the first primer extension product, the first extension. At least partial substitution of the controller oligonucleotide from binding to the product can be characterized by being simultaneously achieved by strand substitution.

In addition, the method according to any one of the aforementioned embodiments is modified such that step (g) of the method involves substitution of the controller oligonucleotide from binding to a first primer extension product under the control of a polymerase. It can be characterized by being done.

Further, in the method according to any one of the above-described embodiments, the step (h) of the method is that the controller oligonucleotide is complementary to the segment of the first specific extension product of the first oligonucleotide primer. Binding of the controller oligonucleotide to the uncopied polynucleotide tail of the first extension oligonucleotide primer and substitution of the second primer extension product from binding to the first primer extension product while forming a chain. It may be characterized by being modified to include.

Further, the method according to any one of the above-described embodiments can be characterized in that the step repetition is performed under conditions that allow the repetition of steps (a) to (g).

Furthermore, the method according to any one of the above embodiments is characterized by including simultaneous amplification of the first and second primer extension products in an exponential reaction using first and second oligonucleotide primers and controller oligonucleotides. The primer extension product functions as a template for mutual synthesis.

Yields of individual products and intermediates depend on several factors. For example, the higher the concentration of individual oligonucleotide primers, the higher the yield of the product / intermediate in general. In addition, product / intermediate formation is influenced by the choice of reaction temperature and the mutual binding affinity of the individual reactants (the affinity of individual primer binding to complementary primer binding sites within the template strand). It is possible to receive: For example, longer oligonucleotides generally bind better than shorter oligonucleotides at higher temperatures, and CG levels can also play a role in complementary sequence segments: CG Rich sequences also bind more strongly at higher temperatures than AT-rich sequences. In addition, modifications such as MGB or 2-amino-dA or LNA can increase the binding strength of the primers to their respective complementary segments, which also preferentially binds the oligonucleotide primers at elevated temperatures.

Therefore, it can be inferred that by designing the sequence segments of the individual primer sequences, it is possible to influence the yield of specific products / intermediates and thus their enrichment during amplification.

In addition, the nucleic acid amplification method according to the invention (FIGS. 1, 5, 55-57) can be constructed as a result of the following steps:
a) First amplification step, including the next step:
-Step of providing the starting nucleic acid 1.1 containing the target sequence-The first oligonucleotide primer (P1.1) is amplified with the target sequence (either the starting nucleic acid 1.1 or P2.1-Ext). In the step of hybridizing to nucleic acid, the first oligonucleotide primer (P1.1) contains the following regions:
The first region (P1.1.1), which can be sequence-specifically bound to the region of nucleic acid to be amplified (P2.1E1),
A second region (P1.1.2) adjacent to the 5'end of the first region or connected via a linker, the second region can be attached by a controller oligonucleotide and selected. It remains essentially uncopied by the polymerase used under the reaction conditions that were used;
-A first primer extension containing a synthetic region (P1.1E1 to P1.1E4) that is essentially complementary to the amplified nucleic acid or target sequence in addition to the first oligonucleotide primer (P1.1). It is a step of extension of the first oligonucleotide primer (P1.1) by the first polymerase to obtain the product (P1.1-Ext), and the first primer extension product and the amplified nucleic acid are two. Exists as a main chain;
-A step of binding the controller oligonucleotide (C1.2) to the first primer extension product (P1.1-Ext), the controller oligonucleotide (C1.2) contains the following regions;
The first region (C1.2.1), which can bind to the second region (overhang) of the first primer extension product (P1.1E6),
The second region (C1.2.2), which is complementary to the first region of the first oligonucleotide primer (P1.1.1), and the first primer extension product (P1.1E4). A third region (C1.2.3) that is essentially complementary to at least a portion of the synthetic region; and where the controller oligonucleotide (C1.1) is the first oligonucleotide primer (P1.1). Does not function as a template for primer extension, and the controller oligonucleotide (C1.1) binds to and is amplified by the first primer extension products (P1.1E6, P1.1E5, P1.1E4). Replaces complementary regions of nucleic acids (P2.1E1, P2.1E2);
-The step of hybridizing the second oligonucleotide primer (P1.1) to the first primer extension product, the second oligonucleotide primer (P2.1) is the first primer extension product (P1.1E1). ) Includes a region (P2.1.1) that can be sequence-specifically bound to the synthetic region.
-A second primer extension product (P2.1E4 to P2.1E1) containing a synthetic region (P2.1E4 to P2.1E1) that is essentially identical to the nucleic acid or target sequence to be amplified in addition to the second oligonucleotide primer (P2.1). A step of extension of the second oligonucleotide primer (P2.1) by the first polymerase to obtain P2.1-Ext), the first primer extension product (P1.1-Ext) and the second. The primer extension product (P2.1) forms the first double-stranded amplification product; and-the first amplification step is repeated until the desired amount of the first amplification product is synthesized.
b) Second amplification step, including the next step:
− The step of hybridizing the third oligonucleotide primer (P3.2) to the second primer extension product (P1.1-Ext), the third oligonucleotide primer (P3.2) is the second primer. A first region (P3.1.1) capable of sequence-specific binding to the region of the primer extension product (P2.1E1) is included.
-In addition to the third oligonucleotide primer (P3.2), a synthetic region (P3.1E5 to P3.1E2) that is essentially complementary to the second primer extension product (P2.1-Ext) or target sequence. Alternatively, it is a step of extension of the third oligonucleotide primer (P3.2) by the second polymerase to obtain a third primer extension product (P3.2-Ext) having P3.1E5 to P3.1E1). The second primer extension product (P2.1) and the third primer extension product (P3.1) are present as double strands;
-A step of hybridizing a fourth oligonucleotide primer (P4.2) to a first primer extension product (P1.1-Ext) and / or a third primer extension product (P3.1-Ext). , The fourth oligonucleotide primer (P4.2) can bind specifically to the synthetic region of the first primer extension product (P1.1E1) and the third primer extension product (P3.1E2). Includes region (P4.1.1),
-From the synthetic region (from P4.1E5) that is essentially complementary to the first primer extension product (P1.1-Ext) or essentially identical to the target sequence, in addition to the fourth oligonucleotide primer. A fourth oligonucleotide primer (P4.2) with a second polymerase to obtain a fourth primer extension product (P4.2-Ext) containing P4.1E2 or P4.1E5 to P4.1E1). In the extension step, the first primer extension product (P1.1-Ext) and the fourth primer extension product (P4.1-Ext) exist as double strands;
-Separate the double strand by the first primer extension product (P1.1-Ext) and the fourth primer extension product (P4.2-Ext), and separate the second primer extension product (P2.1-Ext) and The double strand by the third primer extension product (P3.1-Ext) is separated, and by the fourth primer extension product (P4.1-Ext) and the third primer extension product (P3.1-Ext). Steps to separate double strands;
-The third oligonucleotide primer (P3.2) is hybridized to the fourth primer extension product (P4.2-Ext), and the third oligonucleotide primer (P3.2) is extended by the second polymerase. Steps; and-The fourth oligonucleotide primer is hybridized to the third primer extension product (P3.2-Ext) and the fourth oligonucleotide primer (P4.2) is extended by the second polymerase. Step.

Furthermore, the present invention includes the following aspects:

The method of aspect 1, wherein the nucleic acid fragment comprises a single-stranded form of the target sequence.

The method according to aspect 1, wherein the nucleic acid fragment comprising the target sequence is provided in double-stranded form (including the first target sequence, the starting nucleic acid strand, and a nucleic acid fragment complementary thereto), and the double-stranded nucleic acid. Fragments are converted to single-stranded form prior to the first amplification.

In the method according to aspect 1, the second amplification is carried out using the third and fourth primers as the polymerase chain reaction (PCR).

In the method according to aspect 1, the separation of the synthesized strand during the first amplification is sequence-specific, mainly with the involvement of the controller oligonucleotide.

The method according to aspect 1, wherein the separation of the synthesized strands during the second amplification is performed using a temperature that can cause the separation of the synthesized strands. For example, such temperatures include a temperature range of 80 ° C to 105 ° C.

In the method according to aspect 1, hybridization and primer extension during the second amplification result in predominantly complementary binding of the primer to the complementary sequence segment of the synthesized strand, and primer extension by the second polymerase. It is done at a temperature that allows it. For example, such temperatures include a temperature range of 20 ° C to about 80 ° C.

In the method according to the first aspect, the first and second primer extension products synthesized during the first amplification are separated before the second amplification, and both primer extension products are single-stranded. Become.

In the method according to aspect 1, the first oligonucleotide primer comprises a sequence segment in its first region capable of complementarily binding to the first target sequence of its 3'segment.

The method according to aspect 1, wherein the second oligonucleotide primer contains a sequence segment in its 3'region that is essentially identical to a portion of the first target sequence, the portion of which is the 5'segment of the target sequence. Located in.

In the method according to aspect 1, the controller oligonucleotide contains a sequence segment in its second and third regions that is essentially identical to a portion of the target sequence.

In the method according to aspect 1, a first target sequence and a strand complementary to the first target sequence are contained on both sides of a primer pair containing a first oligonucleotide primer and a second oligonucleotide primer.

The method according to aspect 1, wherein the third oligonucleotide primer comprises a sequence segment in its 3'region that is capable of binding essentially complementaryly to the second primer extension product of the first amplified fragment 1.1.

In the method according to aspect 1, the third oligonucleotide primer comprises a sequence segment in its 3'region that is capable of binding essentially complementary to the second primer extension product of the first amplified fragment 1.1. The 3'region contains a segment complementary to the first target sequence.

The method according to aspect 1, wherein the fourth oligonucleotide primer comprises a sequence segment in its 3'region that is capable of binding essentially complementaryly to the first primer extension product of the first amplified fragment 1.1.

In the method according to aspect 1, the fourth oligonucleotide primer comprises a sequence segment in its 3'region that is capable of binding essentially complementaryly to the first primer extension product of the first amplified fragment 1.1. Further, the 3'region contains a segment that is essentially identical to a portion of the first target sequence, which portion is in the 5'region of the target sequence.

In the method according to aspect 1, the first target sequence and the strand complementary to the target sequence are formed at least partially on both sides of the primer pair formed by the third oligonucleotide primer and the fourth oligonucleotide primer. included.

In the method according to the first aspect, the first oligonucleotide primer and the third oligonucleotide primer are the same.

In the method according to the first aspect, the first oligonucleotide primer and the third oligonucleotide primer are the same, and the primer extension products (P4.1-Ext part1) and (P4.1-) generated during the second amplification. Ext) is the same.

In the method according to the first aspect, the first oligonucleotide primer and the third oligonucleotide primer are the same, but the second and fourth primers are different.

In the method according to the first aspect, the first oligonucleotide primer and the third oligonucleotide primer are the same, and the primer extension products (P4.1-Ext part1) and (P4.1-) generated during the second amplification. Ext) is the same, but the primer extension products of the second and fourth primers are different.

In the method according to the first aspect, the second oligonucleotide primer and the fourth oligonucleotide primer are the same.

In the method according to the first aspect, the second oligonucleotide primer and the fourth oligonucleotide primer are the same, and the primer extension products (P3.1-Ext part1) and (P3.1-) generated during the second amplification. Ext) is the same.

In the method according to the first aspect, the second oligonucleotide primer and the fourth oligonucleotide primer are the same, but the first and third oligonucleotide primers are different.

In the method according to the first aspect, the second oligonucleotide primer and the fourth oligonucleotide primer are the same, and the primer extension products (P3.1-Ext part1) and (P3.1-) generated during the second amplification. Ext) is the same, but the primer extension products of the first and third oligonucleotide primers are different.

In the method according to the first aspect, the first oligonucleotide primer, the third oligonucleotide primer, and the second and fourth oligonucleotide primers are the same.

A primer extension product (P4) obtained during the second amplification in which the first oligonucleotide primer, the third oligonucleotide primer, and the second and fourth oligonucleotide primers are the same in the method according to the first aspect. .1-Ext part1) and (P4.1-Ext) are the same, and the obtained primer extension products (P3.1-Ext part1) and (P3.1-Ext) are the same.

In the method according to aspect 1, the second region of the first oligonucleotide primer comprises a polynucleotide tail that does not bind to the first target sequence.

The aforementioned embodiments with respect to aspect 1 can be combined with each other.

The nucleic acid amplified by the first amplification comprises a first target sequence. Prior to initiating the first amplification, at least one starting nucleic acid chain 1.1 containing the target sequence and acting as a template for the synthesis of the first amplified fragment is added to the reaction.

The synthetic primer extension product of the first amplification (first amplification fragment 1.1) comprises the target sequence. The first amplified fragment 1.1 can function as a starting nucleic acid chain 2.1 having a template function for the second amplification.

The primer extension products (P3.1-Ext and P4.1-Ext) synthesized in the second amplification (second amplification fragment 2.1) contain at least a portion of the first target sequence.

The term "essentially complementary" in the context of the present disclosure means that two nucleic acids, in particular their mutually complementary regions, do not disagree with each other more than 5, 4, 3, 2, or 1. means.

In a particular embodiment of the method according to the invention, the first amplification step is that the first amplification fragment (1.1) is 10 to 1E12, especially 100 to 10E9, especially 1,000 to 10E9 (10E9 = 1,). It is provided that it is repeated until it exists with a copy number in the range of Million (,000,000).

In certain embodiments of the method according to the invention, it is provided that the first amplification step is repeated at least twice.

In certain embodiments of the method according to the invention, the second amplification begins with a copy number of the first amplified fragment (1.1) in the range of 10 to 1E12, particularly 100 to 1E10, particularly 10E3 to 10E9. That is provided.

In a particular embodiment of the method according to the invention, the second amplification step is repeated until the amplified fragment (2.1) is present in copy numbers in the range 10E5 to 1E16, in particular 10E5 to 1E14, in particular 10E6 to 1E14. Is provided.

Certain embodiments of the method according to the invention provide that the second amplification step is repeated at least twice.

In a further embodiment, the detection system comprises at least one probe oligonucleotide labeled with a fluorescent reporter.

In a further embodiment, the detection system comprises at least one probe oligonucleotide labeled with a fluorescent reporter and a fluorescent quencher.

In a further embodiment, the detection system comprises at least one probe oligonucleotide labeled with a donor fluorochrome molecule capable of forming a FRET pair with the fluorescent reporter.

In a further embodiment, the probe oligonucleotide is essentially complementary to at least one of the primer extension products formed (P1.1-Ext, P2.1-Ext, P3.1-Ext, P4.1-Ext). Sequence segment, which can hybridize to at least one of the formed primer extension products under suitable conditions (hybridization conditions).

In a further embodiment, the sequence segment is complementary to the first and / or third primer extension products.

In a further embodiment, the sequence segment is complementary to the first and / or third primer extension products, and the probe oligonucleotide is a 3'segment of each primer extension product that is not complementarily bound by the controller oligonucleotide. Can be complementarily combined with.

In a further embodiment, the sequence segment is complementary to a second and / or fourth primer extension product.

In a further embodiment, the sequence segment of the probe oligonucleotide comprises a length in the range of 10 nucleotides to 50, in particular 15-40, in particular 15-30 nucleotides.

In a further embodiment, the sequence segment of the probe oligonucleotide does not include a sequence segment that is essentially complementary to the controller oligonucleotide.

In a further embodiment, the sequence segment of the probe oligonucleotide comprises a sequence segment that is essentially complementary to the controller oligonucleotide, the length of which segment is less than 20 nucleotides, particularly less than 15 nucleotides, particularly. Contains less than 10 nucleotides.

In a further embodiment, the sequence segment of the probe oligonucleotide does not include a sequence segment that is essentially complementary to one of the oligonucleotide primers.

In a further embodiment, the sequence segment of the probe oligonucleotide comprises a sequence region that is essentially complementary to one of the oligonucleotide primers, and the length of the segment is less than 20 nucleotides, particularly less than 15 nucleotides. In particular, it contains less than 10 nucleotides, especially less than 5 nucleotides.

In a further embodiment, the sequence segment of the probe oligonucleotide does not include a sequence region that is essentially identical to the sequence of the third region of the controller oligonucleotide.

In a further embodiment, the sequence segment of the probe oligonucleotide comprises a sequence region that is essentially identical to the sequence of the third region of the controller oligonucleotide, and the length of the segment is less than 20 nucleotides, in particular. Includes less than 15 nucleotides, especially less than 10 nucleotides.

In one embodiment, the controller oligonucleotide comprises one of the following components (fluorescent reporter and / or fluorescent quencher and / or donor fluorochrome molecule), at least one of the components being a third of the controller oligonucleotides. Located in the area of.

In one embodiment, the probe oligonucleotide is at least partially cleaved by a 5'-3'nuclease.

In a further embodiment, the probe oligonucleotide comprises a sequence segment that is essentially complementary to the target sequence or portion thereof contained by one of the amplification products, the segment length being 5 to 50 nucleotides, particularly 10 to 40. Nucleotides, especially in the range of 15-30 nucleotides.

In a further embodiment, the probe oligonucleotide does not contain a sequence segment that is essentially complementary to the target sequence or its complementary strand.

In a further embodiment, the probe oligonucleotide comprises a sequence segment that is not essentially complementary to the target sequence or its complementary strand.

Binding of probe oligonucleotides to their respective complementary segments of primer extension products formed under appropriate hybridization conditions results in the formation of double strands. Hybridization conditions exist between the methods.

At the appropriate time between methods, the reaction is irradiated with light of the appropriate wavelength to generate a fluorescent signal, the fluorescent signal is detected with a fluorescent reporter, and the intensity of the fluorescent signal is measured.

Fluorescence signal detection is performed by appropriate optical / physical means by which an increase in fluorescence signal or a decrease in fluorescence signal is measured. Using the appropriate sensor, after proper calibration, the intensity of the fluorescent signal can be converted to a measurement that can determine if the desired target sequence is present in the reaction mixture.

An important aspect of the fluorescence detection system is a pair of reporter and quencher. If the quencher is in close proximity to the reporter, no signal will be emitted when exposed to excitation light. When the reporter molecule and the quencher molecule are closer together by the complementary stem sequence, no fluorescent signal is generated even under illumination. However, if the fluorescence system is modified so that the nucleotide sequence between the reporter and the quencher can hybridize with the target sequence, the reporter molecule emits fluorescent radiation when the reaction mixture is irradiated with an excitation source. The reporter and the quencher are brought to a spatial distance so that the quencher is far away from. The distance between the reporter and the quencher depends on the molecule used. Generally, when the distance between the quencher and the reporter is less than 25 nucleotides, the emission of light after irradiation with the fluorescent reporter is reduced or almost completely eliminated. This distance can be provided either by the nucleotide sequence, or by a particular molecule that provides the distance, such as a linker or spacer.

In a further embodiment, the fluorescent reporter can form a particular reporter-donor pair (FRET pair) with a donor fluorochrome molecule that can transfer the absorbed energy to the fluorescent reporter by fluorescence resonance energy transfer (FRET). it can.

For a reporter-donor pair that contains a fluorescent reporter and a matching donor fluorochrome molecule and forms a fluorescence resonance energy transfer pair, only one such FRET pair partner is attached to the probe oligonucleotide and the other partner It can become bound to a controller oligonucleotide. Both the fluorescent reporter and the donor fluorochrome molecule are bound to their respective oligonucleotides in the non-hybridized state of the probe oligonucleotide to the extent that the energy absorbed by the donor fluorochrome molecule cannot be transferred to the fluorescent reporter.

In one embodiment, the controller oligonucleotide is a component of the detection system and comprises a fluorescent reporter and / or a fluorescent quencher and / or a donor fluorescent dye molecule. Binding by simultaneously hybridizing the controller oligonucleotide to the synthesized first or third primer extension product and simultaneously hybridizing the probe oligonucleotide to the 3'segment of the same first and / or third primer extension product. Will occur at adjacent sequence positions of the first and / or third primer extension products, resulting in spatial proximity of the donor fluorochrome molecule and the fluorescent reporter. As a result, the distance between the fluorescent reporter and the donor fluorescent dye molecule is shortened to the extent that FRET is executed from the donor fluorescent dye molecule to the fluorescent reporter. This leads to the generation of the fluorescent signal of the fluorescent reporter, resulting in a detectable increase in the fluorescent signal of the fluorescent reporter.

The distance between the donor fluorochrome molecule and the fluorescent reporter after hybridization should be less than 25 nucleotides, especially less than 15 nucleotides, especially less than 5 nucleotides. The wavelength of light at the time of excitation is absorbed by the donor fluorochrome molecule and delivered to the reporter, which emits detectable light. As far as the placement of the detection system in the amplification product is concerned, the following embodiments are preferred, where either the fluorescent reporter or fluorescent quencher or the donor fluorochrome molecule is attached to a third region of the controller oligonucleotide, particularly the controller oligonucleotide. Ru:
-Binding with the controller oligonucleotide is preferably done in the third region of the controller oligonucleotide.
-Binding with the controller oligonucleotide is preferably carried out at or near the 5'end or the third region, eg, 2 to about 10 nucleotides from the 5'end of the third region.
− Binding to the probe oligonucleotide is preferably carried out in the central region of the probe oligonucleotide.
-Binding to the probe oligonucleotide is preferably carried out in the 3'segment of the probe oligonucleotide.

In the application of the present invention, the following embodiments have been found to be particularly advantageous:
-The distance between the two elements of the FRET pair in the hybridized state of the oligonucleotide hybridized to the first primer extension product is less than about 30 nucleotides.
-Probe oligonucleotides containing a 3'segment complementary to one of the primer extension products are modified so that the polymerase cannot extend the 3'end.
-The probe oligonucleotide contains a 3'segment complementary to one of the primer extension products so that the polymerase can extend the 3'end, and the probe oligonucleotide serves as a PCR primer.
-A method of detecting amplification in which two or more nucleic acid strands are amplified, a specific detection system is used for each amplified nucleic acid strand.
-A method for detecting amplification that amplifies two or more nucleic acid chains to be amplified, and detection of amplification of two or more nucleic acid chains to be amplified is performed by a uniform detection system.
-Amplification involves one asymmetric amplification of the primer extension product.
-Excitation of the fluorescence reporter and measurement of the fluorescence signal of the fluorescence reporter are performed during amplification.
-Excitation of the donor fluorochrome molecule and measurement of the fluorescence signal of the fluorescence reporter are performed during amplification.
-Excitation of the fluorescence reporter and measurement of the fluorescence signal of the fluorescence reporter are performed after amplification.
-Excitation of the donor fluorescent dye molecule and measurement of the fluorescent signal of the fluorescent reporter are performed after amplification.
-The probe oligonucleotide is a DNA oligonucleotide.
-The probe oligonucleotide is a DNA oligonucleotide and the 3'end is blocked so that the polymerase cannot be extended.
-The probe oligonucleotide contains a complementary sequence segment to the first and / or third primer extension product, said sequence segment having a length of 8 to about 60 nucleotides.
-The probe oligonucleotide contains a sequence segment complementary to the 3'segment of the 1st and / or 3rd primer extension products, the complementary sequence segment is not bound by the controller oligonucleotide, and the segment is 8 to about 40 nucleotides. Including the length of.

The probe oligonucleotide is at least one addition selected from the following groups: linkers (HEG, C3, C6, etc.), phosphate sugar backbone modifications (PTO, 2'-O-Me, RNA, PNA, LNA modifications, etc.) Includes modification.

In certain embodiments of the method according to the invention, the third oligonucleotide primer and / or the fourth oligonucleotide primer is adjacent to the 5'end of the first region or is connected via a linker. It is provided that it has a region of 1 and cannot be complementarily bound to a first primer extension product or a second primer extension product.

In certain embodiments of the method according to the invention, the second region of the third and / or fourth oligonucleotide primer is oriented in the 5'direction of the first region of the third and / or fourth oligonucleotide primer. Provided to be located.

In certain embodiments of the method according to the invention, it is provided that the third oligonucleotide primer and / or the fourth oligonucleotide primer comprises a barcode sequence.

In certain embodiments of the method according to the invention, double strand separation from the first primer extension product and the fourth primer extension product, and two from the second primer extension product and the third primer extension product. It is provided that chain separation is carried out by thermal denaturation, especially at temperatures in the range of 85 ° C to 105 ° C.

In certain embodiments of the method according to the invention, it is provided that the first polymerase is capable of strand substitution during synthesis.

In certain embodiments of the method according to the invention, it is provided that the second polymerase is a thermostable polymerase.

In certain embodiments of the method according to the invention, it is provided that the first polymerase and the second polymerase are identical.

In certain embodiments of the method according to the invention, it is provided that the first oligonucleotide primer and the third oligonucleotide primer are essentially identical.

In certain embodiments of the method according to the invention, it is provided that the second oligonucleotide primer (P2.1) and the third oligonucleotide primer (P4.2) are essentially identical.

The term "essentially identical" in the context of the present disclosure specifically means that the two nucleic acid sequences do not have more than 5, 4, 3, 2, or 1 mismatch with each other.

In certain embodiments of the method according to the invention, it is provided that the reaction conditions for the first amplification are selected so that spontaneous dissociation of the first amplification product cannot occur.

In certain embodiments of the method according to the invention, the second amplification is performed at two or more temperatures, at which the first and / or first and / or first of the third and / or fourth oligonucleotide primers are at the first temperature. It is provided that hybridization and extension to the second and / or third and / or fourth oligonucleotide primer extension products occur and the resulting double-stranded separation occurs at the second temperature.

In certain embodiments of the method according to the invention, the second amplification takes place at three or more temperatures, at the first temperature the first and / or second and / or second and / or fourth oligonucleotide primers of the third and / or fourth oligonucleotide primers. Hybridization to the / or third and / or fourth oligonucleotide primer extension product occurs, at the second temperature extension of the third and / or fourth primer occurs, and at the third temperature the gain It is provided that the resulting double-stranded separation occurs.

In certain embodiments of the method according to the invention, it is provided that the first amplification and the second amplification are carried out in the same reaction batch.

In certain embodiments of the method according to the invention, a second polymerase, a third oligonucleotide primer and / or a fourth oligonucleotide primer can be activated and / or the controller oligonucleotide is absent. It is provided that it can be activated.

The activable polymerase can be, for example, a thermostable polymerase (hot start polymerase) or a reversible inactivating polymerase such as an antibody or a polymerase that is reversibly inactivated by chemical modification.

The activable primer can be a reversible inactivating primer, eg, an oligonucleotide having a protected 3'-OH group, and the protecting group can be removed particularly by a polymerase having 5'-exonuclease activity. Here, it would be advantageous to use a polymerase without 5'-exonuclease activity in the first amplification.

The controller oligonucleotide can be inactivated or cleaved, for example, by a polymerase having 5'exonuclease activity. Alternatively, the controller oligonucleotide can also be cleaved by other enzymatic or chemical methods prior to the second amplification and thus removed.

In certain embodiments of the method according to the invention, it is provided that the first polymerase is inactivated before the second amplification takes place. This can be achieved, for example, by thermal denaturation when the first polymerase is a non-thermostatic polymerase.

In certain embodiments of the method according to the invention, it is intended that the first amplification is performed in the first reaction batch and the second amplification is performed in the second reaction batch.

In certain embodiments of the method according to the invention, it is intended that an aliquot of the first reaction mixture is added to the second reaction mixture.

In certain embodiments of the method according to the invention, it is intended that the complete first reaction mixture is added to the second reaction mixture.

In certain embodiments of the method according to the invention, the third region of the controller oligonucleotide is that portion of the synthetic region of the first primer extension product immediately adjacent to the oligonucleotide primer portion of the first oligonucleotide extension product. Is provided to be essentially complementary to. This has the advantage of improving sequence-specific substitutions of the amplified nucleic acid.

In certain embodiments of the method according to the invention, the controller oligonucleotide is intended to include a fourth region that is sequence-specific and essentially capable of binding to the 3'region of the third oligonucleotide primer. .. The fourth region can be located within the second and / or third region of the controller oligonucleotide, or can contain sequence segments of the second and / or third region, and the fourth region. The region has a length of 9 to 30 nucleotides in particular, 10 to 20 nucleotides in particular, or 12 to 16 nucleotides in length.

The term "essentially sequence-specific" in the context of the present specification refers to 5, 4, 3, 2 or one region of an amplified oligonucleotide primer and nucleic acid that is essentially complementary to each other. Means that it does not include any discrepancies that exceed.

In certain embodiments of the invention, it is provided that the controller oligonucleotide comprises one or more nucleotide modifications adapted to prevent extension of the primer bound to the controller oligonucleotide. For example, multiple 2'-O-alkyl modifications can prevent or prevent such extension. In particular, the plurality of nucleotide modifications are located in the second and / or third region of the controller oligonucleotide. In particular, the controller oligonucleotide contains at least 5 such modifications, and better at least 10 such modifications.

In certain embodiments, the controller oligonucleotide consists entirely of nucleotide modifications.

In certain embodiments of the method according to the invention, the portion of the region of the first primer extension product synthesized by the polymerase, which is essentially complementary to the third region of the controller nucleotide, is 5 to 70 nucleotides. Has a range length.

In certain embodiments, the third single-stranded region of the controller oligonucleotide is completely complementary to the 5'segment of the first primer extension product, the length of the complementary sequence segment being the following length: S: It can have at least 3 to 70 nucleotides, or at least 5 to 50 nucleotides, especially 5 to 40 nucleotides, 5 to 30 nucleotides, especially 5 to 20 nucleotides.

In certain embodiments of the method according to the invention, it is intended that the first amplification is essentially isothermal.

In certain embodiments of the method according to the invention, the second amplification takes place at three temperatures, at which the hybridization of the third or fourth oligonucleotide primer and optionally the initial extension of the primer. It is provided that, at a second temperature, complete elongation of the primers takes place, and at a third temperature, separation of the formed primer extension products from their respective template chains takes place. .. In particular, the first temperature is in the range of 25 ° C. to 65 ° C., the second temperature is in the range of 65 ° C. to 80 ° C., and the third temperature is in the range of 85 ° C. to 105 ° C.

In certain embodiments of the method according to the invention, the amplified nucleic acid is provided to have a length in the range of 20 nucleotides to 300 nucleotides.

In certain embodiments of the method according to the invention, it is provided that the first segment of the oligonucleotide primer has a length in the range of 15 to 100 nucleotides.

In certain embodiments of the method according to the invention, it is provided that the second oligonucleotide primer has a length in the range of 15 to 100 nucleotides.

In certain embodiments of the method according to the invention, the controller oligonucleotide is provided to have a length in the range of 20 to 100 nucleotides.

In certain embodiments of the method according to the invention, the third oligonucleotide primer is provided to have a length in the range of 15 to 60 nucleotides.

In certain embodiments of the method according to the invention, the fourth oligonucleotide primer is provided to have a length in the range of 15 to 60 nucleotides.

In certain embodiments of the method according to the invention, it is provided that the third oligonucleotide primer has at least one nuclease-resistant nucleotide modification, such as a PTO (phosphothioate) or 2'-O-alkyl modification.

In certain embodiments of the method according to the invention, it is provided that the fourth oligonucleotide primer has at least one nuclease-resistant nucleotide modification, such as a PTO or 2'-O-alkyl modification.

In certain embodiments of the method according to the invention, the first oligonucleotide primer is a copy of the second region of the polymerase used immediately after the first region of the second region, particularly the first region of the first oligonucleotide primer. It is provided to have one or more modifications to stop / prevent. It is advantageous that only the first region of the first oligonucleotide primer is copied.

This can be achieved in certain embodiments by using a nucleotide modification that can form a complementary bond with the first primer extension product, and the nucleotide modification is not allowed as a template by the polymerase. Examples of such nucleotide modifications are nucleotide compounds having a modified phosphate-sugar backbone moiety, such as, for example, 2'-O-alkylRNA modification (such as 2'-OMe), LNA modification or morpholino modification. In general, the presence of such modifications on strands prevents DNA-dependent polymerases from transcribing such strands. The number of such modifications can vary, and usually some modifications (between 1 and 20) are sufficient to prevent the polymerase from reading such strands. Such nucleotide modifications can be used, for example, at or around the binding site of the first oligonucleotide primer to the controller oligonucleotide and / or as a component of the second region of the first oligonucleotide primer.

Thanks to the use of such modifications, polymerase function is locally impeded so that certain segments of the structure used are not copied by the polymerase and remain predominantly single-stranded. In the single chain form, they can bind to additional reaction components and thus perform their function.

Those skilled in the art are aware that the features can be combined with more specific embodiments, as long as the individual features of the method according to the invention are described as present in a particular embodiment. This applies to all features discussed in the context of this description, unless it is clear that their combination is meaningless (eg, in the case of overlapping parameters that are mutually exclusive).

According to another aspect of the invention, there is provided a kit for carrying out the method according to the invention according to the above aspects or specific embodiments or alternatives thereof. The kit according to the invention includes:
-The first oligonucleotide primer, which contains the following regions:
A first region that can be sequence-specifically bound to the region of nucleic acid to be amplified,
A second region that is close to the 5'end of the first region or is connected via a linker, the second region can be bound by the first controller oligonucleotide and selected reaction conditions. It remains essentially uncopyed by the polymerase used for amplification below;
Here, the first oligonucleotide primer can be extended by the polymerase to a synthetic region and a first primer extension product containing the first oligonucleotide primer;
-A second oligonucleotide primer that can bind sequence-specifically to the synthetic region of the first primer extension product and is synthesized by a polymerase in addition to the second oligonucleotide primer. A second primer extension product containing a region containing a region that can be extended.
− A controller oligonucleotide that contains the following regions:
A first region that can bind to a second region of the first primer extension product,
A second region that is essentially or completely complementary to the first region of the first oligonucleotide primer; and that is essentially complementary or completely complementary to at least a portion of the synthetic region of the primer extension product. Third area of interest;
Here, the controller oligonucleotide does not function as a template for primer extension of the first oligonucleotide primer, and the controller oligonucleotide becomes a primer extension product while substituting the complementary region of the second primer extension product. Can be combined,
− Third oligonucleotide primer, the third oligonucleotide primer can bind sequence-specifically to the segment of the second primer extension product, and the polymerase can be used to bind the synthetic region and the third oligonucleotide primer. The containing third primer extension product (P3.1-Ext) contains an extendable first region; and-a fourth oligonucleotide primer, the fourth oligonucleotide primer is a first primer extension. It contains a first region that can be sequence-specifically attached to the synthetic region of the product and can be extended by the polymerase to the synthetic region and a fourth primer extension product containing a fourth oligonucleotide primer.

In certain embodiments of the kit according to the invention, the kit further comprises at least one polymerase.

In certain embodiments of the kits of the invention, the kit is intended to extend the first polymerase and the third and fourth primers intended to extend the first and second oligonucleotide primers. Contains a second polymerase.

In certain embodiments of the kit according to the invention, the first polymerase does not have 5'exonuclease activity and the second polymerase does not have 5'exonuclease activity. In particular, the second polymerase can be a thermostable polymerase.

In certain embodiments of the kit according to the invention, a second polymerase, a third oligonucleotide primer and / or a fourth oligonucleotide primer can be activated and / or the controller oligonucleotide is absent. It is provided that it can be activated.

According to another aspect of the invention, the use of the kit according to the above aspects is provided to carry out the method according to the invention according to the above aspects or specific embodiments or alternatives thereof.

Further details and advantages of the present invention shall be explained in a non-limiting manner by means of figures and detailed description of selected embodiments.

The combination according to the invention includes two amplification methods performed one after the other. During the first partial amplification (first amplification method), the nucleic acid chain containing the target sequence is amplified. This gives the amplified nucleic acid chain, which is then used as a template in the second partial amplification (second amplification method). The second partial amplification is often conventional PCR. During the second partial amplification, amplification is continued from the target nucleic acid amplified in the first partial amplification or the nucleic acid containing the target nucleic acid. The PCR fragment is amplified. The PCR fragment specifically comprises a target sequence or a portion of the target sequence. In addition, certain changes can be made to the amplifyable nucleic acid strand during the second amplification procedure. For example, target sequences can be flanked by the use of barcoding primers or detection-specific oligonucleotide primers. Detection using a PCR probe (such as the so-called Taqman probe) can also be detected. In addition, during the PCR step, immobilization of the target sequence to the solid phase using immobilized oligonucleotide primers can be used, so that the PCR is performed as a solid phase PCR.

The first amplification method according to the invention should be able to synthesize or amplify a nucleic acid chain having a defined sequence composition, and the product produced will start with a template function for subsequent PCR. It can act as a nucleic acid chain and PCR amplification is performed using at least one particular PCR primer.

An object of the present invention is further solved by providing a first amplification method (first partial amplification) and corresponding means for carrying it out. The implementation of the first amplification procedure is already described in PCT application PCT / EP2017 / 0711011 and European application 16185624.0. See the application above for details on performing amplification.

The second amplification method (second partial amplification) can be carried out specifically as PCR, and at least one of the primers used has a different composition and / / from the oligonucleotide primer used in the first amplification method. Or has a structure. This results in amplification of at least one nucleic acid strand containing the specific target sequence or portion thereof.

The PCR amplification itself usually produces a well-characterizable amplification product from about 1,000 copies or more of the initial template, or better, about 100,000 copies or more, or a specific signal in real-time mode. It is possible to generate. If the copy number is reduced to less than 1000 (such as 100 or 10 copies), the PCR needs to include more synthetic cycles and defects in the amplification product or signal can be increased. This requires PCR to amplify sequence variants that are present in small amounts in the batch (eg, sequence mutations or allelic variants) and the presence of large amounts of other sequence variants (eg, wild-type sequences or allelic variants). This is especially true when PCR amplification is performed below. Although many methods have already been developed to improve the specificity of PCR, amplification-dependent detection methods can benefit from further improving the specificity of amplification methods, for example in the field of liquid biopsies. it can. Especially in clinical diagnosis, it is necessary to improve the specificity of the measurement method.

Due to the widespread use of PCR techniques, many modifications have been made to the techniques or extensions of the essential PCR methods used in many areas of molecular biology. By upstream connection of another first amplification method, for example, signal specificity or PCR product specificity can be improved in the application.

The present invention describes a combination of the highly specific amplification method described above with downstream PCR. This combination amplifies the initial copy number of the nucleic acid strand to the desired amount under highly selective amplification conditions of the first amplification method, and then, if necessary, further under less specific PCR amplification conditions. It can be amplified. As described above, the first amplification method (first partial amplification) has a highly specific first amplification function. The amount of copies synthesized in the first segment includes, for example, a range of about 10 copies to about 1E11 copies, better preferably about 100 copies to about 1.0E10 copies, preferably about 1000 to about 10E8 copies. The amount of target sequence is increased by the first amplification. The amplification (based on the initial amount of target sequence in the batch) is, for example, in the range of about 2-fold to about 1E10-fold, better about 10-fold to about 10E9-fold, and even better about 10 times. It is about 10 times to about 10E8 times, preferably about 10 times to about 10E7 times, and particularly preferably about 100 times to 10E6 times. Based on the volume of the reaction, the following concentration ranges are achieved: from about 10 amol / l to about 10 nmol / l, better from about 1 fmol / l to about 1 nmol / l, and even better from about 10 fmol / l to about. 0.1 nmol / l, especially from about 10 fmol / l to about 10 pmol / l.

The second amplification method (used as a second partial amplification, PCR method or a variant thereof) functions as a second amplification and NGS library preparation or solid phase in combination with a particular product of the first amplification. Existing PCR-based methods such as probe insertion or barcoding or sequence coding in PCR can be used.

The advantage of the combination, on the other hand, is the reduction in the number of PCR cycles required to perform amplification to the desired amount of the final product. This reduces the probability or degree of synthesis of defective products by PCR. Therefore, the synthetic result can have higher specificity than the case of PCR amplification alone.

In certain embodiments, the first amplification with the required components of the first amplification system is at least one non-existence of the essential components of the second amplification system in the same reaction batch during the first amplification reaction. First executed in the presence. For example, PCR primers or thermostable polymerases are added only prior to PCR amplification.

After completion of the first amplification reaction, the reaction mixture is brought into contact with the components of the second amplification system for PCR amplification.

In certain embodiments, the reaction components of a particular amplification system are present in separate reaction batches and / or separate reaction vessels prior to the initiation of amplification.

For example, the first amplification reaction is carried out in one reaction vessel, then the compound of the reaction is transferred (completely or partially) to the second reaction vessel, and then the second amplification is carried out. ..

In a further embodiment, first, the components of the first amplification system are added, and the reaction components are added sequentially so that the first amplification reaction is carried out. Next, the components of the second amplification system are added so that the second amplification can be performed. In a further embodiment, the components of the second amplification system are added to the first amplification system so that the reaction takes place in essentially the same reaction vessel. Therefore, the sequences of both amplification methods are measured by the temporal order of addition of the individual components.

Both reactions can be carried out, especially in closed systems, to reduce the potential for contamination. Such a system can include, for example, at least two separate reaction vessels. The transfer of batches from one reaction vessel to another is also carried out using a closed system in certain embodiments. Such separation of the two reactions in a closed reaction vessel system is known as a separate reaction vessel combined to form, for example, a cartridge system, or an array system, or a microfluidic system.

In certain embodiments, the first amplification approach, which comprises the amplified nucleic acid of the first amplification, is used for the second amplification without splitting the first approach. Therefore, the first batch is essentially completely transferred to the second amplification reaction. The amount of copy synthesized from the first amplification procedure is then transferred to a second amplification system, eg, about 10 copies to about 1E11 copies, better about 100 copies to about 1E10 copies, preferably about. Includes a range of 1000 to about 10E9 copies. Thereby, the amount of target sequence is increased by the first amplification. The increase (compared to the initial amount of target sequence in batch) is, for example, in the following range, from about 2-fold to about 1E11-fold, and better from about 10-fold to about 1,000,000,000. Double, even better, about 10 to about 1,000,000, preferably about 10 to about 1,000,000,000,000, especially preferably about 100 to 1,000,000. is there. The following concentration range can be achieved in relation to the volume of the reaction: from about 10 amol / l to about 10 nmol / l, better from about 1 fmol / l to about 1 nmol / l, and even better from about 10 fmol / l to about. 0.1 nmol / l, preferably from about 10 fmol / l to about 10 pmol / l.

In a further specific embodiment, only a portion (alliquot or partial amount) of the first amplification batch is preferably used in the second amplification reaction. Amplification by the first amplification system results in an increase in the amount of nucleic acid chains containing at least one target sequence. In the second amplification reaction, a part of the above amount can be used as a starting material. The portion can be transferred or added to, for example, a second reaction batch. This portion is, for example, relatively small and can be essentially dependent on the requirements of the desired application. Due to the amplification effect of PCR, the second amplification can be initiated, especially with more than about 1000 copies of sequence-specifically amplified nucleic acid chains in the first amplification procedure.

Therefore, PCR amplification can be initiated when a sufficient amount of amplified nucleic acid strand is synthesized in the first amplification procedure. The amount of the synthesized nucleic acid chain can include the following ranges: for example, from about 10,000 copies to about 1E11 copies, better from about 10,000 copies to about 1E10 copies, especially about 10,000 copies. It is in the range of about 1E9 copy. The amount of target sequence is increased as desired by the first amplification. The amplification (compared to the initial amount of target sequence in batch) can be measured as N times the initial amount, eg, within the following range: about 2 times to about 1E11 times, better about about. It is 10 times to about 1E10 times, and even better, about 10 times to about 1E9 times, particularly about 10 times to about 1E8 times, particularly about 100 times to about 1E6 times. Based on the volume of the reaction, the following concentration ranges are achieved: from about 10 amol / l to about 10 nmol / l, better from about 1 fmol / l to about 1 nmol / l, and even better from about 10 fmol / l to about. 0.1 nmol / l, especially from about 10 fmol / l to about 10 pmol / l.

Since more than 1000 copies of a particular nucleic acid chain containing the target sequence can be produced in the first amplification reaction, the amount of fraction (or aliquot or portion) can be used in the second amplification reaction.

For example, PCR can be initiated from an amount that includes the following ranges: for example, from about 100 copies to about 1E11 copies, better from about 1,000 copies to about 1E10 copies, especially about 1,000 to about 1E9. copy. The amount can be understood as a sub-amount of the synthesized nucleic acid chain containing the target sequence amplified by the first amplification.

The provision of the amplified synthetic product of the first amplification comprises the following concentration range with respect to the amount of reaction: from about 10 amol / l to about 100 nmol / l, better from about 1 fmol / l to about 10 nmol / l, and further. Better yet, from about 10 fmol / l to about 1 nmol / l, especially from about 10 fmol / l to about 10 pmol / l. PCR amplification can be initiated from the concentration of the product of the first amplification. In the process of PCR amplification, further amplification of the product takes place, with final concentrations of PCR fragments ranging from 0.01 nmol / l to 5 μmol / l, and better preferably in the range of 1 nmol / l to 1 μmol / l.

Further, in the second amplification method, only the fraction of the nucleic acid chain specifically synthesized by the first amplification method can be used. In certain embodiments, therefore, the first amplification provides a copy amount that indicates a relative surplus with respect to the required amount of copy required or desired as the starting material for PCR.

A part of the first amplification reaction is transferred to the second amplification reaction, and the synthesized nucleic acid chain contained therein serves as a template for initiating the PCR reaction.

For example, a portion of the first reaction batch can be made by diluting the first reaction batch and transferring a specific volume portion containing the synthesized nucleic acid chain to the second reaction. A portion of the amount of specifically synthesized nucleic acid chains transferred thereby acts as a template for generating PCR fragments, which can include the following ranges (in the first amplification step): Calculated based on the total amount of synthetic products): Approximately 1E-7% to approximately 90%, better from approximately 1E-6% to approximately 90%, and even better from approximately 0.0001% to approximately 50%, particularly approximately 0. 001% to about 50%, or about 0.01% to about 50%, especially 0.1% to about 20%.

This not only results in dilution of the synthesized nucleic acid chain containing the target sequence, but also in dilution of the components of the first amplification system, such as primers and controller oligonucleotides. Any by-product that may have been produced is diluted. The reduction resulting from the concentration of the components of the first amplification system is acceptable and may even have a positive effect on PCR performance. For example, dilution of primers and controller oligonucleotides can result in the oligonucleotide primer or oligonucleotide probe used during the PCR reaction being present at a higher concentration than the transferred oligonucleotide primer of the first amplification system. .. This can facilitate the PCR reaction and minimize interference with the amplification procedure.

In addition, said controller oligonucleotides are present in the batch during the second amplification reaction, but are reduced in concentration during the second amplification reaction, causing their binding to synthetic products or reaction components to be sufficiently slow or complementary. Dilution of the controller oligonucleotide has a positive effect on the PCR reaction so that the interaction with the sequence segment is diminished or inadequate and therefore its effect on the overall reaction sequence is diminished by the dilution effect. It is possible to give.

Therefore, when providing a nucleic acid chain for the second amplification, the dilution of the reaction component of the first amplification system can generally be performed in parallel with the dilution of the specific nucleic acid chain provided. The relative amount of the component of the first amplification system transferred to the second amplification reaction can be shown as a part. The said portion refers to the amount (100%) of the component used in the first amplification reaction. The transferred portion of the components of the first amplification system in the second amplification reaction includes, for example, the following ranges: about 1E-7% to about 50%, better about 1E-6% to about 30. %, Even better about 0.0001% to about 20%, especially about 0.001% to about 10%, particularly preferably about 0.1% to about 10%. Therefore, the concentrations of the components of the first amplification system are reduced or significantly reduced so that their effects in the second amplification step are reduced or negligible.

The transferred portion of the first reaction batch can refer, for example, to the concentration or volume portion used. For example, transferring 1 microliter (1 μl) of a batch containing the first amplification system to 49 μl of the batch containing the second amplification system (after completion of amplification) may result in a 2% (v / v) dilution or 1: It can be diluted to 50. Higher dilution levels can be achieved using the dilution series.

For example, the concentration of controller oligonucleotide in the first amplification system can be reduced from about 10 μmol / l at a dilution of 1: 1000 to about 10 nmol / l. Therefore, the transferred portion is only 0.01%. With such dilution, the same proportion of synthesized nucleic acid chains containing the target sequence can be transferred simultaneously. For example, in the first amplification reaction, an amount of about 10,000,000 copies was amplified, and the amount of product transferred to the PCR reaction at a dilution of 1: 1000 was a partial amount of about 10,000 copies. become. The initial copy amount is usually sufficient for PCR to be stable and to carry out a sufficiently specific amplification reaction.

Dilution of nucleic acid strands that may interfere (eg, wild-type molecules or allelic variants) is equally advantageous. During the sequence-specific first amplification, the target molecule is mainly specifically amplified. Other nucleic acid chains are not amplified or significantly not amplified.

When diluted, the dilution effect is also applied to the sequences that may interfere with the PCR reaction. For example, the amount of wild-type sequences is reduced from 100,000 to about 100. This can have a positive effect on the specificity of the PCR reaction.

In certain embodiments, in particular both methods are performed in one approach (as a "uniform assay"). For the above purpose, the components of both amplification systems need to be available in one batch at the start of the first amplification. Therefore, the components required for both amplification systems are added to the reaction batch before the first amplification begins.

In certain embodiments, the first amplification with the required components of the first amplification system presents at least one of the essential components of the second amplification system in the same reaction batch during the first amplification reaction. Is executed first.

Integrating both amplification systems in one reaction batch may require adjustment of individual components or concentrations and reaction conditions.

The presence of components of the second amplification system should not interfere with the first amplification reaction.

In certain embodiments, the length of the 3'segment of the third oligonucleotide primer, which can form a complementary bond with the controller oligonucleotide, is selected so that the segment does not interfere with strand substitution by the controller oligonucleotide. Will be done. This is because under the reaction conditions of strand substitution with the controller oligonucleotide (eg, 65 ° C. step during the first amplification), the stability of the double-stranded complex of the 3'segment of the controller oligonucleotide and the third primer is , The binding of the third primer to the controller oligonucleotide is only temporary and is essentially achieved by being low enough not to interfere with strand substitution. This can be influenced, for example, by the length of the 3'segment of the third primer, which can form such a complex with the controller oligonucleotide. For example, the complex is 8 to 20 nucleotides in length. In addition, the 3'segment of the third primer can have one or more discrepancies with the sequence composition of the controller oligonucleotide of the corresponding segment, which can destabilize the complex. In particular, the 1 to 3 discrepancies are located at positions -10 to -20 with respect to the 3'terminal nucleotide of the third primer. This does not significantly affect the primer function of the third primer, but reduces the stability of the complex with the controller oligonucleotide.

In addition, the polymerase of the second amplification system and the controller oligonucleotides of both primers should not be used as templates. This is of particular importance for the third oligonucleotide primer as it contains sequence segments that can complementarily bind to the controller oligonucleotide. Therefore, the structure of the controller oligonucleotide in the segment of its possible complementary binding to the third oligonucleotide primer can be designed similar to the first primer in the first set. In certain embodiments, primer extension of the third oligonucleotide primer is prevented by the controller oligonucleotide, which uses nucleotide modifications to prevent the polymerase from extending the third primer bound to the controller oligonucleotide. For example, some 2'-O-alkyl modifications can mediate such blocking effects.

In order to avoid unnecessary interference between the two amplification systems, in the first partial amplification, the components of the second amplification system are completely or partially "inactivated" or "inactivated" or "reversibly". It may also be advantageous to keep it in a "non-activated" or "non-reactive" state. Only for the performance of the second amplification, such components need to be transferred to an active, i.e., functional state.

In certain embodiments, at least one of the PCR components is reversibly inactivated during the first amplification so that the component is not involved in the first amplification reaction, or its involvement is not important. It exists in a form. The reversibly inactivated after the completion of the first amplification so that the inactivation is removed and the components are present in the active form in the second amplification reaction and thus can play their role in the amplification. The components are then activated first.

For example, reversibly inactivated PCR primers or reversibly inactivated thermostable polymerases (so-called hot start polymerases) are used. For example, the conversion step from the first amplification to the second amplification uses reaction conditions that allow activation of the component of the inactive form. In other embodiments, activation of the components is carried out continuously under PCR conditions.

Some examples of reversibly inactivated primers are known to those of skill in the art (eg, thermal instability primers called CleanAmp primers (Trilink-Technologies)). Such primers can be activated by heating so that the polymerase can initiate synthesis from said primers. Other examples of reversibly inactivated primers include primers with 3'terminal nucleotides containing modified sugar residues, eg, blocked 3'-OH groups (eg, at the 3'position). Primers with C3 linkers or terminal dideoxynucleotides, Sambrook et al., NAR 1998 p. 3073).

In certain embodiments, such primers show a 3'end mismatch with the template. When such a primer is used, a polymerase that does not exhibit 3'-5'exonuclease activity (eg, Bst polymerase or a modified product thereof) is used for the first amplification. Therefore, such primers remain inactive during the first step. Therefore, if such primers are used in the PCR step, they need to be activated. Activation is usually carried out by enzymatically cleaving the 3'terminal nucleotide with the modified sugar residue. For the above reasons, it is advantageous to use such primers in combination with thermostable polymerases containing 3'-5'exonuclease activity (so-called proofreading polymerases). In certain embodiments that include primers with 3'end mismatches, such end mismatches promote the function of the polymerase's 3'-5'exonuclease. After initiation of PCR amplification, such "inactivated" primers bind to complementary positions on the template. After binding to the template, exonuclease activity (with polymerase) can remove terminal nucleotides containing blocking groups. This gives a free 3'-OH group that can be extended by the polymerase and a complementary binding oligonucleotide primer. Such "activated" primers can then be extended by the polymerase so that strands complementary to each template can be synthesized. Polymerases capable of activating such primers under PCR conditions include Vent polymerase, Deep Vent polymerase, Pfu polymerase, and Phusion polymerase. Activation of such primers by designing PCR primers that have a mismatch at the 3'end (or, for example, at the -1 or -2 position with respect to the 3'end nucleotide) for each template. Can usually be accelerated (eg, Pfu polymerase or Phusion polymerase). Other thermostable polymerases can also cleave modified terminal nucleotides with blocked 3'-OH groups, even if they are complementary to the template (eg, Vent polymerase or Deep Vent polymerase). To limit the degradation effect of polymerases on PCR primers, such primers are 3'-5'nuclease non-cleavable bindings, eg, one or more phosphorothioate (PTO) modifications (eg, "minus 2" or "minus". The position of "3" or the position of "minus 2" to "minus 7") may be included. This prevents excessive degradation of the primer.

Reversible activating polymerases include, for example, those that are reversibly inactivated by an antibody or reversibly inactivated by a chemical modification (Amplitaq polymerase). Some commercial suppliers are developing such "hot start" polymerases for PCR (Qiagen, Thermo Fisher, Roche). In particular, Taq polymerase and its modifications have been provided as so-called hot-start Taq polymerases in various reversibly inactivated states. Particularly preferred is, for example, a hot start polymerase that is converted to the active form by first heating the preparation to 95 ° C. for 1-10 minutes. Because the antibody can shield different epitopes of the polymerase, it can reversibly inactivate different activities of the polymerase to varying degrees (Scalice et al., J. Immunol. Methods, 1994, p. 147-, Lyamicev et et al., PNAS. , 1999, p. 6143).

In a particular type of amplification of uniform form, the polymerase that has undergone exponential amplification of the nucleic acid strand amplified in the first amplification reaction is inactivated by heating prior to the initiation of the second amplification. The inactivation can be achieved, for example, by heating the preparation above 80 ° C., preferably above 90 ° C., especially to 95 ° C. for 1-10 minutes. In this way, the mesophilic polymerase (such as Bst polymerase or Bsm polymerase, or Gst polymerase) can be inactivated. Such inactivation facilitates the transition from the process of the first amplification reaction to the second amplification reaction. Such a temperature step allows the inactivation of the polymerase of the first amplification reaction and the activation of the polymerase of the second amplification reaction to occur simultaneously.

Due to such thermal inactivation of the first polymerase, the first polymerase can no longer bind to the primer or template to the same extent. Therefore, during the second amplification reaction, the primer-template complex is used specifically by the polymerase that synthesizes in the second amplification step.

Switching from one polymerase to another thus "switches off" other activities related to the polymerase, such as strand substitution with Bst polymerase, or "switches" the 5'-3'exonuclease activity of Taq polymerase. It is also possible to "turn it on".

In certain embodiments, the 5'segment of controller oligonucleotide hybridized to the first primer extension product is a 5'-3 nuclease of polymerase (eg, 5'-3' of Taq polymerase) during the second amplification. It can be cleaved by nuclease). The process is well known and is often used for probe degradation and real-time monitoring of PCR reactions (US Pat. No. 5,210,015). Here, the polymerase can complementarily extend the fourth PCR primer by incorporating the nucleotide under appropriate reaction conditions using the third primer extension product, and at the same time, the polymerase can extend the third primer. The controller oligonucleotide can be enzymatically cleaved in its 5'segment of the third region hybridized to the product. During synthesis, a structure capable of recognizing the 5'-3'nuclease activity of Taq polymerase is formed under the reaction conditions used, and the strand extended by the polymerase is at least so that the structure can be cleaved. One 3'terminal nucleotide forms a particular overlap with the controller oligonucleotide. 5'-3'-exonuclease activity results in the cleavage of phosphodiester bonds between nucleotides in the third region of the controller oligonucleotide. 5'-3'-exonuclease induced cleavage occurs specifically in the DNA strand, but some glycophosphate skeleton modifications such as 2'-OMe ribonucleotide or PTO modification or PNA delay or further prevent such cleavage. To do. For the above reasons, a combination of a polymerase containing 5'-3'exonuclease activity (eg, Taq polymerase) and a controller oligonucleotide is particularly used in said embodiments and of the 5'segment of the third region. Controller oligonucleotides mainly contain nucleotides that can be cleaved by nucleases or their analogs (eg, DNA). The length of the 5'segment of the controller oligonucleotide includes, for example, 5 to 50, especially 10 to 30 nucleotides.

By degrading the controller oligonucleotide bound to the first primer extension product, the polymerase can extend the second primer even without strand substitution properties, thus synthesizing the second primer extension product.

In certain embodiments, the presence of components of the second amplification system during the first amplification is taken into account as follows:

For one thing, the effects of the components of the second amplification system can be reduced by their reversible inactivation prior to the first amplification. On the other hand, the individual sequence elements should not appear as a non-specific matrix for primer extension during the first amplification. For the above reasons, the primers of the first and second amplification systems should be confirmed for the presence of self-complementary structures to avoid the formation of primer dimers.

The third PCR primer usually contains a sequence segment in its 3'segment, which contains a region complementary to the controller oligonucleotide. This allows the third PCR primer to complementarily bind to the controller oligonucleotide. To perform the first amplification, the third PCR primer should not prevent strand substitution by the controller oligonucleotide during the first amplification. For this purpose, the sequence length or sequence composition of the 3'segment of the third PCR primer is adjusted so that it does not essentially bind to the controller oligonucleotide under the reaction conditions of the strand replacement step (eg 65 ° C.). .. This is because, for example, the melting temperature of the complex consisting of the controller oligonucleotide and the 3'segment of the third oligonucleotide primer is essentially below the reaction temperature of the chain replacement step, eg, between 45 ° and 60 ° C. Such a third PCR primer can still bind its 3'segment to its template and initiate a primer extension reaction. This can be achieved, for example, the 3'segment of the third PCR primer, which can complementarily bind to the controller oligonucleotide, is between 9-30 nucleotides in length, better 12-20. Includes regions between nucleotides, especially between 12-16 nucleotides. The 3'segment of the third PCR primer may contain one or more discrepancies with the corresponding position of the controller oligonucleotide such that the binding strength continues to decrease.

The sequence segment of the controller oligonucleotide that is essentially complementary to the third PCR primer points to the fourth region of the controller oligonucleotide. The fourth region of the controller oligonucleotide may include sequence segments of a second region and / or a third region.

A fourth region of the controller oligonucleotide comprises a region of length between 9-30 nucleotides, better 12-20 nucleotides, particularly 12-16 nucleotides. The fourth region of the controller oligonucleotide can contain one or more discrepancies with the 3'segment of the third PCR primer. In particular, the fourth region comprises a nucleotide modification that uses a controller oligonucleotide as a template to prevent the third PCR primer from being extended by the polymerase of the first and / or second amplification system. Such modifications have already been described for the combination of the first primer and the controller oligonucleotide. They include, for example, a sequence segment with a 2'-O-alkyl modification, the length of which segment can be 6-30 modifications and is complementary to the controller oligonucleotide against the 3'end nucleotide of the third PCR primer. Nucleotides specifically include such modifications themselves, and at least three nucleotides with such modifications are flanked on both sides. This ensures that the controller oligonucleotides of the third and fourth primers and the polymerase of the second amplification system are not used as templates.

The production of first and second primer extension products with defined lengths plays a role in the separation of the two products using controller oligonucleotides. Therefore, possible premature extension of the 3'end (of the first and second primer extension products, respectively) by the third or fourth primer of the second amplification system as a template during the first amplification reaction. It is advantageous to prevent it.

Reduction or prevention of unwanted excessive extension of the 3'end of the first primer extension product using the fourth PCR primer as a template can be achieved in a variety of ways. In certain embodiments, this is complete with the 3'terminal nucleotide of the synthesized first primer extension product when the fourth PCR primer binds to the 3'segment of the first primer extension product. Achieved by the inability to form matching complementary bonds. This should prevent or at least reduce the over-extension of the 3'terminal nucleotides of the first primer extension product.

In certain embodiments, this is a complement that perfectly matches at least one of the synthesized first terminal nucleotides when the fourth PCR primer binds to the 3'segment of the first primer extension product. Achieved by the fact that no target can be formed. The resulting at least one discrepancy position is particularly at positions -1 to -5 with respect to the terminal nucleotide of the first primer extension product.

Using the third PCR primer as a template, reducing or preventing unwanted over-extension of the 3'end of the second primer extension product can be achieved in a variety of ways. In certain embodiments, this is complete with the 3'terminal nucleotide of the synthesized first primer extension product when the third PCR primer binds to the 3'segment of the second oligonucleotide extension product. Achieved by the fact that complementary bonds that match are not formed. This should prevent or at least reduce the over-extension of the 3'terminal nucleotides of the second primer extension product.

In certain embodiments, this is a complement that perfectly matches at least one of the synthesized second terminal nucleotides when the third PCR primer binds to the 3'segment of the second primer extension product. It is achieved by the inability to form a primer. The resulting at least one discrepancy position is particularly at positions -1 to -5 with respect to the terminal nucleotide of the second primer extension product.

The presence of components of the first amplification system during the second amplification is considered as follows:
For one thing, diluting them before the second amplification can reduce the effects of the components of the first amplification system. On the other hand, the individual elements should not appear as non-specific templates for primer extension during the second amplification. For the above reasons, the primers of the first and second amplification systems should be confirmed for the presence of self-complementary structures to avoid the formation of primer dimers.

In certain embodiments, the second amplification is performed in the presence of the controller oligonucleotide of the first amplification system. For the above reasons, the controller oligonucleotide design, the second set of primers and reaction conditions must be adapted so that the second amplification reaction is not significantly disturbed by the presence of the controller oligonucleotide.

First, the polymerase of the second amplification system and the controller oligonucleotides of both primers should not be used as templates. This is of particular importance for the third oligonucleotide primer as it contains a sequence segment that can complementarily bind to the controller oligonucleotide in the fourth region. The structure of the controller oligonucleotide in the fourth region specifically comprises modifications that prevent possible primer extension. For example, some 2'-O-alkyl modifications can mediate such synthetic inhibitory effects.

The controller oligonucleotide can bind to the third primer extension product during the second amplification and thus form a complementary strand with the third primer extension product. Such double-stranded fragments are such that under certain circumstances, the polymerase starting with the fourth PCR primer synthesizes the fully complementary strand, including copying the 5'segment of the third primer extension product. Can be prevented. Therefore, the choice of polymerase and reaction conditions must be such that binding of the controller oligonucleotide does not prevent the synthesis of the fourth primer extension product.

The length of the segment of the third primer extension product that can complementarily bind to the controller oligonucleotide is determined, for example, by the placement of the third primer. In particular, the third primer has the length of the fully complementary portion of the third primer extension product expected for the controller oligonucleotide in the following range: about 15 nucleotides to about 60 nucleotides, or It is arranged to be between about 20 nucleotides and about 40 nucleotides, especially about 20 and about 30 nucleotides.

In certain embodiments, the thermostable polymerase is selected, in particular in the second amplification reaction, which comprises chain substitution activity and uses, for example, a thermostable polymerase such as bentexominus polymerase or pyrophage polymerase. Can be done. This allows the controller oligonucleotide to be separated from the third primer extension product during the second amplification and the fourth primer extension product to be synthesized in full length.

In certain embodiments, in particular, a polymerase capable of cleaving the controller oligonucleotide by the 5'-3'exonuclease activity of the polymerase is selected in the second amplification reaction, resulting in simultaneous primer extension (see above). Controller oligonucleotides that can be cleaved by 5'-3'exonuclease in at least the 5'segment are used. Degradation incrementally shortens the controller oligonucleotide hybridized to the third primer extension product, which under suitable reaction conditions (eg, temperatures between about 65 ° C and 75 ° C) make this binding unstable. And finally leads to the separation of binding to the third primer extension product. Therefore, the fourth primer extension product can be synthesized in full length.

In certain embodiments, the concentration of the controller oligonucleotide and the concentration of the fourth primer in the second primer set are more primers than the binding of the controller oligonucleotide to the third primer extension product under selected reaction conditions. It is selected for faster binding and primer extension. For example, concentrations of controller oligonucleotides in the range 0.01 μmol / l to 0.3 μmol / l are used. At the same time, the concentration of the fourth oligonucleotide primer in the range of about 0.5 μmol / L to 2 μmol / L is used. In particular, a polymerase having a high processing capacity (for example, Performance polymerase) is used in the second amplification reaction. Primer binding and its extension are usually faster than controller oligonucleotide binding so that the primer extension reaction has kinetic advantages.

In certain embodiments, the primer extension reaction is carried out at two or more temperatures in the second amplification reaction. Initially, the temperature is generally low, for example in the range of 45 ° C to 65 ° C, with complementary binding to the fourth primer and its complementary segment in the 3'segment of the third primer extension, and by the polymerase. The first elongation occurs. This is a third such that after raising the temperature in the second range, for example, from about 70 ° C to about 80 ° C, the partially extended third primer extension product is extended by the polymerase. To produce a partially extended primer extension product that is sufficiently stable with the primer extension product of. At said temperature, the controller oligonucleotide can spontaneously dissociate from binding to the third primer extension product, so that the polymerase contains the 5'segment of the third primer extension product and is copied by the polymerase. Until then, the synthesis of the fourth primer extension product can be continued. In particular, a polymerase having a high processing capacity (for example, fusion polymerase) is used in the second amplification reaction.

The method of working with the composition of the reaction mixture in "homogeneous form" is when the division of the first reaction batch is not very complicated practically or technically, for example, "micro or nanodrop reaction". Especially suitable (also called digital PCR).

A solid phase containing an oligonucleotide that is suitable for specific binding of the formed product, eg, and / or also occurs as an immobilization primer, can be added for amplification, resulting in solid phase PCR being performed. The obtained primer extension product is immobilized on a solid phase. Contact with the solid phase containing the particular oligonucleotide can be made before, during or after the first amplification.

Prior to the initiation of the first amplification reaction, the reaction batch can be divided into multiple reaction volumes (partitions) so that the amplification of the reaction batch is simultaneously divided into about 100 or 1000 or more equal volume partitions. The partition can be done in the form of droplets (eg, as an emulsion). Amplification can be performed in parallel on the large number of droplets (eg, as digital PCR).

The sequence of amplification reactions (first sequence-specific amplification followed by PCR only) plays an important role: sequence-specific amplification using controller oligonucleotides (first amplification) is particularly pre-PCR amplification. Is done in. This is intended to avoid PCR-based deformations and side reactions of the target sequence prior to the first amplification. For the above reasons, PCR-based amplification is performed only as a second step.

Therefore, in certain embodiments, the DNA fragment produced by PCR or any other amplification method is not used as the starting nucleic acid strand of the first amplification reaction.

In the following, the necessary components of the first amplification method (first partial amplification) and the first amplification system will be schematically described, followed by the second amplification method (second partial amplification) and the second. The explanation of the components of the amplification system follows. Next, a combination of the first and second amplification methods will be described and advantageous embodiments will be presented. The first amplification is followed by a second amplification procedure (also called PCR).

In particular, the first amplification is performed as an exponential amplification and the newly synthesized product of both primers (primer extension product) is used as a template for further synthesis steps. Therefore, the primer sequences are at least partially copied to form complementary primer binding sites, which are present as double-stranded sequence segments immediately after synthesis. In the first amplification method, the synthesis step of both strands and the double-strand opening step of the newly synthesized sequence segment are performed alternately. Sufficient double-strand separation after synthesis is an important prerequisite for further synthesis. Overall, alternating such synthesis and double-stranded separation steps can lead to exponential amplification.

In the first amplification method according to the present invention, the double-stranded opening of the main product of amplification (amplification of the nucleic acid chain containing the target sequence) is, in particular, by an oligonucleotide called a controller oligonucleotide. The controller oligonucleotide specifically comprises a sequence segment corresponding to the target sequence.

In particular, strand separation according to the invention is a controller oligo having a respective predetermined sequence that specifically separates newly synthesized duplexes consisting of both specific primer extension products by sequence-dependent nucleic acid mediated strand separation. Achieved by using nucleotides. The resulting single-stranded segment of the primer extension product can serve as the target sequence, as well as the binding site for additional oligonucleotide primers, such that exponential amplification of the amplified nucleic acid chain is achieved. including. In essence, the primer extension reaction and the chain substitution reaction preferably occur at the same time. Amplification is particularly carried out under reaction conditions that do not allow the natural separation of both specific synthesized primer extension products.

The particular exponential first amplification of the target sequence-containing nucleic acid strand comprises repeating the synthesis step and the double-strand opening step (primer binding site activation step) as essential prerequisites for the growth of the nucleic acid strand.

The synthesized double-stranded opening is performed as a reaction step that is sequence-specifically affected by the controller oligonucleotide. The opening can be done completely or partially up to the dissociation of both complementary primer extension products.

According to the present invention, the controller oligonucleotide includes a sequence segment capable of interacting with the target sequence and additional sequence segments that induce, tolerate, or improve the interaction, respectively. In the process of interaction with the controller oligonucleotide, the double-stranded site of the synthesized primer extension product is converted to a single-stranded form by sequence-specific strand substitution. The process is sequence-dependent: sufficient double-stranded openings occur only if the synthesized double-stranded sequence has a certain amount of complementarity with the corresponding sequence of controller oligonucleotides, resulting in sufficient double-stranded openings. Sequence segments essential for continued synthesis, such as the primer binding site, are converted to a single-stranded form corresponding to the "active state". Therefore, the controller oligonucleotide specifically "activates" the newly synthesized primer extension product containing the target sequence for further synthetic steps.

In contrast, sequence segments that do not contain the target sequence are not converted to the single-stranded state and remain double-stranded, which corresponds to the "inactive" state. Possible primer binding sites for such double strands are disadvantageous or completely impeded in interaction with the new primer, and as a result, further synthetic steps on such "inactivated" strands are generally It doesn't happen. The lack or reduction of activation (ie, conversion to a single-stranded state) of the synthesized nucleic acid strand after the synthesis step results in a successful primer extension reaction with only the reduced amount of primers in the subsequent synthesis step. Brings the fact that you can participate.

The first exponential amplification of the main product (amplified nucleic acid strand containing the target sequence) combines several synthesis steps and activation steps (double strand cleavage steps) in one amplification method, respectively. It is performed or repeated until the desired amount of a particular nucleic acid chain is provided.

The reaction conditions (eg, temperature) in the process of the first amplification method are designed so that spontaneous separation of complementary primer extension products in the absence of the controller oligonucleotide is unlikely or significantly slowed down. To.

Therefore, the increased specificity of amplification is due to the sequence dependence of the separation of the complementary primer extension product containing the target sequence: the controller oligonucleotide is consistent with a given sequence segment of the primer extension product of that sequence segment. As a result, the double-stranded separation is possible or advantageous. The match is verified after each synthesis cycle by the controller oligonucleotide. Exponential amplification results in good iterations from the synthetic process by controller oligonucleotides and sequence-specific strand substitutions, i.e., "activation" of the newly synthesized primer extension products (corresponding primer binding sites). It results in a double-strand opening / double-strand separation / strand replacement process) that results in a single-stranded form.

Therefore, the use of pre-determined controller oligonucleotides allows sequence-dependent validation of the content of primer extension products between individual synthetic steps during exponential amplification, and sequence selection or sequence selection in subsequent synthetic steps. Sorting can be obtained. Here, the "active" single-stranded state of the newly synthesized specific primer extension product as a result of successful interaction with the controller oligonucleotide and the defective interaction with the controller oligonucleotide and / or It is possible to distinguish the "inactive" double-stranded state of the newly synthesized non-specific primer extension products as a result of inadequacy and / or reduction and / or deceleration.

Exponential amplification has the following effects.
Under non-denaturing conditions, the separation of specifically synthesized strands is carried out by the coordination of controller oligonucleotides.

Exponential amplification of the target sequence-containing nucleic acid strand is sequence-controlled (main reaction). The sequence control is performed after each synthesis step and includes a sequence segment that exists between the primers and contains the target sequence. Confirmation of the synthesis results after each successful synthesis step is the precondition of a further specific synthesis step, the separation of both specific primer extension products.

During the first amplification, the initial production of non-specific primer extension products cannot be ruled out essentially (by-product). Due to template dependence, such non-specific primer extension products are generally present in double-stranded form immediately after synthesis. However, the interaction with the controller oligonucleotide is either completely unsuccessful or restricted, resulting in no chain separation or slowed chain separation over the main reaction. Therefore, there is no erroneous transfer of sequence information from one synthesis cycle to the next.

Therefore, the choice of reaction conditions and the design of the controller oligonucleotide can specifically affect the efficiency of regeneration of the correct nucleic acid strand template during a single synthetic step in the amplification method. In general, the more successful the separation of the synthesized product and the regeneration of the correct template from one synthesis step to the next, the degree of agreement between a given sequence of controller oligonucleotides and the synthesized sequence. It gets higher. On the other hand, the difference in the sequence of the by-products results in inadequate regeneration of the template chain, thus delaying the initiation of synthesis and reducing the yield in each subsequent cycle. The overall exponential amplification of the by-products either progresses more slowly, does not occur at all, and / or remains at undetectable levels.

Therefore, this method allows verification of the synthesized sequence in real time, ie without stopping the reaction, and thus all components of the assay are already present in the reaction mixture at the beginning of the reaction. Show potential for development.

As a result of the first partial amplification, a sufficient amount of highly specific amplification fragments are provided, which serve as a template for the second partial amplification (PCR).

After a sufficient reaction time or number of cycles, the first amplification method can then be terminated or the reaction conditions can be modified to initiate the second amplification method (PCR). In the second amplification method, the target sequence is amplified using the nucleic acid strand produced by the first amplification method and at least one additional PCR-specific component (eg, oligonucleotide primer and / or PCR polymerase). The number of specific nucleic acid chains is already high enough at the beginning of the second amplification, so that the PCR process requires fewer cycles to obtain the desired total amount of product. This produces a PCR product with less error overall.

By sequentially switching between the highly specific first amplification method at the start of amplification and the PCR only thereafter, the overall specificity of the PCR product produced is conventional PCR that works under PCR conditions from the start. Can be improved compared to the method.

In particular, the components of both amplification methods are already available at the beginning of the amplification procedure.

In certain embodiments, the components of the first amplification system are different from the components of the second amplification system (PCR).

In certain embodiments, the components of the first amplification system are different from the components of the second amplification system, and the PCR components (of the second amplification system) are at least under the reaction conditions of the first amplification method. Partially inactive form (eg, as a hot start polymerase). Therefore, the change from the first amplification to the second amplification requires an additional activation step of the PCR component (eg, polymerase).

In certain embodiments, the polymerase of the first amplification system is inactivated after the completion of the first amplification, so that a different polymerase is used for the second amplification (PCR).

In certain embodiments, the components of the first amplification system are also partially used by the second amplification system and are not part of the first amplification system, at least one additional PCR-specific component (eg, eg). Additional oligonucleotide primers or polymerases) are used.

The present invention describes some embodiments of both methods, the arrangement of oligonucleotide primers, which are advantageous for carrying out both amplification methods. Appropriate placement of individual elements of the first and second amplification systems on the nucleic acid strand containing the target sequence can construct a uniform assay with higher overall specificity.

Terms and Definitions Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by the average expert. Standard methods are used for molecular biology or biochemical methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. et al. And Ausube et al., Short Protocols in Molecular Biology (1999), 4th Edition, John Wiley & Sons, Inc.)

If the sequences shown in the description and the sequences shown in the sequence listing are different from each other, the sequences shown in this description apply in case of doubt.

In the context of the present invention, the terms used have the following meanings:
As used herein with respect to primers, controller oligonucleotides, probes, and amplified nucleic acid chains, the term "oligonucleotide" refers to two or more, preferably more than three deoxyribonucleotides and / or ribonucleotides and / or nucleotides. Defined as a molecule containing modified and / or non-nucleotide modifications. Its length includes, for example, a region between 3 and 300 nucleotide units or analogs thereof, in particular a region between 5 to 200 nucleotide units or analogs thereof. Its exact size depends on several factors that depend on the ultimate function or use of the oligonucleotide.

As used herein, the term "primer" does not relate to an oligonucleotide whether it is naturally occurring, eg, purified restricted cleavage, or synthetically produced. Primers are used under conditions that induce the synthesis of primer extension products that are complementary to the nucleic acid strand, i.e. in the presence of nucleotides and inducers, such as DNA polymerase at the appropriate temperature and pH value. If so, it can serve as a starting point for synthesis. Preferably, the primer is single strand for maximum potency in amplification. The primer needs to be long enough to initiate the synthesis of the extension product in the presence of the inducer. The exact length of the primer depends on several factors, such as reaction temperature, primer source, and application of the method. For example, the length of the oligonucleotide primer in a diagnostic application is between 5-100 nucleotides, preferably 6-40 nucleotides, particularly preferably 7-30 nucleotides, depending on the complexity of the target sequence. Short primer molecules generally require a lower reaction temperature to perform the primer function to form a sufficiently stable complex with the template, or allow the formed primer template complex to be sufficiently long. It is necessary to increase the concentration of other reaction components such as DNA polymerase.

The primers used herein are selected to be "essentially" complementary to the various strands of each specific sequence to be amplified. This means that the primers need to be sufficiently complementary to hybridize with their respective strands and initiate the primer extension reaction. Thus, for example, the primer sequence need not reflect the exact sequence of the target sequence. For example, a non-complementary nucleotide fragment can be attached to the 5'end of the primer and the remaining primer sequence is complementary to the strand. In another embodiment, a single non-complementary base or a longer non-complementary sequence can be inserted into the primer, provided that the primer sequence is sufficient with the sequence of the amplified strand to hybridize with it. If it has great complementarity to, it produces a primer template complex capable of synthesizing the extension product.

During the enzymatic synthesis of the template-complementary strand, a primer extension product that is completely complementary to the template strand is produced.

Tm-Melting Temperature Complementary or partially complementary double-stranded melting temperatures are generally measurements of reaction temperatures in which approximately half of the strands are present as double strands and the other half are present as single strands. Is understood to be. This system (chain binding and dissociation) is in equilibrium.

Tm of nucleic acid amplified by several factors that can affect double-stranded Tm (eg, sequence length, sequence CG level, buffer conditions, divalent metal cation concentration, etc.) Is measured under the same conditions as the desired amplification reaction.

Since the measurable melting temperature depends on multiple reaction parameters, such as the respective buffer conditions and the respective concentration of the reaction partner, the melting temperature is double-stranded in the same reaction buffer as exponential amplification. Concentrations of both complementary components measured at a concentration of about 0.1 μmol / l to about 10 μmol / l, preferably about 0.3 μmol / to about 3 μmol / l, preferably about 1 μmol / l. It is intended to be. Each value of melting temperature is a guide value that correlates with the stability of the corresponding double strand.

Deoxyribonucleoside triphosphate Deoxyribonucleoside triphosphate (dNTP) dATP, dCTP, dGTP, and TTP (or dUTP, or dUTP / TTP mixture) are added to the synthetic mixture in appropriate amounts. In certain embodiments, in addition to dNTPs, at least one additional type of dNTP analog can be added to the synthetic mixture. In certain embodiments, the dNTP analog comprises, for example, a characteristic label that can be incorporated into a nucleic acid chain (eg, biotin or a fluorescent dye). In another embodiment, the dNTP analog confers at least one modification of the sugar phosphate ratio of the nucleotide, eg, alpha-phosphorothioate-2'-deoxyribonucleoside triphosphate (or other nucleic acid chain nuclease resistance). Modifications), including 2', 3'-dideoxy-ribonucleoside triphosphates, acyclo-nucleoside triphosphates (or other modifications that result in cessation of synthesis). In certain embodiments, the dNTP analog comprises at least one modification of the nucleobase, such as isocytosine, isoguanosine (or other modifications of the nucleobase of the extended genetic alphabet), 2-aminoadenosine, 2-. Contains thiouridine. Inosine, 7-deraza-adenosine, 7-deraza-guanosine, 5-me-cytosine, 5-propyl-uridine, 5-propyl-cytosine (or nucleic acids that can be incorporated by the polymerase as compared to the naturally occurring nucleobases. It also includes other modifications of the base, resulting in changes in chain stability). In certain embodiments, the dNTP analog comprises both nucleobase modification and sugar phosphate ratio modification. In certain embodiments, at least one additional type of dNTP analog is added to the synthetic mixture in place of at least one natural dNTP substrate.

Nucleic acid synthesis inducers can be enzymes or systems that act to result in the synthesis of primer extension products.

Suitable enzymes for the first amplification for the above purpose include, for example, DNA polymerases such as Bst polymerase and its modifications, Vent polymerase, and especially thermostable DNA polymerases that allow the incorporation of nucleotides in the correct manner. Primer extension products are formed that are complementary to, but not limited to, each nucleic acid strand synthesized. Synthesis usually begins at the 3'end of each primer and then proceeds in the 5'direction along the template strand until synthesis is complete or interrupted.

In particular, strand-substitutable template-dependent DNA polymerases are used in the first amplification. These include large fragments of Bst polymerase or modifications thereof (eg, Bst 2.0 DNA polymerase), Klenow fragment, bent exo-minus polymerase, deep bent exo-minus DNA polymerase, large fragments of Bsu DNA polymerase, and Bsm DNA polymerase. Contains a large fragment of.

In certain embodiments, polymerases that do not exhibit 5'-3'exonuclease activity or 5'-3'FEN activity are used, in particular.

In particular, a thermostable template-dependent DNA polymerase is used for the second amplification. For example, Taq polymerase or a modified product thereof (Ampli-Taq, etc.), or a polymerase having a calibration function: Pfu and its modified product, or Vent polymerase and its modified product. A polymerase fused with another protein, such as Phusion polymerase, can be used to increase processing power. Many polymerases are commercially available.

A combination of polymerases can also be used, for example OneTaq-polymerase (NEB) is a combination of Taq polymerase and vent polymerase. Such a combination of polymerases can contribute to higher synthesis accuracy in the second amplification.

In certain embodiments, at least two different polymerases are used, eg, a polymerase capable of strand substitution and a polymerase having 3'-5'proofreading activity.

In certain embodiments, polymerases with a hot start function are used and can only perform their function when a certain temperature is reached.

The first amplification system comprises the components required to perform a particular first amplification: a first oligonucleotide primer, a second oligonucleotide primer, a controller oligonucleotide and a first polymerase.

In addition, nucleotide mixtures and buffer systems can also be included to the extent necessary to perform a specific first amplification (eg, a specific buffer system for the first polymerase).

The first amplification system is the first amplification fragment 1.1 (or the first amplification product 1.1, the nucleic acid of the first amplification to be amplified) in the first amplification (also referred to as the first partial amplification). Supports the synthesis of (also called strands).

The second amplification system contains the components required to perform a specific second amplification: a third oligonucleotide primer, a fourth oligonucleotide primer and a second polymerase.

In addition, nucleotide mixtures and buffer systems can also be included as long as they are necessary to perform a specific second amplification (eg, a particular buffer system for a second polymerase).

The second amplification system supports the synthesis of the second amplification fragment 2.1 (or second amplification product 2.1) in the second amplification (also referred to as the second partial amplification).

First oligonucleotide primer: (component of the first amplification system)
The first oligonucleotide primer (FIGS. 12-16) includes a first primer region and a second region. The first primer region can bind to an essentially complementary sequence within the amplified nucleic acid or equivalent and initiate a primer extension reaction. The second region is capable of binding the controller oligonucleotide, thereby causing sufficient proximity between the controller oligonucleotide and the rest of the first primer extension product and initiating chain substitution with the controller oligonucleotide. Includes a polynucleotide tail that can be. The second region of the first oligonucleotide primer further comprises at least one modification (nucleotide-modified or non-nucleotide-modified) that prevents the polymerase from copying the polynucleotide tail by inhibiting the continuation of polymerase-dependent synthesis. .. The modification is located, for example, at the transition between the first and second regions of the first oligonucleotide primer. As a result, the first primer region of the oligonucleotide primer can be copied by the polymerase, so that complementary sequences to the region can be generated by the polymerase during the synthesis of the second primer extension product (detailed below). Explained to). The polynucleotide tail of the second region of the first oligonucleotide primer is not specifically copied by this polymerase. In certain embodiments, this is achieved by modifying a second segment that arrests the polymerase before the polynucleotide tail. In certain embodiments, this is achieved by nucleotide modification in the second region, and the entire polynucleotide tail is essentially from such nucleotide modification and is therefore not copyable to this polymerase.

In certain embodiments, each first oligonucleotide primer is specific for one nucleic acid to be amplified.

In certain embodiments, each first oligonucleotide primer is specific for at least two nucleic acids to be amplified, each containing an essentially different sequence.

In certain embodiments, the first oligonucleotide primer is a characteristic label, eg, a fluorescent dye (eg, TAMRA, fluorescein, Cy3, Cy5) or an affinity marker (eg, biotin, digoxigenin) or an additional sequence fragment (eg, biotin, digoxigenin). For example, for detection or immobilization, or for binding of specific oligonucleotide probes for bar code labeling).

The first oligonucleotide primer is particularly used as a primer in the first amplification.

In certain embodiments, it can be used as a primer in both the first and second amplifications.

Second oligonucleotide primer: (component of first amplification system)
Oligonucleotides with 3'segments can bind to essentially complementary sequences within the amplified nucleic acid or equivalent and initiate a specific second primer extension reaction. Therefore, the second oligonucleotide primer can bind to the 3'segment of the first specific primer extension product of the first oligonucleotide primer and initiate polymerase-dependent synthesis of the second primer extension product. it can.

The length of the second oligonucleotide primer can be between 15-100 nucleotides, especially 20-60 nucleotides, especially 30-50 nucleotides.

In certain embodiments, each second oligonucleotide primer is specific for one nucleic acid to be amplified.

In certain embodiments, each second oligonucleotide primer is specific for at least two amplified nucleic acids, each containing an essentially different sequence.

In certain embodiments, the second oligonucleotide primer is a characteristic label, eg, a fluorescent dye (eg, TAMRA, fluorescein, Cy3, Cy5) or an affinity label (eg, biotin, digoxigenin) or an additional sequence fragment (eg, biotin, digoxigenin). For example, for detection or immobilization, or for binding of specific oligonucleotide probes for bar code labeling).

The second oligonucleotide primer is specifically used as a primer in the first amplification. In certain embodiments, it can be used as a primer in both the first and second amplifications.

Primer extension products Primer extension products (also called primer extension products) are formed by enzymatic (polymerase-dependent) extension of oligonucleotide primers as a result of polymerase-catalyzed template-dependent synthesis.

Primer extension products include sequences of 5'segment oligonucleotide primers and sequences of extension products synthesized by template-dependent polymerases (also referred to as extension products). The extension product synthesized by the polymerase is complementary to the template strand from which it was synthesized.

Specific primer extension products of the first amplification (main product) (eg, P1.1-Ext and P2.1-Ext) contain the sequence of the nucleic acid chain to be amplified. This is the result of specific synthesis or accurate execution of the intended primer extension reaction in which the specifically amplified nucleic acid strand acts as a template. In certain embodiments, the sequence of the synthesized primer extension product exactly matches the expected sequence of the amplified nucleic acid. In another embodiment, the resulting sequence differences from the theoretically expected sequences can be tolerant. In certain embodiments, the degree of sequence match resulting from amplification with the theoretically expected sequence of nucleic acid for amplification is, for example, between 90% and 100%, and in particular the degree of match is 95. More than%, ideally more than 98% match (measured by the ratio of synthesized bases).

The extension product length of the specific primer extension product can be between 10 and 300 nucleotides, especially 10 to 180 nucleotides, especially 20 to 120 nucleotides, especially 30 to 80 nucleotides.

For example, in the second amplification, the product of the specific template-dependent primer extension reaction of the third and fourth primers is the main product and their respective intermediates: partial third primer extension product (P3.1-Ext part1). , FIGS. 15-18), or complete primer extension products (P3.1-Ext, FIGS. 15-18), partial fourth primer extension products (P4.1-Ext part 1, FIGS. 15-18), or complete. Primer extension product (P4.1-Ext, FIGS. 15-18). The product, in certain embodiments, comprises a target sequence or an equivalent thereof. In another embodiment, the second primer extension product comprises a target sequence and the first primer extension product comprises an equivalent of the target sequence, i.e. a complementary strand. In certain embodiments, the fourth primer extension product comprises a target sequence and the third primer extension product comprises an equivalent of the target sequence, i.e. a complementary strand. In certain embodiments, the fourth primer extension product comprises a portion of the target sequence and the third primer extension product comprises an equivalent of said portion of the target sequence, i.e. a complementary strand.

The first primer extension product (P1.1-Ext) and the second primer extension product (P2.1-Ext) are also referred to as the first amplification fragment 1.1 (also referred to as amplification product 1.1) of the first amplification. ) Together (Fig. 1).

Both the third primer extension product (P3.1-Ext) and the fourth primer extension product (P4.1-Ext) are the second amplification fragment 2.1 (second amplification product 2) of the second amplification. (Also called .1) (Fig. 1).

The third primer extension product (P3.1-Ext) can also be referred to as a complete third primer extension product. The product is obtained by using a fourth primer extension product (P4.1-Ext) as a template. The fourth primer extension product (P4.1-Ext) can also be referred to as the complete fourth primer extension product. The product is produced by using a third primer extension product (P3.1-Ext) as a template (FIGS. 15-18).

In contrast, the partial third primer extension product (P3.1-Ext-part1) is produced as an intermediate using the second primer extension product (P2.1-Ext) as a template. The partial fourth primer extension product (P4.1-Ext-part1) is produced as an intermediate product using the first primer extension product (P1.1-Ext) as a template (FIGS. 15-18). ..

Non-specific primer extension products (by-products) include, for example, sequences that are the result of non-specific or inaccurate or unintended primer extension reactions. These include, for example, primer extension products that are the result of inaccurate initiation events (wrong priming) or other side reactions (polymerase-dependent sequence changes such as base substitutions, deletions, etc.). The degree of sequence difference of non-specific primer extension products generally exceeds the ability of controller oligonucleotides to successfully replace such double-stranded by-products from the template, resulting in slow amplification of such by-products. Becomes or completely disappears. The tolerance or tolerance of the difference depends, for example, on the reaction temperature and the type and method of sequence difference. Examples of non-specific primer extension products are primer dimers or sequence variants that do not correspond to the nucleic acid being amplified, such as sequences that do not contain a target sequence.

Assessing the full specificity of amplification is often associated with the task at hand. In many amplification methods, some nonspecificity of the amplification reaction can be tolerated as long as the desired result can be achieved. In certain embodiments, the proportion of nucleic acid chains amplified in the total result of the reaction is greater than 1%, particularly greater than 10%, and particularly greater than 30%, as measured in the total amount of newly synthesized strands.

Amplified Nucleic Acid (Expected Result of First Amplification)
The nucleic acid to be amplified represents a nucleic acid strand that is sequence-specific, or at least primarily sequence-specific, amplified by the polymerase in the first amplification using primers and controller oligonucleotides. The nucleic acid to be amplified contains both strands of the first amplified fragment 1.1 (also referred to as the first amplification product 1.1) (FIG. 1). It can be used as the starting nucleic acid strand 2.1 and at least one of the strands can be used to initiate a second amplification.

The length of the nucleic acid to be amplified can be 20 to 300 nucleotides, especially 30 to 200 nucleotides, especially 40 to 150 nucleotides, especially 50 to 100 nucleotides.

The amplified nucleic acid strand may contain one or more target sequences or their equivalents. In addition, the amplified nucleic acid may comprise a sequence that is essentially complementary to the target sequence, which is amplified in the amplification reaction with similar efficiency to the target sequence and comprises the target sequence or a segment thereof. In addition to the target sequence, the amplified nucleic acid encodes a strand by an additional sequence segment, such as a primer sequence, a sequence having a primer binding site and / or a sequence segment for binding the detection probe, and / or a bar code sequence. It may include a sequence segment of the sequence and / or a sequence segment for binding to a solid phase. The oligonucleotide primer sequences or their sequence portions, as well as the primer binding sites or their sequence portions, can belong, for example, to the sequence segment of the target sequence.

In certain embodiments, the amplified nucleic acid corresponds to the target sequence.

In another embodiment, the target sequence forms part of the sequence of the amplified nucleic acid chain of the first amplification. Such target sequences can be flanked by the 3'and / or 5'sides of additional sequences. The additional sequence may include, for example, the binding site of an oligonucleotide primer or a portion thereof, and / or the primer sequence or a portion thereof, and / or the binding site of a detection probe, and / or solid. It may include an adapter sequence for complementary binding to a phase (eg, in the context of microarray or bead-based analysis) and / or a digital signature bar code sequence.

In order to initiate amplification, a nucleic acid strand must be added to the reaction mixture at the initiation of the reaction, which serves as an initial template for the synthesis of the nucleic acid strand to be amplified. The nucleic acid strand is called the starting nucleic acid strand. The starting nucleic acid strand defines the arrangement of individual sequence elements that are important for the formation / synthesis / exponential amplification of the nucleic acid strand to be amplified.

In certain embodiments, the initial template (starting nucleic acid chain) fed to the amplification reaction at initiation or added to the reaction mixture corresponds to the sequence composition of the amplified nucleic acid chain.

In the initial and subsequent steps of the amplification reaction, each primer binds to the corresponding binding site of the starting nucleic acid strand and initiates the synthesis of a specific primer extension product. Such specific primer extension products accumulate exponentially during amplification and gradually take over the role of template for the synthesis of complementary primer extension products in exponential amplification.

Therefore, the iterative template-dependent synthetic process in exponential amplification forms the nucleic acid strand to be amplified.

Towards the end of the amplification reaction, the main product of the reaction (amplified nucleic acid) can be predominantly single-stranded or predominantly complementary double-stranded. This can be determined, for example, by the relative concentration of both oligonucleotide primers and the corresponding reaction conditions.

Amplified nucleic acid equivalents include nucleic acids that have essentially the same information content. For example, the amplified nucleic acid complementary strands have the same information content and can be called equivalent.

Target Sequence In a particular embodiment, a target sequence is a segment of an amplified nucleic acid chain that can function as a characteristic sequence of the amplified nucleic acid. The target sequence can serve as a marker for the presence or absence of another nucleic acid. Thus, said other nucleic acids serve as a source of target sequences, eg, genomic DNA or RNA or parts of genomic DNA or RNA (eg, mRNA), or equivalents of biological genomic DNA or RNA (eg, eg, mRNA). It can be a modified RNA such as cDNA, rRNA, tRNA, microRNA), or a defined change in the genomic DNA or RNA of an organism, such as a gene mutation (eg, deletion, insertion, substitution, addition, sequence propagation, eg, microsatellite). Includes repetitive transmission in the context of instability), splice mutations, rearrangement mutations (eg, T cell receptor mutations, etc.). Individual target sequences may represent phenotypic characteristics such as antibiotic resistance or prognostic information and are therefore involved in diagnostic assays / tests. As a source / origin of the target sequence, such nucleic acids may include, for example, the target sequence as a sequence element of its strand. Therefore, the target sequence can serve as a characteristic marker for the specific sequence content of another nucleic acid.

The target sequence can be single-stranded or double-stranded. It may be essentially identical to the amplified nucleic acid of the first amplification, or may represent only a portion of the amplified nucleic acid.

Equivalents of the target sequence include nucleic acids having essentially the same information content. For example, the complementary strand of the target sequence has the same information content and can be called an equivalent, and the RNA and DNA mutants of the sequence are also examples of the equivalent information content.

During material preparation prior to the amplification reaction, such target sequences can be isolated from their original sequence environment and prepared for the amplification reaction.

In certain embodiments, the amplified nucleic acid comprises a target sequence. In certain embodiments, the target sequence corresponds to the nucleic acid being amplified. In another particular embodiment, the starting nucleic acid chain 1.1 comprises a target sequence. In certain embodiments, the target sequence corresponds to the starting nucleic acid chain 1.1.

The target sequence may be completely or partially included as part of the first amplified fragment 1.1 during the first amplification. For example, the second primer extension product contains the target sequence or a portion thereof, and thus the second primer extension product contains a strand complementary to the target sequence. This chain has the same information content.

The target sequence may be completely or partially included as part of the second amplified fragment 2.1 during the second amplification. For example, the fourth primer extension product contains the target sequence or a portion thereof, and thus the third primer extension product contains a strand complementary to the target sequence. This chain has the same information content.

Initiation In order to initiate the first amplification of the nucleic acid strand, it is necessary to add the nucleic acid strand to the reaction mixture at the initiation of the reaction, which serves as an initial template for the synthesis of the nucleic acid strand to be amplified (FIG. 1). .. The nucleic acid chain is called the starting nucleic acid chain of the first amplification (starting nucleic acid chain 1.1). The starting nucleic acid strand identifies the arrangement of individual sequence elements that are important for the formation / synthesis / exponential amplification of the nucleic acid strand to be amplified.

Such initiation nucleic acid strands can be single or double strands at the onset of the reaction. When the complementary strands of the starting nucleic acid strand are separated from each other, the strands serve as a template for the synthesis of specific complementary primer extension products, regardless of whether the nucleic acid is originally double-stranded or single-stranded. Can function.

In certain embodiments, the starting nucleic acid chain 1.1 comprises a target sequence.

The starting nucleic acid strand further comprises at least one predominantly single-stranded sequence segment in which at least one of the primers of the amplification system is capable of predominantly complementary binding to its 3'segment, resulting in the starting nucleic acid strand. When hybridized, the polymerase used can extend such primers in a template-specific manner by incorporating dNTPs.

The starting nucleic acid strand may also contain a segment that is not the target sequence but can bind to an oligonucleotide primer.

To initiate the second amplification, at the beginning of the second amplification reaction, a nucleic acid strand is present in the reaction mixture that serves as an initial template for the synthesis of the second amplified fragment, or a nucleic acid strand is added to it. It is necessary (Fig. 1).

The starting nucleic acid strand 2.1 identifies the arrangement of individual sequence elements that are important for the formation / synthesis / exponential amplification of the nucleic acid strand to be amplified.

Here, the first amplified fragment 1.1 (also referred to as the amplified nucleic acid strand) produced during the first amplification functions as the starting nucleic acid strand 2.1 of the second amplification. Thus, the starting nucleic acid chain 2.1 comprises or consists of a first primer extension product produced by the first amplification. In certain embodiments, the starting nucleic acid chain 2.1 comprises a target sequence.

PCR amplification (also called second amplification)
PCR is a polymerase chain reaction commonly used to amplify DNA fragments. Many variants of the PCR reaction are known in this area. Among other things, allogeneic PCR, genotyping PCR, asymmetric PCR, solid phase PCR, digital PCR (partitioning / microdroplet PCR), multiplex PCR, real-time PCR using probes or insert dyes (eg, molecular beacons or Two oligonucleotide probes with a 5'-3'exonuclease or FRET pair), clamping PCR using a blocking probe, quantitative PCR, nested PCR, and the like. Those skilled in the art refer to numerous evaluations that describe individual PCR methods.

Generally, nucleic acid fragments defined by the position of oligonucleotide primers are amplified during PCR. In general, at least two temperature ranges: a cold range for binding primers primarily to their specific sequence segments and expanding them to form complementary strands, and a newly synthesized primer extension product strand from the template. At least one temperature range is used, which is predominantly separated by sequence non-selective separation so that it is separated.

Many techniques have been developed to influence the course of PCR, such as with hot start polymerases, hot start oligonucleotides, and activable primers (eg, due to the 3'-5'proofing activity of the polymerase). is there.

PCR can be performed in liquid phase, in solid phase, in microfluidic systems, or in situ using PCR test tubes / microtiter plates.

PCR primer pair (3rd and 4th oligonucleotide primers)
(Component of the second amplification)
Usually, a PCR primer pair contains two oligonucleotides that can support the amplification of nucleic acid fragments by the PCR method.

Generally, PCR primer pairs include third and fourth oligonucleotide primers. Such oligonucleotide primers are well known in the scientific community.

For clarity, PCR primers are commonly referred to herein as P3 and P4 (for controller-dependent amplifications P1 and P2).

Many variants of PCR primers are known to those of skill in the art. Usually, they are oligonucleotides about 15 to about 60 nucleotides in length, and at least the 3'segment can be attached to the complementary segment of the target sequence, using the target sequence as a template and template dependent by the polymerase. Contains sequences that can initiate sex synthesis.

Dye labeling (eg, FAM, TAMRA, Cy5) or quenching (eg, BHQ1), affinity labeling (eg, biotin, digoxigenin), or additional sequences, such as sequence bar coding (use of specific tag sequences) or library creation. The use of sequences for, etc. is known to those of skill in the art. PCR primers can be used in solution or attached to a solid phase (eg, beads, microtiter plate). The mutant is also well known to those of skill in the art.

Controller oligonucleotide:
The controller oligonucleotide (FIG. 1, C1.1) is a predefined essentially complementary segment of the first primer extension product that is specifically produced during amplification of the nucleic acid to be amplified (first amplification). It is a single-stranded nucleic acid chain containing a specific sequence. This allows the controller oligonucleotide to bind to the first oligonucleotide primer in an essentially complementary manner, at least to the 5'segment of the specific extension of the first oligonucleotide primer. .. In certain embodiments, the controller oligonucleotide comprises a nucleotide modification in its internal sequence segment, which is templated by the polymerase when the first and / or third oligonucleotide primers are complementary to the controller oligonucleotide. As a controller oligonucleotide is used to prevent the synthesis of complementary strands. In certain embodiments, it is advantageous for the controller oligonucleotide to consist entirely of modified nucleotides that block template function. In certain embodiments, it is advantageous that the controller oligonucleotide consists of only a portion of the modified nucleotide that blocks template function. The controller oligonucleotide can also completely or partially replace the second specific primer extension product from binding to the first specific primer extension product through strand substitution under selected reaction conditions. it can. Thereby, the controller oligonucleotide having its complementary region binds itself to the first specific primer extension product. If the binding between the controller oligonucleotide and the first specific primer extension product is successful, this will result in a second specific primer extension product 3'suitable for binding the first oligonucleotide primer. It results in the restoration of the single-stranded state of the containing segment, and as a result, a new primer extension reaction can occur. During the synthesis of the second primer extension product, the controller oligonucleotide can be separated from the binding to the first primer extension product, for example by polymerase and / or strand replacement with the second oligonucleotide primer.

Detection-system:
The detection system includes at least one oligonucleotide probe and at least one fluorescent reporter (fluorescent dye molecule). The detection system is a first and / or second and / or third and / or fourth primer extension product (P1.1-Ext, P2.1-Ext, P3.1-Ext, P4.1-Ext). Must be able to detect the synthesis of. This is done by using oligonucleotide probes that can bind to each primer extension product, thereby producing a specific signal or causing a change in the signal. The change can be an increase or decrease in fluorescence intensity. The detection system may also contain additional components. Such components include, among other things, fluorescent quenchers and / or donor fluorescent dye molecules. In addition, the detection system may also include controller oligonucleotides. Binding events to the first primer extension product can be detected by the placement of donor fluorescent dye molecules, fluorescent reporters, fluorescent quenchers and controller oligonucleotides on or on the oligonucleotide probe containing the oligonucleotide probe.

Fluorescent Reporter A fluorescent dye molecule is a chemical or molecule that, when excited by electromagnetic radiation, can emit electromagnetic radiation (light) (emission). The radiation (emission) emitted by the fluorescent dye molecule can be detected as a fluorescent signal by appropriate technical means. Such reporters can be covalently attached to oligonucleotides. Many fluorescent dye molecules that can bind to oligonucleotides are known (eg, FAM, TAMRA, HEX, ROX, Cy dye, Alexa dye).

Fluorescent quencher A quencher is a chemical compound / molecule that can reduce the emission of fluorescent dye molecules by direct contact (contact quenching) or energy transfer (eg, as FRET). In principle, the quencher should be placed in close proximity to the fluorochrome to make the signal reduction significant.

Advantageous in the significant signal reduction of the fluorescent reporter by the quencher is that the distance between the reporter and the quencher is less than 25 nucleotides, especially less than 15 nucleotides, especially less than 5 nucleotides.

In order to overcome the signal reduction of the fluorescent reporter due to the quencher, the distance between the components needs to be increased accordingly. It is advantageous if the distance between the reporter and the quencher increases by more than 15 nucleotides, especially more than 20 nucleotides, especially more than 40 nucleotides. In this case, the distance caused by the nucleotide sequence needs to be due to the extended nucleotide sequence. For example, once the hairpin structure is formed, the spatial distance can no longer exist.

Especially in FRET-based quenchers, sufficient spectral overlap between the emission spectrum of the fluorochrome molecule and the absorption spectrum of the quencher is beneficial. Therefore, a pair of fluorescent dye molecule quenchers with more than 25% spectral overlap is preferred (eg, FAM / TAMRA).

Donor Fluorophore Molecular A fluorescent dye molecule absorbs electromagnetic radiation and excites the fluorescent dye molecule into another fluorescent dye molecule (acceptor) by energy transfer (eg, as FRET), resulting in itself. A chemical compound / molecule that can be transmitted to produce luminescence. A fluorescent signal is generated during light emission. Usually, the donor and acceptor form a fluorescence resonance energy transfer pair (FRET pair). As a general rule, donors need to be placed in close proximity to acceptors (fluorescent dye molecules) in order to generate the signal generation to a significant extent.

Beneficial to signal generation of fluorescent reporters as a result of FRET from donor fluorochrome molecules is that the distance between the reporter and the donor is less than 25 nucleotides, especially less than 15 nucleotides, especially less than 5 nucleotides.

Elimination of the FRET effect from the donor to the acceptor is usually achieved by increasing the distance between the FRET pair. It is beneficial when the distance between the reporter and the donor increases by more than 15 nucleotides, especially by more than 20 nucleotides, especially by more than 40 nucleotides.

In addition, sufficient spectral overlap between the donor emission spectrum and the acceptor absorption spectrum is usually advantageous. Therefore, a FRET pair with a spectral overlap of greater than 25% (eg, FAM / Cy3) is preferred.

According to the present invention, the spatial distance between the individual components is provided, among other things, by the fact that the two components are connected via a nucleotide sequence that can hybridize to a particular portion of the target sequence. Is done.

Chain substitution:
In the process that, by the action of appropriate means, results in the complete or partial separation of the first duplex (eg, consisting of the A1 and B1 strands) and the simultaneous / parallel formation of the new second duplex. There, at least one of the strands (A1 or B1) is involved in the formation of the new second strand described above. Here, a distinction can be formed between the two forms of chain substitution.

In the first form of strand substitution, the existing complementary strand can be used to form a new second duplex, which is usually in the single strand form at the onset of the reaction. In this case, the means of strand substitution (eg, preformed single strand C1 with a sequence complementary to strand A1) acts on the first already formed duplex (A1 and B1). , Entering a complementary bond with chain A1, thereby replacing chain B1 from the bond with chain A1. When the substitution of B1 is completed, a new double strand (A1: C1) and a single strand B1 are generated as a result of the effect of C1. If the replacement of B1 is incomplete, the result depends on several factors. For example, a partially double-stranded A1: B1 and A1: C1 complex may be present as an intermediate.

In the second form of strand substitution, the formation of a new second duplex can occur under co-enzymatic synthesis of complementary strands, with one strand of the first preformed duplex being a polymerase. Acts as a synthetic template. Thereby, the strand substitution agent (eg, a polymerase capable of strand substitution) acts on the double strands (A1 and B1) already formed first to synthesize a new complementary strand D1 into the strand A1, and the strand B1 becomes the strand B1. At the same time, it is replaced from the bond with chain A1.

The term "nucleic acid-mediated strand substitution" is in equilibrium with each other, resulting in a temporary or permanent opening of the first preformed duplex (consisting of complementary strands A1 and B1), and a new second. Used to describe the sum / series of intermediate steps that result in the formation of the duplex (consisting of complementary strands A1 and C1) of, A1 and C1 are complementary to each other.

The essential structural conditions for initiating strand substitution are the double-strand end (pre-formed first duplex of A1 and B1) and the single strand (C1) initiating the strand substitution. It is known to establish spatial proximity between them (A1 and C1 form complementary strands). Such proximity can be achieved specifically by single-stranded overhangs (see the above literature, known in the literature called "toehold" for examples of short overhangs), which are temporary or permanent in a complementary fashion. It binds to the single-stranded chain (C1) so that the complementary segments of chains C1 and A1 are sufficiently close together so that the substitution of chain B1 is successfully initiated. The efficiency of initiating nucleic acid-mediated strand substitution is generally higher as the complementary segments of strands A1 and C1 are placed closer to each other.

Another important structural precondition for the efficient continuation of nucleic acid-mediated strand substitutions in the inner segment is the high degree of complementarity between strands to form new duplexes (such as between A1 and C1). ). For example, a single nucleotide mutation (within C1) can lead to disruption of strand substitution (eg, described as bifurcation).

The present invention uses the ability of complementary nucleic acids to form sequence-dependent nucleic acid-mediated strand substitutions.

Specific embodiments of the present invention are further described in the figures and examples.

Reaction Conditions for Individual Embodiments First Amplification Reaction conditions include, among other things, buffer conditions, temperature conditions, reaction times, and concentrations of the respective reaction components.

During the reaction, the amount of specifically produced nucleic acid to be amplified accumulates in an exponential manner. Reactions involving the synthesis of elongation products can be performed to generate the desired amount of specific nucleic acid sequence, if desired. The method according to the invention is particularly continuous. In a preferred embodiment, the amplification reaction proceeds at the same reaction temperature, especially between 50 ° C and 70 ° C. In another embodiment, the reaction temperature can also be variably controlled so that each single step of amplification proceeds at a different temperature.

The reagents required for exponential amplification are already present, especially at the start of the reaction in the same batch. In another embodiment, reagents can be added at a later stage of the method.

In particular, no helicase or recombinase is used in the reaction mixture for the separation of the newly synthesized double strands of the amplified nucleic acid.

In certain embodiments, the reaction mixture is free of biochemical energy-imparting compounds such as ATP.

The amount of amplified nucleic acid present at the start of the reaction can be between several copies and billions of copies in one batch. For diagnostic applications, the amount of nucleic acid strands amplified can be unknown.

In the reaction, there may also be additional nucleic acids that are not the subject of amplification. The nucleic acid may be derived from natural DNA or RNA or their equivalents. In certain embodiments, the control sequence is present in the same batch that needs to be amplified in parallel with the nucleic acid being amplified.

In particular, a reaction mixture containing a template chain for the synthesis of an amplified nucleic acid strand with a molar excess of ^{about 10 3} : 1 to about 10 ¹⁵ : 1 (primer: template ratio) of the primers and controller oligonucleotides used. Is added to.

When the method according to the invention is used for diagnostic purposes, the amount of target nucleic acid may be unknown and therefore the relative amount of primer and controller oligonucleotide relative to the complementary strand cannot be reliably determined. When the sequence to be amplified is contained in a mixture of complex long-stranded nucleic acid chains, the amount of primer added is generally in molar excess relative to the amount of complementary strand (template). Large amounts of excess moles are preferred to improve the efficiency of the method.

The concentrations of Primer 1, Primer 2 and the controller oligonucleotide used are, for example, between 0.01 μmol / l and 100 μmol / l, particularly between 0.1 μmol / l and 100 μmol / l, especially between 0.1 μmol / l and 50 μmol / l. l, especially in the range between 0.1 μmol / l and 20 μmol / l. The higher the concentration of the component, the higher the amplification factor can be. The concentration of each of the individual components can be varied independently to achieve the desired reaction result.

The concentration of the polymerase is in the range of 0.001 μmol / l to 50 μmol / l, preferably 0.01 μmol / l to 20 μmol / l, particularly 0.1 μmol / l to 10 μmol / l.

The concentration of the individual dNTP substrates is in the range of 10 μmol / l to 10 mmol / l, especially in the range of 50 μmol / l to 2 mmol / l, preferably in the range of 100 μmol / l to 1 mmol / l. is there. The concentration of dNTPs can act on the concentration of divalent metal cations. Optionally, this is adjusted accordingly.

As the divalent metal cation, for example, Mg ²⁺ is used. As the corresponding anion Cl, for example, acetate, sulfate, glutamic acid and the like can be used.

The concentration of the divalent metal cation is adapted, for example, to the optimum range for the corresponding polymerase, in the range of 0.1 mmol / l to 50 mmol / l, better between 0.5 mmol / l and 20 mmol / l. , Preferably in the range of 1 mmol / l to 15 mmol / l.

Generally, enzyme synthesis is carried out in a buffered aqueous solution. As the buffer solution, a dissolved conventional buffer substance such as Tris HCl, Tris acetic acid, potassium glutamate, HEPES buffer solution, and monosodium glutamate at a general concentration can be used. The pH value of the solution is usually between 7 and 9.5, preferably between about 8 and 8.5. Buffer conditions can be applied, for example, according to the manufacturer's recommendations for the polymerase used.

Other substances such as so-called Tm depressants (DMSO, betaine, TPAC, etc.) can be added to the buffer. Such substances may lower the double-stranded melting temperature (“Tm lowering agent”) and thus have a positive effect on the double-stranded opening. Polymerase stabilizing components such as Tween 20 or Triton 100 can also be added to the buffer in normal amounts. Heavy metals can be compounded by adding EDTA or EGTA in conventional amounts. Polymerase stabilizers such as trehalose or PEG6000 can also be added to the reaction mixture.

In particular, the reaction mixture does not contain any inhibitors of the chain substitution reaction and does not contain inhibitors of polymerase-dependent primer elongation.

In certain embodiments, the reaction mixture contains a DNA binding dye, particularly an insertion dye such as EvaGreen or SybrGreen. Such dyes may allow detection of the formation of new nucleic acid chains.

The reaction mixture can also contain, for example, proteins or other substances that are derived from the original material and do not affect amplification.

The temperature of a particular embodiment of the reaction conditions has a significant effect on double-stranded stability.

In certain embodiments, no temperature conditions are used during the amplification reaction that essentially lead to the separation of the double strands of the nucleic acid that is amplified in the absence of the controller oligonucleotide. This confirms that the double-stranded separation of the amplified nucleic acid strand depends on the presence of the controller oligonucleotide throughout the amplification procedure.

Spontaneous separation of both strands of the amplified nucleic acid occurs at a temperature approximately equal to the measured melting temperature (Tm) of the amplified nucleic acid, thereby affecting the effect of the controller oligonucleotide on the separation of the synthesized strands. However, the effect of controller oligonucleotides on the sequence specificity of amplification is therefore minimized and limited.

For exponential amplification, which requires low sequence specificity (ie, low controller oligonucleotide dependence), the reaction temperature is near the melting temperature of the amplified nucleic acid (ie, Tm plus / minus 3 ° -5 ° C). Can be. At such temperatures, sequence differences between the controller oligonucleotide and the synthesized primer extension product can generally be well tolerated in the strand substitution reaction.

Even in the temperature range of about (Tm −3 ° C.) to about (Tm −10 ° C.), spontaneous chain separation of the synthesized primer extension product still occurs, but the efficiency is reduced. The effect of the controller oligonucleotide on the sequence specificity of the amplified nucleic acid is higher under temperature conditions near the melting temperature (Tm) of the amplified nucleic acid chain.

The separation of the strands occurs as the reaction temperature decreases, mainly due to the interaction between the newly synthesized duplex and the controller oligonucleotide. However, the double strands of the primer at extension temperature can also be spontaneously degraded under the above conditions, i.e., without sequence-dependent strand substitution by the controller oligonucleotide. For example, the reaction temperature for reduced sequence-specific amplification ranges from about (Tm -3 ° C) to about (Tm -10 ° C), especially from about (Tm -5 ° C) to about (Tm -10 ° C). is there. At such temperatures, sequence differences between the controller oligonucleotide and the synthesized primer extension product are less tolerable in the strand substitution reaction.

The high sequence specificity of amplification of this method is predominantly achieved when the newly synthesized strand of the amplified nucleic acid cannot spontaneously dissociate into a single strand under reaction conditions. In such cases, sequence-specific strand substitution by the controller oligonucleotide plays a decisive role in sequence-specific strand separation and plays a major role in the sequence specificity of the amplification reaction. This can generally be achieved in the absence of helicases or recombinases, where the reaction temperature is well below the melting temperature of both strands of the amplified nucleic acid and no other components are used for strand separation. For example, in sequence-specific amplification, the reaction temperature is between about (Tm -10 ° C) and about (Tm -50 ° C), especially between about (Tm -15 ° C) and about (Tm -40 ° C), especially about. It ranges from (Tm −15 ° C.) to approximately (Tm −30 ° C.).

In certain embodiments of amplification, the maximum reaction temperature does not rise above the melting temperature of the nucleic acid chains amplified during the total amplification reaction.

In certain embodiments of amplification, the reaction temperature can be raised at least once above the melting temperature of the amplified nucleic acid chain. For example, the temperature is raised at the start of the amplification reaction and the double strand of genomic DNA can result in denaturation. It should be noted that during such steps, the dependence of double strand separation on the action of the controller oligonucleotide is eliminated, or at least significantly reduced.

The reaction temperature of each step of the amplification reaction can be in the range of about 15 ° C to about 85 ° C, preferably in the range of about 15 ° C to about 75 ° C, particularly in the range of about 25 ° C to about 70 ° C.

In Examples 2 and 3 below, a reaction temperature of 65 ° C. was used for the amplification reaction and the Tm of the amplified nucleic acid was between about 75 ° C. and about 80 ° C. Therefore, the double strand of the amplified nucleic acid was stable under the reaction conditions, and the amplification reaction was sequence-specific.

In general, the reaction temperature can be optimally adjusted for each individual reaction step, so that such temperature can be achieved for each reaction step. Therefore, the amplification reaction includes cyclically repeated changes in temperature. In an advantageous embodiment of the process, the reaction conditions of some reaction steps are standardized so that the number of temperature steps is less than the number of reaction steps. In such a particular embodiment of the invention, at least one of the amplification steps is performed at a reaction temperature different from the reaction temperature of the other steps of amplification. Therefore, the reaction does not proceed isothermally and the reaction temperature changes cyclically.

For example, at least two temperature ranges that are alternately induced during amplification are used (periodic changes in temperature between the individual temperature ranges). In certain embodiments, the lower temperature range includes, for example, between 25 ° C. and 60 ° C., particularly between 35 ° C. and 60 ° C., particularly between 50 ° C. and 60 ° C., and the upper temperature range includes. For example, it includes a temperature between 60 ° C. and 75 ° C., particularly between 60 ° C. and 70 ° C.

In certain embodiments, the lower temperature range includes, for example, between 15 ° C. and 50 ° C., particularly between 25 ° C. and 50 ° C., particularly between 30 ° C. and 50 ° C., and the upper temperature range includes, for example, 50 ° C. Includes temperatures between ° C. and 75 ° C., especially between 50 ° C. and 65 ° C.

In certain embodiments, lower temperature ranges include, for example, temperatures between 15 ° C. and 40 ° C., particularly between 25 ° C. and 40 ° C., especially between 30 ° C. and 40 ° C., and upper temperature ranges include. For example, it includes a temperature between 40 ° C. and 75 ° C., particularly between 40 ° C. and 65 ° C.

The temperature can be kept constant in each range or varied as a temperature gradient (down or up).

Further description of the temperature setting is given in detail in the following sections of the embodiment.

Each induced temperature can be maintained for a specific time, thus resulting in an incubation step. Therefore, the reaction mixture can be incubated for a specific period of time during amplification at the selected temperature. The time can be different for each incubation step and can depend on the progress of each reaction at a given temperature (eg, primer extension or chain substitution). The duration of the incubation step may include the following range: 0.1 seconds to 10,000 seconds, especially 0.1 seconds to 1000 seconds, especially 1 second to 300 seconds, especially 1 second to 100 seconds. it can.

By varying the temperature as described above, the individual reaction steps can be carried out at a particularly selected temperature. In this way, the yield from each reaction step can be improved. Within the synthesis cycle, temperature changes or temperature switching between individual ranges can be performed several times, as needed. Therefore, the synthesis cycle can include at least one temperature change. Such temperature changes can be performed periodically on a PCR device / temperature cycler, for example as a time program.

In certain embodiments, amplification methods are preferred in which at least one of the steps involving chain substitution and at least one of the steps involving the primer extension reaction are performed simultaneously or in parallel under the same reaction conditions. In such embodiments, for example, the primer extension reaction of at least one oligonucleotide primer (eg, the first oligonucleotide primer) can be performed under temperature conditions, especially in the lower temperature range. In contrast, strand substitution is carried out in cooperation with the controller oligonucleotide, and further primer extension reactions (eg, of the second oligonucleotide primer) occur, especially in the reaction step in the upper temperature range.

In certain embodiments, amplification methods are preferred in which at least one step involving chain substitution with a controller oligonucleotide and at least one step involving a primer extension reaction are performed at different temperatures. In such embodiments, for example, the primer extension reaction of at least one oligonucleotide primer (eg, the first oligonucleotide and / or the second oligonucleotide primer) is carried out, especially under temperature conditions in a lower temperature range. It can be carried out. On the other hand, strand substitution involving controller oligonucleotides occurs especially in the reaction step in the upper temperature range.

In another particular embodiment, all steps of the amplification reaction are carried out under the same reaction conditions.

In such an embodiment, the amplification procedure can be performed under isothermal conditions, i.e. no temperature change is required to carry out the process. In such a particular embodiment of the invention, the entire amplification reaction is carried out under constant temperature, i.e., the reaction is isothermal. The time of such a reaction includes, for example, the following range: 100 seconds to 30,000 seconds, particularly 100 seconds to 10,000 seconds, particularly 100 seconds to 1000 seconds.

The "Examples" section shows that it is possible to match the structure of individual reaction components and corresponding reaction steps so that an isothermal reaction is possible.

The sum of all the method steps that doubles the amount of nucleic acid strands amplified is called the synthetic cycle. Such cycles can be isothermal or can be characterized by temperature changes in the process. Temperature changes are repeated cycle by cycle and can be identical.

When more than 5 nucleotides in the third region of the controller oligonucleotide can enter complementary binding to the first primer extension product, especially more than 10, especially more than 20 nucleotides of the controller oligonucleotide are the first. Amplification methods in which the maximum achieved temperature allows only chain separation with the involvement of the controller oligonucleotide are particularly advantageous when entering into binding with the oligonucleotide extension product of 1. In general, the longer the required bond between the controller oligonucleotide and the complementary strand of the first primer extension product, the more specific the amplification reaction is before the synthesized strand separates under reaction conditions. Specifically, the degree of specificity desired can be determined by extending or shortening a third portion of the controller oligonucleotide.

The steps of the process can be repeated at a constant temperature or at different temperatures over the entire duration of the process.

The steps of the individual methods can be performed one after another by adding individual components. In certain embodiments, all reaction components required for the amplification procedure are present in the reaction mixture at the beginning of the amplification procedure.

The initiation of the amplification reaction can be initiated by adding a component, eg, a nucleic acid strand containing the target sequence (eg, the initiation nucleic acid strand), or a polymerase or divalent metal ion, or inducing the reaction conditions required for amplification. This can be done, for example, by setting the reaction temperature required for one or more process steps.

Amplification can be performed until the amount of nucleic acid to be amplified reaches the desired amount. In another embodiment, the amplification reaction is carried out for a period of time sufficient to obtain a sufficient amount of nucleic acid to be amplified. In another embodiment, the amplification reaction is carried out over a sufficient number of synthetic cycles (doubling time) that appear to be sufficient to obtain a sufficient amount of nucleic acid to be amplified.

The reaction can be stopped by various interventions. For example, by changing the temperature (eg, cooling or heating that interferes with the function of the polymerase), or by adding a substance that arrests the polymerase reaction, such as EDTA or formamide.

After amplification, the amplified nucleic acid strand can be used for further analysis. The synthesized nucleic acid strand can be analyzed by various detection methods. For example, a fluorescently labeled oligonucleotide probe can be used, or a sequencing method (Sanger sequencing or next-generation sequencing), solid phase analysis such as microarray or bead array analysis, and the like can be used. The synthesized nucleic acid chain can be used as a substrate / template for further oligonucleotide extension reactions.

In certain embodiments, the progress of the synthetic reaction is monitored during the reaction. This can be done, for example, by using an insertion dye such as Sybrgreen or Evagreen, or by using a labeling primer (eg, Lux-oligonucleotide primer or Scorpion-oligonucleotide primer, etc.), or by using a fluorescently labeled oligonucleotide probe. It can be done by using it.

Detection of fluorescence changes during amplification is performed in the detection step of this procedure. The temperature and duration of the steps can be adapted to the respective requirements of the oligonucleotide probe. For example, the temperature of the detection step includes between 20 ° C. and 75 ° C., especially between 40 and 70 ° C., especially between 55 and 70 ° C.

During the detection step, the reaction is irradiated with light of a wavelength capable of exciting the fluorochrome molecules used in the detection system (donor or fluorescent reporter). Signal acquisition is usually performed in parallel with excitation to detect a specific fluorescent signal and quantify its intensity.

Amplification methods can be used to confirm the presence of a target nucleic acid chain in a biological or diagnostic material as part of a diagnostic procedure.

Specific Embodiment of Reaction Conditions for Second Amplification The reaction conditions for PCR are well known to those of skill in the art.

Therefore, some specific embodiments are presented herein by exemplary means.

In certain embodiments, the temperatures shown in FIGS. 3-11 include the following ranges:
T1 = 55 ° C (+/- 5 ° C)
T2 = 65 ° C (+/- 5 ° C)
T3 = 95 ° C (+/- 5 ° C)
T4 = 45 ° C (+/- 5 ° C)
T5 = 70 ° C (+/- 5 ° C)

In certain embodiments, the temperatures shown in FIGS. 3-11 include the following ranges:
T1 = 55 ° C (+/- 10 ° C)
T2 = 65 ° C (+/- 10 ° C)
T3 = 95 ° C (+/- 10 ° C)
T4 = 45 ° C (+/- 10 ° C)
T5 = 70 ° C (+/- 10 ° C)

In certain embodiments, the temperatures shown in FIGS. 3-11 include the following ranges:
T1 = 55 ° C (+/- 15 ° C)
T2 = 65 ° C (+/- 10 ° C)
T3 = 95 ° C (+/- 10 ° C)
T4 = 45 ° C (+/- 20 ° C)
T5 = 70 ° C (+/- 10 ° C)

In certain embodiments, the temperatures shown in FIGS. 3-11 include the following ranges:
T1 = 55 ° C (+/- 5 ° C)
T2 = 65 ° C (+/- 3 ° C)
T3 = 95 ° C (+/- 10 ° C)
T4 = 45 ° C (+/- 5 ° C)
T5 = 70 ° C (+/- 3 ° C)

The incubation time during a single temperature step can be between 0.1 seconds and 60 minutes, especially between 1 second and 10 minutes, especially between 1 second and 5 minutes in some runs.

Periodic temperature changes are often referred to as PCR cycles. For example, the PCR cycle involves a complete change in temperature from T1, T2 to T3.

The temperature range used is primarily intended to support the next step in the second amplification:
Temperatures of T1, T2, T4, and T5: Hybridization of third and fourth primers to complementary segments of their respective template strands, and extension of third and fourth primer extension products.

The T3 temperature is used to separate double strands containing at least one of the primer extension products. All nucleic acid strands present as double strands (eg, double strand target sequence, first amplified fragment 1.1, second amplified fragment 2.1, and P1.1-Ext and P4.1-Ext- Part1 or an intermediate product containing P2.1-Ext and P3.1-Ext-part1) is converted to a single-stranded form by the action of temperature.

In addition, the T3 temperature can serve to activate the hot-started form of the second polymerase (eg, inactivated by the antibody) or to inactivate the first polymerase. In addition, the thermal instability controller oligonucleotide can be cleaved by the temperature (T3). In addition, heat-activated oligonucleotide primers 3 and 4 can be activated.

Therefore, different temperatures can be used for reaction control.

In addition, the choice of reaction temperature in combination with the respective PCR primers (third and fourth primers of the second amplification) will affect the production / intermediate formation of the product during the second amplification. Is possible.

The degree of yield of individual products and intermediates depends on several factors. For example, higher concentrations of individual primers generally favor product / intermediate yield yields. In addition, product / intermediate formation is influenced by reaction temperature and mutual binding / affinity of individual reactants (affinity of individual primer binding to complementary primer binding sites within the template chain). It is possible to receive: In general, for example, longer oligonucleotides bind better than shorter oligonucleotides at higher temperatures, and levels of CG can also play a role in complementary sequence segments: CG. Primer-rich sequences also bind more strongly at elevated temperatures than AT-rich sequences. In addition, modifications such as MGB or 2-amino-dA or LNA can increase the binding strength of the primers to their respective complementary segments, which allows the oligonucleotide primers to preferentially bind at elevated temperatures.

For example, longer oligonucleotide primers with relatively high Tm with complementary segments (eg 60-70 ° C.) are used for PCR (eg nucleotides between 25-60, CG levels 40%). Exceeds). This allows a second amplification hybridization step and a primer extension step to be performed at a higher temperature (such as T2 or T5). Therefore, in the relatively short combination of the first and second primers (from the first amplification), the primer extension products P4.1-Ext and P3.1-Ext are preferentially formed and P1 during PCR amplification. The reaction can be controlled so that the co-amplification of the primer extension products starting with .1 and P2.1 can be significantly suppressed.

For example, when using third and fourth primers that contain only short 3'segments capable of binding to the target sequence or its equivalent (eg, the segment has a nucleotide length between 6 and 15). It may be advantageous to carry out several cycles first at low temperatures (steps 10A and B, A2.1). A similar situation can be applied, for example, to PCR primers containing a mismatch of only the essentially complementary first amplified fragment 1.1.

During the relatively cold steps (T1 and T4), primer binding to the complementary segment of the first amplified fragment of the third and / or fourth primer occurs, resulting in shorter length complementary segments and / Or despite existing discrepancies, polymerases starting from said primers hybridized to individual strands of the first amplified fragment can perform template-dependent primer extension and third and fourth oligonucleotide primers. Primer extension products containing the above can be synthesized.

The PCR cycle is repeated at least twice so that complementary strands can also be synthesized. As the cycle repeats, the third and fourth primers themselves are also copied as templates, resulting in the formation of complete primer binding sites for the third and / or fourth oligonucleotide primers.

The temperature can then be increased so that the third and fourth primers can bind to the fully formed primer binding sites at higher temperatures (step A2.2).

The use of higher temperatures (T2 and T5) is advantageous, for example, in uniform assays (when both amplification systems are in the same reaction batch). This can be used, for example, to reduce the effect of binding of the controller oligonucleotide to the complementary region of the target sequence of the third primer extension product (P3.1-Ext or P3.1-Ext part).

Enzyme synthesis is also generally carried out in buffered aqueous solution in the second amplification. As the buffer solution, a dissolved conventional buffer substance such as Tris-HCl, Tris acetic acid, potassium glutamate, HEPES buffer solution, and monosodium glutamate at a general concentration can be used. The pH value of the solution is usually between 7 and 9.5, especially between about 8 and 8.5. Buffer conditions can be applied, for example, according to the manufacturer's recommendations for the polymerase used.

Additional substances such as so-called Tm depressants (DMSO, betaine, TPAC, etc.) can be added to the buffer. Such substances may lower the double-stranded melting temperature (“Tm lowering agent”) and thus have a positive effect on the double-stranded opening. Also, polymerase stabilizing components such as Tween 20 or Triton 100 can be added to the buffer in normal amounts. Heavy metals can be compounded by adding EDTA or EGTA in conventional amounts. Polymerase stabilizers such as trehalose or PEG6000 can also be added to the reaction mixture.

Specific Embodiment of the Starting Nucleic Acid Chain: The nucleic acid chain used or to be used at the initiation of the first amplification reaction for amplification can be referred to as the starting nucleic acid chain (FIG. 1).

Its function can be understood in that it represents a primer, a synthetic segment between both primers, and an initial template that allows the correct placement of the initiation of the binding and extension processes. In certain embodiments, the starting nucleic acid strand comprises a target sequence.

The first primer extension product is produced by binding the primers to the respective primer binding sites (PBS1 and PBS2) and initiating an appropriate primer extension reaction. These are synthesized as specific copies of the nucleic acid strands present at the onset of the reaction.

In certain embodiments, the nucleic acid strand (initiating nucleic acid strand) used in the reaction mixture prior to the initiation of the amplification reaction can be identical to the nucleic acid strand being amplified. The amplification reaction only increases the amount of such nucleic acid chains.

In certain embodiments, the amplified nucleic acid and the starting nucleic acid strand define the arrangement of the individual sequence elements of the nucleic acid strand from which the starting nucleic acid strand is amplified, whereas the sequence composition of the starting nucleic acid strand is the amplified nucleic acid strand. It differs in that it can differ from the sequence of. For example, in terms of primer binding and extension during amplification, the new sequence content (with respect to the starting nucleic acid strand) can be incorporated into the amplified nucleic acid strand. Moreover, the sequence elements of the amplified nucleic acid strands may differ from such sequence elements of the starting nucleic acid strand in their sequence composition (eg, primer binding site or primer sequence). The starting nucleic acid serves only as an initial template for the specific synthesis of the amplified nucleic acid strand. The initial template can remain in the reaction mixture until the end of amplification. However, due to the exponential properties of amplification, the amount of nucleic acid strands amplified at the end of the amplification reaction exceeds the amount of starting nucleic acid strands added to the reaction.

In certain embodiments, the starting nucleic acid strand can include at least one sequence portion that is not amplified. Therefore, such starting nucleic acid strands are not identical to the sequence to be amplified. Such unamplified segments can indicate, for example, the sequence segment of the starting nucleic acid chain as a result of the sequence preparation step or the previous sequence manipulation step, respectively.

In certain embodiments, the starting nucleic acid chain added to the reaction mixture prior to the initiation of the reaction comprises at least one target sequence.

In certain embodiments, such a starting nucleic acid strand comprises at least one target sequence and an additional sequence that is still a non-target sequence. During amplification, the sequence segment containing the target sequence grows exponentially, so that the other sequence segments either do not grow exponentially at all or grow only partially.

Structure of the starting nucleic acid chain (for the first amplification)
An example of such a starting nucleic acid chain is a nucleic acid chain that comprises a target sequence, as well as a sequence fragment A and a sequence fragment B.

The sequence fragment A of the starting nucleic acid chain has significant homology to one of the sequences of both primers used for this amplification, or is essentially identical to the copyable portion of the 3'segment of one primer. Contains an array. In the synthesis of complementary strands for this segment, complementary sequences representing the respective primer binding sites are generated.

Sequence fragment B of the starting nucleic acid chain comprises a sequence suitable for complementarily binding the corresponding additional primers or 3'segments thereof to form an extendable primer template complex, sequence fragment A and sequence fragment B. Are predominantly / particularly non-complementary to each other.

In certain embodiments, the starting nucleic acid strand is added to the reaction mixture of the amplification method having the following properties:
In particular, sequence fragment A is in the 5'segment of the starting nucleic acid chain. In particular, the sequence fragment A forms a restriction in the 5'direction of the nucleic acid chain.

In particular, sequence fragment B is located 3'from sequence fragment A with respect to the binding site of the first primer and with respect to the polarity of the first primer ("downstream").

In certain embodiments, the sequence fragment B forms a nucleic acid chain restriction in the 3'direction. In certain embodiments, the sequence fragment B does not represent a nucleic acid chain limitation in the 3'direction, but is flanked by additional sequences from the 3'side. In particular, the sequence is not a target sequence and does not participate in exponential amplification.

In certain embodiments, the target sequence comprises at least one of both sequence fragments A or B. In certain embodiments, the target sequence is between sequence fragment A and sequence fragment B.

In certain embodiments (FIG. 50), such initiation nucleic acid strands can serve as templates for the synthesis of the first primer extension product. Here, for example, the starting nucleic acid strand during the primer extension reaction using the second oligonucleotide primer can be provided as a sequence segment of the longer starting nucleic acid strand during the preparatory steps prior to exponential amplification. Can be converted to single-stranded form. For example, the starting nucleic acid strand can be genomic DNA or RNA, which serves as a source of target sequences. The starting nucleic acid chain of the 5'segment of the nucleic acid chain contains a sequence segment of the second oligonucleotide primer and represents a restriction in the 5'direction, a segment 2, a segment 2 containing the target sequence or a part thereof, the first oligonucleotide. It contains a segment 3 containing the primer binding site of the primer and a segment 4 containing a non-target sequence localized in the 3'segment of the starting nucleic acid chain and thus adjacent to the segment 3 on the 3'side.

At the start of amplification, the first primer with at least the 3'segment in the first region can bind to such starting nucleic acid strand in segment 3 and be adequately extended in the presence of polymerase and nucleotides. This produces a first primer extension product that is complementary to the template strand of the starting nucleic acid strand and has a limited length. During the amplification reaction, such primer extension products are sequence-specifically separated from the template strand via the controller oligonucleotide, resulting in a sequence segment corresponding to the 3'segment of the first primer extension product to be synthesized. Can be used as a binding site for the second oligonucleotide primer.

Thus, in certain embodiments, the starting nucleic acid chain comprises the following sequence fragment (FIG. 50):
Sequence segment 1 (called segment 1), which is a sequence that is significantly homologous to the sequence of the second primer, or the copyable portion and essence of the 3'segment of the second primer. Includes the same sequence. The sequence segment 1 is in the 5'segment of the starting nucleic acid strand, and in particular the sequence segment 1 forms a nucleic acid strand restriction in the 5'direction.
Sequence segment 3 (referred to as segment 3), which complementarily binds to the first region of the first primer or its 3'segment to form an extendable primer template complex. Contains sequences suitable for. In particular, the sequence segment 3 is in three directions (“upstream”) of the sequence segment 1.
A target sequence that is partially or wholly between segment 1 and segment 3 (this portion of the target sequence is called segment 2). In certain embodiments, the target sequence comprises at least one of segment 2 and segment 1 and / or segment 3.
• Optionally, the starting nucleic acid strand of the 3'segment comprises an unamplified flanking sequence segment (referred to as segment 4).

In a particular embodiment (FIG. 51), the starting nucleic acid strand can serve as a template for the synthesis of the second primer extension product.

Here, for example, the starting nucleic acid strand during the primer extension reaction using the first oligonucleotide primer can be provided as a sequence segment of the longer starting nucleic acid strand during the preparatory step prior to exponential amplification. It can be converted to a main chain form. For example, the starting nucleic acid strand can be genomic DNA or RNA, which serves as a source of the target sequence (shown as double strand). The starting nucleic acid chain of the 5'segment of the nucleic acid chain comprises a sequence segment of the first oligonucleotide primer and represents a restriction in the 5'direction, a segment 6, a segment 6 containing the target sequence or a portion thereof, a second oligonucleotide. It contains segment 7 containing the primer binding site of the nucleotide primer and segment 8 containing a non-target sequence localized in the 3'segment of the starting nucleic acid chain and thus flanking the segment 7 on the 3'side.

During the initiation of amplification, the first primer with at least the 3'segment of its first region can bind to such starting nucleic acid strand of segment 7 and is properly the first in the presence of polymerase and nucleotides. It can be extended to the stop section of the oligonucleotide primer. This produces a second primer extension product that is complementary to the template strand of the starting nucleic acid strand and has a limited length. During the amplification reaction, such primer extension products are sequence-specifically separated from the template strand via the controller oligonucleotide, resulting in a sequence segment corresponding to the 3'segment of the second primer extension product to be synthesized. Can be used as a binding site for the first oligonucleotide primer.

In the particular embodiment, the starting nucleic acid strand comprises the following sequence fragments:
Sequence segment 5 (called segment 5), which has significant homology to the sequence of this region of the first primer, or with a copyable portion of the first region of the first primer. Contains essentially the same sequence. The sequence segment 5 is in the 5'segment of the starting nucleic acid strand and is flanked by a non-copyable oligonucleotide tail (analog to the first oligonucleotide primer), especially the segment 5 in the 5'direction. Form a limit on the copyable nucleic acid strand.
Sequence segment 7 (referred to as segment 7), which is a sequence suitable for complementarily binding to a second primer or its 3'segment to form an extendable primer template complex. Including. In particular, the segment 7 is in the 3'direction (“upstream”) of the sequence segment 5.
A target sequence that is partially or wholly between segment 5 and segment 7 (in FIG. 23, this portion of the target sequence is referred to as segment 6). In certain embodiments, the target sequence comprises segment 6 and at least one segment 5 and / or segment 7.
• Optionally, the starting nucleic acid strand of the 3'segment comprises an unamplified adjacent sequence segment (referred to as segment 8).

Functional Mode of Initiating Nucleic Acid Chains for First Amplification Reaction At the initiation of the amplification reaction, the initiating nucleic acid strand serves as a template for the initial production of each primer extension product. Therefore, it represents a starting template for the amplified nucleic acid chain. The starting nucleic acid strand does not necessarily have to be the same as the amplified nucleic acid strand. By binding and extending both primers during the amplification reaction, essentially both primers define which sequence on both terminal segments of the amplified nucleic acid chain is produced during amplification.

In certain embodiments of this method, reaction conditions that do not denature the duplex are maintained during the exponential amplification procedure. Therefore, it is advantageous if the starting nucleic acid strand has a polymerase-extensible restriction on the 5'sequence segment, resulting in arrest of enzymatic extension of each primer. Therefore, the length of the primer extension fragment produced under the reaction conditions is limited. This has a beneficial effect on strand substitution by the controller oligonucleotide, which can result in dissociation of each strand, resulting in the primer binding site being converted to a single-stranded step and thus reaching new binding of the primer. become able to.

Specific Embodiment of the First Oligonucleotide Primer (Primer 1) The first oligonucleotide primer (Primer 1) is a nucleic acid chain containing at least the following regions (FIGS. 12-14):
The first primer region of the 3'segment of the first oligonucleotide primer that is essentially sequence-specifically capable of binding to the strand of the amplified nucleic acid chain.
Directly at the 5'end of the first primer region of the first oligonucleotide primer, which contains a polynucleotide tail suitable for binding the controller oligonucleotide and supporting strand substitution by the controller oligonucleotide (step c). Alternatively, in the second region linked via a linker, the polynucleotide tail remains essentially single-stranded under reaction conditions, i.e., without forming a stable hairpin or ds structure. In essence, it is not copied by the polymerase.

The total length of the first oligonucleotide primer is between 10 and 80 nucleotides, especially between 15 and 50 nucleotides, better between 20 and 30 nucleotides, or an equivalent thereof (eg, a nucleotide modifier). The structure of the first oligonucleotide primer is adapted so that it can reversibly bind to the controller oligonucleotide under selected reaction conditions. Furthermore, the structure of the first oligonucleotide primer is compatible with its primer function. In addition, this structure is adapted to allow strand substitution with controller oligonucleotides. Overall, the structures of the first and second regions are adapted to allow exponential amplification.

In an advantageous embodiment of the invention, the first and second regions of the primer are bound in a conventional 5'-3'configuration. In certain embodiments of the invention, the second region has a direction opposite to that of the first region, as the binding of both sections is via a 5'-5'bond.

The connection of regions between each other / between each other is particularly covalent. In certain embodiments, the bond between the first and second regions is a conventional 5'-3'phosphodiester bond (coupling) for DNA. In certain embodiments, it is a 5'-5'phosphodiester bond. In certain embodiments, it is a 5'-3'phosphodiester bond and between adjacent terminal nucleotides or nucleotide modifications of both regions, at least one linker (eg, C3, C6, C12, or HEG linker or none). Base modification) is arranged.

The individual regions can contain different nucleotide modifications. Here, individual elements of nucleotides can be modified: nucleobases and backbones (sugar content and / or phosphate content). In addition, modifications that lack or modify (eg, PNA) at least one component of a standard nucleotide building block can be used.

In certain embodiments, the second region of the first oligonucleotide primer comprises additional sequences that do not bind to the controller oligonucleotide. These sequences can be used for other purposes, such as binding to a solid phase. These sequences are particularly localized at the 5'end of the polynucleotide tail.

In certain embodiments, the first oligonucleotide primer may contain a characteristic label. Examples of such labels are dyes (eg, FAM, TAMRA, Cy3, Alexa488, etc.) or biotin, or other groups capable of specifically binding, such as digoxigenin.

The length of the first primer region sequence of the first oligonucleotide primer is between about 3-30 nucleotides, especially 5-20 nucleotides, and this sequence is predominantly in the 3'segment of the chain of the nucleic acid chain to be amplified. Is complementary to. Specifically, the primer region must be able to specifically bind to the complementary 3'segment of the second primer extension product. The first region can be copied by reverse synthesis and also functions as a template for the second strand. In particular, nucleotide building blocks are linked to each other via a common 5'-3'phosphodiester bond or phosphothioester bond.

The first primer region specifically contains nucleotide monomers that do not or have a slight effect on the function of the polymerase, such as:
• Natural nucleotides (dA, dT, dC, dG, etc.) or their modifications without base pair modification • Modified nucleotides, 2-amino-dA, 2-thio-dT, or other nucleotide modifications with different base pairs. body.

In certain embodiments, the 3'-OH end of the region is unmodified and has a functional 3'-OH group that can be recognized by the polymerase. The first primer region serves as an initiator for the synthesis of the first primer extension product in amplification. In a further specific embodiment, the first region comprises at least one phosphorothioate compound, so degradation of the 3'end of the primer by the 3'exonuclease activity of the polymerase cannot occur.

The sequence of the first region of the first oligonucleotide primer and the sequence of the second region of the controller oligonucleotide are particularly complementary to each other.

In certain embodiments, the first primer region or its 3'segment can bind to the sequence segment of the target sequence.

Second Region of First Oligonucleotide Primer The second region of the first oligonucleotide primer remains unparticularly copied by the polymerase, in particular during the synthetic reaction, and can bind to the first region of the controller oligonucleotide. A nucleic acid sequence containing at least one polynucleotide tail. The segment of the second region that primarily binds to the controller oligonucleotide can be referred to as the polynucleotide tail.

In addition, the second region of the first oligonucleotide primer needs to not only specifically bind the controller oligonucleotide under reaction conditions, but also need to be involved in the process of strand substitution by the controller oligonucleotide. .. Therefore, the structure of the second region needs to be suitable for inducing spatial proximity between the controller oligonucleotide and the corresponding double-stranded terminal (specifically, the 3'end of the second primer extension product). ..

The structural composition of the second region of the first oligonucleotide primer is shown in detail in some embodiments. Here, the arrangement of oligonucleotide segments and the modifications used are taken into account so as to lead to termination of polymerase catalytic synthesis.

The length of the second region is between 3-60 nucleotides, especially 5-40 nucleotides, especially 6-15 nucleotides or equivalent.

The sequence of the second region can be arbitrarily selected. In particular, this sequence is non-complementary to the first region of the amplified nucleic acid and / or second oligonucleotide primer and / or first oligonucleotide primer. Moreover, it does not contain any self-complementary segments, especially hairpins and stem loops.

The sequence of the second region is particularly adapted to the sequence of the first region of the controller oligonucleotide because both sequences can bind under reaction conditions. In certain embodiments, the binding is reversible under reaction conditions: therefore there is an equilibrium between the components bound to each other and the components not bound to each other.

The sequence of the second region of the first oligonucleotide primer has 1-40 complementary bases that can bind to the first region of the controller oligonucleotide, better 3-20, especially 6-15. Especially selected as.

In particular, the function of the second region is to bind the controller oligonucleotide. In certain embodiments, the binding is particularly specific such that the second region of the first oligonucleotide primer can bind to a specific controller oligonucleotide. In another embodiment, the second region can bind to more than one controller oligonucleotide under reaction conditions.

In general, there is no need for an exact match in the sequence between the second region of the first oligonucleotide primer and the first region of this controller oligonucleotide. The degree of complementarity between the second region of the first oligonucleotide primer and the first region of this controller oligonucleotide is 20% to 100%, better 50% to 100%, especially 80%. It can be between ~ 100%. Each complementary region may be placed directly adjacent to each other or may also include non-complementary sequence segments between them.

In certain embodiments, the second region of the first oligonucleotide primer may contain at least one Tm modification. By incorporating such modifications, the stability of binding between the second region of the first oligonucleotide primer and the first region of the controller oligonucleotide can be modified. For example, Tm-elevating modifications (nucleotide-modified or non-nucleotide-modified) such as LNA nucleotides, 2-aminoadenosine or MGB modifications can be used. On the other hand, Tm descending modifications such as inosine nucleotides can also be used. Linkers (eg, C3, C6, HEG linkers) can also be incorporated in the structure of the second region.

For strand substitution, the controller oligonucleotide needs to be spatially close to the double-stranded end of the amplified nucleic acid. The double-stranded end consists of a segment of the first primer region of the first primer extension product and a corresponding complementary 3'segment of the second primer extension product.

The polynucleotide tail primarily binds complementarily to the controller oligonucleotide under reaction conditions, thus causing a temporary proximity of the second region of the controller oligonucleotide to the first region of the extended primer extension product. As a result, complementary binding between the elements begins during the strand replacement process.

In certain embodiments, binding of the controller oligonucleotide to the polynucleotide tail of the first oligonucleotide primer directly guides such contact. This means that the polynucleotide tail and the first primer region of the first oligonucleotide primer need to be attached directly to each other. Due to this arrangement, after the controller oligonucleotide binds to its first region, there is direct contact between the complementary base in the second region of the controller oligonucleotide and the corresponding base in the first primer region. It is possible that chain substitution can be initiated.

In certain embodiments, there is another structure of the second region of the first oligonucleotide primer between the structure of the polynucleotide tail and the structure of the first primer region. Thus, after the controller oligonucleotide binds to the polynucleotide tail, it is not placed directly in the first primer region, but at a distance from it. The structure between the non-copyable polynucleotide tail and the copyable first primer region of the oligonucleotide primer can generate such a distance. The distance has a value between 0.1 and 20 nm, especially between 0.1 and 5 nm, especially between 0.1 and 1 nm.

Such structures represent, for example, segments that are not complementary to a linker (eg, C3 or C6 or HEG linker) or controller oligonucleotide (eg, in the form of non-complementary, non-copyable nucleotide modifications). The length of these structures is usually measurable with chain atoms. The length is between 1 to 200 atoms, especially 1 to 50 chain atoms, especially 1 to 10 chain atoms.

To preserve the polynucleotide tail of the polymerase that cannot be copied under amplification conditions, the second region of the first oligonucleotide primer is usually the second primer after the polymerase has successfully copied the first primer region. Each contains a sequence alignment or structure that leads to the termination of the polymerase in the synthesis of the elongation product. The structure is to prevent the polynucleotide tail of the second region from being copied. Therefore, the polynucleotide tail remains uncopied, especially by the polymerase.

In certain embodiments, such a structure is between the first primer region and the polynucleotide tail.

In certain embodiments, the polynucleotide tail sequence may include nucleotide modifications that result in polymerase termination. In this way, the sequence segment of the second region of the first oligonucleotide primer can contain both functions: it is both the polynucleotide tail and the nucleotide-modified sequence that results in the termination of the polymerase.

Modifications in the second region of the first oligonucleotide primer that result in synthetic arrest and thus leave the polynucleotide tail uncopied in this application are under the term "first blocking unit or arrest region". Combined with.

In the following, further embodiments of the structure that can result in the termination of the synthesis of the second strand are shown.

It is known that some building blocks in oligonucleotide synthesis interfere with the polymerase reading the template, leading to the termination of polymerase synthesis. For example, non-copyable nucleotide or non-nucleotide modifications are known. There are also types / alignments of synthesis of nucleotide monomers within oligonucleotides that lead to polymerase termination (eg, 5'-5 alignment or 3'-3'alignment). Oligonucleotide primers with a non-copyable polynucleotide tail are also known in the prior art (eg, a Corpion-primer structure or a primer for binding to a solid phase). Both primer variants exhibit oligonucleotide primer structures that can initiate strand synthesis, allowing primer extension reactions. The result is a first strand that integrates the primer structure with the tail of the primer extension product. In the synthesis of the complementary strand to the first primer extension product, for example, during the PCR reaction, the second strand is extended to the "blocking unit / stop structure" of the primer structure. Both primer structures shown are designed so that the 5'portion of the oligonucleotide primer remains single-stranded and is not copied by the polymerase.

In certain embodiments, the second region of the oligonucleotide primer has a conventional alignment with a total length of 5'to 3'and includes a polynucleotide tail containing a non-copyable nucleotide modification. Such non-copyable nucleotide modifications are, for example, 2'-O-alkylRNA modifications, PNA, morpholino. The modifications can be distributed separately in the second primer region.

The portion of the non-copyable nucleotide modification in the polynucleotide tail can exceed 20% to 100%, especially 50%, of the nucleotide building block. In particular, these nucleotide modifications are in the 3'segment of the second region and thus flank the first region of the first oligonucleotide primer.

In certain embodiments, the non-copyable nucleotide-modified sequence is at least partially complementary to the sequence of the template strand, so that the binding of the primer to the template comprises at least a portion of said nucleotide modification. It is done by. In certain embodiments, the non-copyable nucleotide-modified sequence is non-complementary to the sequence in the template strand.

Non-copytable nucleotide modifications are particularly covalently linked to each other and thus represent the sequence segment of the second region. The length of this segment includes 1-40, especially 1-20 nucleotide modifications, especially 3-10 nucleotide modifications.

In certain embodiments, the second region of the first oligonucleotide primer has a conventional alignment with a total length of 5'-3'and is a non-copyable nucleotide modification (eg, 2'-O-alkyl modification). ) And a polynucleotide tail containing at least one non-nucleotide linker (eg, C3, C6, HEG linker). The function of the non-nucleotide linker is to co-link adjacent nucleotides or nucleotide modifications while site-specifically disrupting the synthesis function of the polymerase.

Such non-nucleotide linkers do not position the structure of the polynucleotide tail and the structure of the first primer region apart from each other. Rather, the polynucleotide tail is spatially close to the first primer region. Non-nucleotide linkers contain modifications of 200 chain atoms or less, more preferably 50 chain atoms or less, particularly preferably 10 chain atoms or less. The minimum length of such a linker can be one atom. An example of such a non-nucleotide linker is a linear or branched chain alkyl linker having an alkyl chain containing at least one carbon atom, particularly at least 2-30, more particularly 4-18. Such linkers are well known in oligonucleotide chemistry (eg, C3, C6 or C12 linkers), between the sequence of the polynucleotide tail and the sequence of the first region of the first oligonucleotide primer. It can be incorporated during solid phase synthesis of oligonucleotides. Another example of such a non-nucleotide linker is a linear or branched polyethylene glycol derivative. A known example in oligonucleotide chemistry is hexaethylene glycol (HEG). A further example of such a non-nucleotide linker is debase modification (eg, THF modification as an analog of dRibose).

If one or more such modifications are integrated into the second region, they can effectively interfere with the copying function of the polymerase during the synthesis of the second primer extension product, and thus such. The segment remains uncopied in the 3'direction after the modification. The number of such modifications in the second region can be between 1-100, especially between 1-10, especially between 1-3.

The location of such a non-nucleotide linker can be at the 3'end of the second region, thus indicating the transition of the oligonucleotide primer to the first and second regions.

Also, the arrangement of non-nucleotide linkers in the central segment of the second region can be used. Therefore, the polynucleotide tail is divided into at least two segments. In this embodiment, the 3'segment of the polynucleotide tail comprises at least one, more particularly, non-copyable nucleotide modification between, for example, between 2 and 20, especially between 2 and 10. These non-copyable nucleotide modifications are particularly present at the transition between the first and second regions of the oligonucleotide primer.

In certain embodiments, the second region of the oligonucleotide primer has a total length of 5'to 3'alignment and contains at least one nucleotide monomer of the "reverse" alignment of 3'to 5'. It contains a nucleotide tail, which is located at the transition between the first and second regions of the first oligonucleotide primer.

In certain embodiments, the second region of the oligonucleotide primer comprises a polynucleotide tail, such a polynucleotide tail from a nucleotide that is inversely aligned and directly adjacent to the first region of the first oligonucleotide primer. It is fully constructed so that the coupling of the first and second regions is achieved by the 5'-5'position. The advantage of such an alignment is that the polymerase encounters a "reverse" alignment of nucleotides after copying the first region, which usually leads to arrest of synthesis at this site.

In the "reverse" alignment of nucleotides over the full length of the polynucleotide tail, especially the 3'terminal nucleotides of the polynucleotide tail should be blocked at the 3'-OH end to prevent side reactions. Alternatively, terminal nucleotides having no 3'OH groups, such as dideoxynucleotides, can be used.

In such embodiments, of course, the corresponding nucleotide alignment within the controller oligonucleotide should also be adapted. In such cases, the first and second regions of the controller oligonucleotide need to be bound in a 3'-3'alignment.

In certain embodiments, the second region of the oligonucleotide primer has a conventional alignment with a total length of 5'to 3'and is synthetically exclusive with the native dNTP (dATP, dCTP, dGTP, dTTP, or dUTP). Includes a polynucleotide tail containing at least one nucleotide modification that does not represent a nucleobase complementary to the polymerase when performed in.

For example, Iso-dG or Iso-dC nucleotide modifications can be incorporated into the second region of the first oligonucleotide primer as a single, but particularly several (at least 2-20) nucleotide modifications. Further examples of nucleobase modifications are various modifications of the extended genetic alphabet. Such nucleotide modifications do not specifically support complementary base pairing with native nucleotides, so that the polymerase (at least in theory) has a set of nucleotides (dATP, dCTP, dGTP, dTTP, or dUTP). Do not insert. However, in practice, essential insertions may occur, especially with high concentrations of dNTP substrate and long incubation times (eg 60 minutes or more). Therefore, some of such nucleotide modifications, especially located at adjacent sites, should be used. Termination of polymerase synthesis is affected by the lack of suitable complementary substrates for these modifications. Oligonucleotides with Iso-dC or Iso-dG can be synthesized by standard methods and are available from several commercial suppliers (eg, Trilink-Technologies, Eurogentec, Biomers GmbH). Alternatively, the sequence of the first region of the controller oligonucleotide can also be adapted to the sequence of such a second primer region. Here, the complementary nucleobases of the extended genetic alphabet can be correspondingly integrated into the first region of the controller oligonucleotide during chemical synthesis. For example, Iso-dG can be integrated into the second region of the first primer nucleotide, and its complementary nucleotide (Iso-dC-5-Me) is at the appropriate site in the first region of the controller oligonucleotide. Can be placed.

In summary, the termination of polymerase synthesis in the second region can be achieved in different ways. However, this blockade preferably occurs only when the polymerase copies the first region of the first oligonucleotide primer. In this way, it is confirmed that the second primer extension product has a suitable primer binding site in the 3'segment. This primer binding site is exposed during strand substitution and is therefore available for new binding of additional first oligonucleotide primers.

In the synthesis of complementary strands to the first primer extension product, the primer extension reaction is stopped in front of the polynucleotide tail. In this way, this polynucleotide tail remains single-stranded for interaction with the controller oligonucleotide and is therefore available for binding, with the corresponding complementary segment of the controller oligonucleotide at the appropriate double-stranded end. By being close to, it supports the initiation of the chain substitution reaction by the controller oligonucleotide. In this way, the distance between the complementary portion of the controller oligonucleotide (second region) and the complementary portion of the extended oligonucleotide primer (first region) is minimized. Such spatial proximity facilitates the initiation of strand substitution.

In light of the schematic of the nucleic acid-mediated strand substitution reaction, the complementary sequence of the controller oligonucleotide is in the vicinity of the appropriate duplex end. This results in competition for binding of the first oligonucleotide primer to the first region between the strand of the controller oligonucleotide and the template strand complementary to the primer. By repeatedly forming base pairs between the primer region and the complementary segment of the controller oligonucleotide (the second region of the controller nucleotide) or the complementary segment of the template strand, the initiation of the nucleic acid-mediated strand replacement process is initiated, respectively. Occur.

In general, the yield of initiation of strand substitution increases as the corresponding complementary sequence portion of the controller oligonucleotide approaches the complementary segment of the primer region. However, as this distance increases, the yield of initiation of chain substitution decreases.

In the context of the present invention, it is not essential that the initiation of chain substitution works in maximum yield. Therefore, the first primer of the first oligonucleotide primer that binds to the complementary strand of the template to form a complementary duplex when bound to the polynucleotide tail of the second region of the first oligonucleotide primer. The distance between the 5'segment of the region and the corresponding complementary sequence portion of the controller oligonucleotide can be in the following range: 0.1-20 nm, especially 0.1-5 nm, especially 0.1-1 nm. .. In certain cases, the distance is less than 1 nm. Expressed in other units, the distance corresponds to tracks of less than 200 atoms, especially less than 50 atoms, especially less than 10 atoms. In certain cases, the distance is one atom. The distance information is for orientation only, indicating that shorter distances between these structures are preferred. In many cases, the distance can only be measured by analyzing the exact structure of the oligonucleotide and evaluating measurements of sequence distance or linker length.

The first primer may also contain additional sequence segments that are not required for interaction with the controller oligonucleotide or template strand. Such sequence segments can be attached, for example, to additional oligonucleotides used as detection probes or immobilization partners in binding to the solid phase.

Primer Function of First Oligonucleotide Primer The first oligonucleotide primer can be used in several separate steps. First, it exerts a primer function in amplification. As a result, the primer extension reaction is carried out using the second primer extension product as a template. In certain embodiments, the first oligonucleotide primer at the initiation of the amplification reaction can use the initiating nucleic acid strand as a template. In certain embodiments, the first oligonucleotide primer can be used to design / provide the starting nucleic acid strand. During amplification, the first primer functions as an initiator in the synthesis of the first primer extension product, using the second primer extension product as a template. The 3'segment of the first primer contains sequences that are primarily complementary to the second primer extension product. Enzymatic extension of the first oligonucleotide primer using the second primer extension product as a template results in the formation of the first primer extension product. Such a first primer extension product comprises a target sequence or a portion thereof. In the process of synthesizing the second primer extension product, the sequence of the copyable portion of the first oligonucleotide primer is recognized as a template by the polymerase, and the corresponding complementary sequence is synthesized, and therefore each primer binding site is Occurs for the first oligonucleotide primer. Synthesis of the first primer extension product is carried out and includes up to the 5'segment of the second oligonucleotide primer. Immediately after the synthesis of the first primer extension product, this product binds to the second primer extension product to form a double-stranded complex. The second primer extension product is sequence-specifically substituted from the complex by a controller oligonucleotide. Thereby, the controller oligonucleotide binds to the first primer extension product. After successful strand substitution with the controller oligonucleotide, the second primer extension product can then serve as the template itself for the synthesis of the first primer extension product. Now that the 3'segment of the first primer extension product is free, additional second oligonucleotide primers can be attached and therefore new synthesis of the second primer extension product can be initiated.

In addition, the first oligonucleotide primer can function as an initiator for the synthesis of the first primer extension product starting from the starting nucleic acid strand at the start of amplification. In certain embodiments, the sequence of the first primer is completely complementary to the corresponding sequence segment of the starting nucleic acid strand. In certain embodiments, the sequence of the first oligonucleotide primer is only partially complementary to the corresponding sequence segment of the starting nucleic acid strand. However, the above-mentioned difference complementarity does not prevent the first oligonucleotide primer from initiating a predominantly sequence-specific primer extension reaction. The respective differences in the complementarity of the first oligonucleotide primer to each position of the starting nucleic acid strand are particularly in the 5'segment of the first region of the first oligonucleotide primer, and therefore the main in the 3'segment. It is possible to pair bases complementary to and initiate synthesis. For the initiation of synthesis, for example, the first 4-10 positions of the 3'segment need to be completely complementary to the template (starting nucleic acid strand). The remaining nucleotide positions can differ from perfect complementarity. Therefore, the degree of perfect complementarity in the remaining 5'segments of the first region of the first oligonucleotide primer includes the range between 50% and 100% of the base composition, especially between 80% and 100%. obtain. According to the length of the first region of the first oligonucleotide primer, the sequence differences are at positions 1 to at most 15, especially 1 to at most 5. Therefore, the first oligonucleotide primer can initiate the synthesis of the starting nucleic acid chain. In subsequent synthesis of the second primer extension product, the copyable sequence segment of the first oligonucleotide primer is copied by the polymerase, which in turn results in a fully complementary primer binding site in the subsequent synthesis cycle. It is formed in the second primer extension product for binding of one oligonucleotide primer and becomes available in subsequent synthetic cycles.

In certain embodiments, the first oligonucleotide primer can be used in the preparation of the starting nucleic acid chain. Thereby, such a first oligonucleotide primer binds primarily / specifically to a nucleic acid (eg, single-stranded genomic DNA or RNA containing the target sequence or its equivalent) in a sequence-specific manner and in the presence of the polymerase. The template-dependent primer extension reaction can be started at. The binding position is selected such that the primer extension product contains the desired target sequence. Extension of the first oligonucleotide primer results in a nucleic acid strand having a sequence complementary to the template. Such chains can be separated from the template (eg, by heat or alkali) and thus converted to a single chain form. Such a single-stranded nucleic acid strand can function as a starting nucleic acid strand at the start of amplification. In the 5'segment such initiation nucleic acid strand comprises a sequence portion of the first oligonucleotide primer, which further comprises a target sequence or equivalent thereof and a primer binding site for the second oligonucleotide primer. The detailed procedure is described in the section "Initiation of Nucleic Acid Chains".

The synthesis of the first primer extension product is a primer extension reaction, forming individual steps of amplification. The reaction conditions during this step are adjusted accordingly. The reaction temperature and reaction time are selected so that the reaction can occur continuously. The preferred temperature in this step depends on the strength of the polymerase used and the binding strength of each first oligonucleotide primer to its primer binding site, eg, 15 ° C to 75 ° C, especially 20 to 65 ° C, especially 25 ° C to. Includes the 65 ° C range. The concentration of the first oligonucleotide primer comprises the range of 0.01 μmol / l to 50 μmol / l, particularly 0.1 μmol / l to 20 μmol / l, particularly 0.1 μmol / l to 10 μmol / l.

In certain embodiments, all steps of amplification proceed under stringent conditions that prevent or delay the formation of non-specific products / by-products. Such conditions exceed, for example, higher temperatures, especially 50 ° C.

When two or more specific nucleic acid chains are amplified in one batch, in certain embodiments, particularly sequence-specific oligonucleotide primers are used to amplify the corresponding target sequence.

In particular, the sequences of the first oligonucleotide primer, the second oligonucleotide primer and the controller oligonucleotide are adapted to each other so that side reactions, such as primer dimer formation, are minimized. Thus, for example, the sequences of the first and second oligonucleotide primers prevent both oligonucleotide primers from initiating or supporting the amplification reaction in the absence of the appropriate template and / or target sequence and / or starting nucleic acid strand, respectively. Fitted to each other. This can be achieved, for example, by the second oligonucleotide primer not containing the primer binding site of the first oligonucleotide primer and the first oligonucleotide primer not containing the primer binding site of the second oligonucleotide primer. .. Furthermore, it should be avoided that the primer sequence contains an extended and extended self-complementary structure (self-complementary).

In certain embodiments, the synthesis of the first and second primer extension products proceeds at the same temperature. In certain embodiments, the synthesis of the first and second primer extension products proceeds at different temperatures. In certain embodiments, the synthesis of the first primer extension product and the strand substitution with the controller oligonucleotide proceed at the same temperature. In certain embodiments, the synthesis of the first primer extension product and the chain substitution with the controller oligonucleotide proceed at different temperatures.

Specific Embodiments of Controller Oligonucleotides Controller oligonucleotides (FIGS. 19-26) include:
A first single-stranded region that can bind to the polynucleotide tail of the second region of the first oligonucleotide primer,
A second single-stranded region that can bind essentially complementaryly to the first region of the first oligonucleotide primer,
A third single-stranded region that is essentially complementary to at least one segment of the extension product of the first primer extension product.
And, the controller oligonucleotide does not function as a template for primer extension of the first or second oligonucleotide primer.

In general, the sequence of the third region of the controller oligonucleotide is adapted to the sequence of the nucleic acid to be amplified, as it serves as a template for the order of the nucleotides in the extension of the first primer. The sequence of the second region of the controller oligonucleotide is matched to the sequence of the first primer region. The structure of the first region of the controller oligonucleotide is adapted to the sequence of the second region of the first oligonucleotide primer, especially the nature of the polynucleotide tail.

The controller oligonucleotide may also contain additional sequence segments that do not belong to the first, second or third region. These sequences can be linked, for example, as sequences flanking the 3'and 5'ends. In particular, these sequence segments do not interfere with the function of the controller oligonucleotide.

The structure of the controller oligonucleotide has the following characteristics in particular:
The individual regions are covalently bonded to each other. For example, the bond can be made via a conventional 5'-3' bond. For example, phosphodiester bonds or nuclease-resistant phosphodiester bonds can be used.

The controller oligonucleotide can be attached to the polynucleotide tail of the first oligonucleotide primer by its first region, and the binding is primarily mediated by hybridization of complementary bases. The length of the first region is 3 to 80 nucleotides, particularly 4 to 40 nucleotides, particularly preferably 6 to 20 nucleotides. The degree of sequence matching between the sequence of the first region of the controller oligonucleotide and the sequence of the second region of the first oligonucleotide primer is 20% to 100%, especially 50% to 100%, especially 80%. It can be between ~ 100%. Binding of the first region of the controller oligonucleotide should be particularly specific to the second region of the first oligonucleotide primer under reaction conditions.

The sequence of the first region of the controller oligonucleotide has 1-40 complementary bases, particularly 3-20, particularly 6-15, that can complementarily bind to the second region of the first oligonucleotide primer. Especially selected as.

Controller oligonucleotides do not show a polymerase template and may therefore include nucleotide modifications that do not support polymerase function, which can be both base modifications and / or glycophosphate backbone modifications. The controller oligonucleotide in the first region contains nucleotides and / or nucleotide modifications selected from the following list: DNA, RNA, LNA (locked with 2'-4'bridged binding to sugar residues. Nucleic acid), UNA (“unlocked nucleic acid” without binding between 2'-3'atoms of sugar residues), PNA (“peptide nucleic acid” analog), PTO (phosphorothioate), morpholino analog, 2'-O-alkylRNA modification (2'-OMe, 2'-O propargyl, 2'-O- (2-methoxyethyl), 2'-O-propyl-amine, etc.), 2'-haloRNA, 2'-haloRNA, 2 '-Amino RNA, etc. These nucleotides or nucleotide modifications are attached to each other, for example, by conventional 5'-3'bonds or 5'-2' bonds. For example, phosphodiester bonds or nuclease-resistant phosphodiester bonds can be used.

The controller oligonucleotide in the first region can include nucleotides and / or nucleotide modifications, and the nucleobase is selected from the list below: adenine and its analogs, guanine and its analogs, cytosine and its analogs, uracil. And their analogs, thymine and its analogs, inosins or other universal nucleotides (eg, nitroindole), 2-amino-adenine and its analogs, isothytosine and its analogs, isoguanine and its analogs.

The controller oligonucleotide in the first region can include a non-nucleotide compound selected from the following list: it may affect the binding strength between the controller oligonucleotide and the first oligonucleotide primer. Intercalating substances (MGB, nucleotides, etc.). The same elements can also be used in the second region of the first primer.

Controller oligonucleotides in the first region can include non-nucleotide compounds, such as linkers such as C3, C6, HEG linkers capable of linking individual segments of the first region to each other.

The controller oligonucleotide can be attached to the first primer region of the first oligonucleotide primer by its second region, and the binding is mediated by hybridization of essentially complementary bases.

The length of the second region of the controller oligonucleotide is adapted to, in particular, the length of the first region of the first oligonucleotide primer. It is between about 3-30 nucleotides, especially 5-20 nucleotides. The sequence of the second region of the controller oligonucleotide is particularly complementary to the first region of the first oligonucleotide primer. The degree of complementarity agreement is 80% to 100%, especially 95% to 100%, especially 100%. The second region of the controller oligonucleotide specifically comprises a nucleotide modification that interferes with the polymerase in the extension of the first oligonucleotide primer, but does not prevent or essentially prevent the formation of complementary double strands, eg, 2'-O-alkylRNA analogs (eg, 2'-O-Me, 2'-O- (2-methoxyethyl), 2'-O-propyl, 2'-O-propargyl nucleotide modification), LNA, PNA , Or a morpholinonucleotide modification. The individual nucleotide monomers are specifically linked via 5'-3'bonds, but 5'-2'bonds between nucleotide monomers can also be used.

The sequence lengths and properties of the first and second regions of the controller oligonucleotide such that binding of the region to the first oligonucleotide primer under reaction conditions in at least one reaction step of the method is reversible. Especially selected for. That is, although the controller oligonucleotide and the first oligonucleotide primer can reliably bind to each other, this binding does not result in the formation of a complex of both elements that is permanently stable under reaction conditions.

Rather, the equilibrium between the bound complex form of the controller oligonucleotide and the first oligonucleotide primer and the free form of the individual components is intended or possible under reaction conditions in at least one reaction step. Should be. In this way, it is guaranteed that at least a portion of the first oligonucleotide primer under reaction conditions is present in free form and can interact with the template to initiate the primer extension reaction. On the other hand, in this way, it is guaranteed that each sequence region of the controller oligonucleotide can be used for binding to the extended oligonucleotide primer.

By selecting the temperature during the reaction, the free single-stranded portion, and thus the reactive component portion, can be acted upon by lowering the temperature, thereby binding the first oligonucleotide primer to the controller oligonucleotide. , Both involved bind complementary double-stranded complexes. In this way, the concentration of the single chain form of the individual components can be reduced. As the temperature rises, both components can result in dissociation into a single-stranded form. Within the melting temperature range of the complex (controller oligonucleotide / first oligonucleotide primer), about 50% of the components are present in single-stranded form and about 50% are present as double-stranded complex. Therefore, the concentration of the single chain form in the reaction mixture can be affected by using the appropriate temperature.

In embodiments of amplification methods involving temperature changes between individual reaction steps, the desired reaction conditions can be achieved during each reaction step. For example, by using the temperature range of the approximate melting temperature of the controller oligonucleotide / first oligonucleotide primer complex, it is possible that parts of the free form of the individual components are affected. Here, the temperature used destabilizes the complex containing the controller oligonucleotide / first oligonucleotide primer, thereby at least temporarily one individual complex component during this reaction step. It becomes a chain and thus can interact with other reaction partners. For example, the first sequence region of the controller oligonucleotide can be released from a double-stranded complex with a non-extended first primer and thus with the second sequence region of the extended first oligonucleotide primer. They can interact and therefore initiate chain substitutions. On the other hand, the release of the first non-extending oligonucleotide primer from the complex containing the controller oligonucleotide / first oligonucleotide primer makes the first primer region single-stranded and thus capable of interacting with the template. Allows the primer to initiate elongation by the polymerase.

The temperature used does not have to correspond exactly to the melting temperature of the controller oligonucleotide / first oligonucleotide primer complex. It is sufficient that the temperature in one reaction step be used within the approximate melting temperature range. For example, the temperature in one of the reaction steps includes a range of Tm ± 10 ° C., particularly Tm ± 5 ° C., particularly Tm ± 3 ° C. of the controller oligonucleotide / first oligonucleotide primer complex.

Such temperatures can be adjusted, for example, during reaction steps involving sequence-specific chain substitutions with controller oligonucleotides.

In an embodiment of an amplification method in which amplification proceeds under isothermal conditions without involving temperature changes between individual reaction steps, the reaction conditions are the composite form of the controller oligonucleotide and the first oligonucleotide primer and the individual components. Equilibrium with the free form is possible.

The ratio between the complex form of the controller oligonucleotide and the first oligonucleotide primer and the free form of the individual components depends on both the reaction conditions (eg, temperature and Mg ²⁺ concentration) and the structure and concentration of the individual components. Can be affected.

The sequence lengths and properties of the first and second regions of the controller oligonucleotide in a particular embodiment determine the first oligonucleotide under given reaction conditions (eg, in the reaction step of chain substitution with the controller oligonucleotide). The ratio between the portion of the free controller oligonucleotide and the portion of the controller oligonucleotide in the complex with the primer ranges from 1: 100 to 100: 1, especially 1:30 to 30: 1, especially 1:10. Selected to include 10: 10. The ratio of the free first oligonucleotide primer portion to the first oligonucleotide primer portion in the complex with the controller oligonucleotide is 1: 100-100: 1, especially 1:30-30: 1. In particular, it includes the range of 1:10 to 10: 1.

In certain embodiments, the concentration of the first oligonucleotide primer is higher than the concentration of the controller oligonucleotide. In this way, an excess of first primer is present during the reaction and the controller oligonucleotide must be released from binding to the first primer by choosing an appropriate reaction temperature for its effect. Must be. Generally, this is done by raising the temperature until a sufficient concentration of free form of the controller oligonucleotide is present.

In certain embodiments, the concentration of the first oligonucleotide primer is lower than the concentration of the controller oligonucleotide. In this way, an excess of controller oligonucleotide is present and the first oligonucleotide primer must be cleaved from binding to the controller oligonucleotide by choosing an appropriate reaction temperature for its effect. .. Generally, this is done by raising the temperature until a sufficient concentration of the free form of the first oligonucleotide primer is present.

Equilibrium exists under isothermal conditions, with certain parts of the first oligonucleotide primer and certain parts of the controller oligonucleotide binding to each other, while the others are present in a single-stranded form in the reaction.

The third region of the controller oligonucleotide allows it to bind to at least one segment of the specifically synthesized extension of the first oligonucleotide primer (FIGS. 13-35). Binding is particularly carried out by hybridization of complementary bases between the controller oligonucleotide and the extension product synthesized by the polymerase.

To support the chain substitution reaction, the sequence of the third region needs to have high complementarity, especially to the extension product. In certain embodiments, 100% of the sequence in the third region is complementary to the extension product.

In particular, the third region binds to the stretch product segment immediately following the first region of the first oligonucleotide primer. Therefore, the extension product segment is in particular in the 5'segment of the total extension product of the first oligonucleotide primer.

In particular, the third region of the controller oligonucleotide is not bound over the full length of the extension product of the first oligonucleotide primer. In particular, one segment at the 3'end of the extension product remains unbound. The 3'end segment is required for binding of the second oligonucleotide primer.

The length of the third region is adapted accordingly so that the third region binds to the 5'-position segment of the extension product but not to the 3'-position segment of the extension product.

The overall length of the third region of the controller oligonucleotide is 2 to 100, especially 6 to 60, particularly preferably 10 to 40 nucleotides or equivalent. The controller oligonucleotide can complementarily bind to a segment of the extension product over this length, thus replacing the 5'-position segment of the extension product from binding to its complementary template strand.

The length of the 3'-position segment of the extension product that is not bound by the controller oligonucleotide includes, for example, the range of 5-60 nucleotides, especially 10-40, especially 15-30 nucleotides.

The 3'-position segment of the extension product is not replaced by the controller oligonucleotide from the binding to the template strand. Also, the fully bound third region of the controller oligonucleotide to the complementary segment of the extension product allows the first primer extension product to remain bound to the template strand via its 3'position segment. it can. The binding strength of the complex is particularly selected so that it can dissociate spontaneously, for example under reaction conditions (step e). This is because, for example, the melting temperature of the complex of the extension product of the first oligonucleotide primer at the 3'position and its template chain is the approximate reaction temperature in each reaction step (reaction step e). It can be achieved if it is within the range or below the reaction temperature. If the complex is less stable in the 3'segment of the extension product, complete binding of the third region of the controller oligonucleotide to the 5'-position segment of the extension product is the template strand of the first primer extension product. Causes rapid dissociation from.

The controller oligonucleotide is fully equipped with the appropriate structure for exerting its function under each reaction condition, and the extended first oligonucleotide primer is sequence-specifically replaced from the binding with the template strand. The template strand can be converted to a single-stranded form by, and thus can be utilized for further binding by the polymerase to the new first oligonucleotide primer and its target sequence-specific extension.

To perform the function of strand substitution, regions 1, 2 and 3 of the controller oligonucleotide will be present primarily in the form of a single strand under reaction conditions. Therefore, double-stranded self-complementary structures (eg, hairpins) in these regions should be avoided if possible, as they can reduce the functionality of the controller oligonucleotides.

In the method according to the invention, the controller oligonucleotide should not be present as a template and therefore the first oligonucleotide primer should not be extended by the polymerase when bound to the controller oligonucleotide under reaction conditions.

This is especially achieved by the use of nucleotide modifications that prevent the polymerase from copying the strands. In particular, when the first oligonucleotide primer binds to the controller oligonucleotide under reaction conditions, the 3'end of the first oligonucleotide primer remains unextended.

The degree of blockade / disturbance / deceleration / complexity of the reaction is the full expression of this property (eg, 100% blockage under certain reaction conditions) and the partial expression of this property (eg, given reaction conditions). Can be between 30-90% blockage). Alone or contiguously (eg, as a sequence fragment consisting of modified nucleotides) that can prevent elongation of the first primer above 70%, especially above 90%, more particularly above 95%, and especially 100%. ) Bound nucleotide modifications are preferred.

Nucleotide modifications may include base modifications and / or sugar phosphate residue modifications. Sugar phosphate modification is preferred because any complementary sequence of controller oligonucleotides can be arranged in combination with conventional nucleobases. Nucleotides with modifications to glycophosphate residues that can interfere with or block the synthesis of polymerases, respectively, include, for example: 2'-O-alkyl modifications (eg, 2'-O-methyl, 2'-O. -(2-Methoxyethyl), 2'-O-propyl, 2'-O-propargyl nucleotide modification), 2'-amino-2'-deoxynucleotide modification, 2'-amino-alkyl-2'-deoxynucleotide modification , PNA, morpholino modifications, etc.

Blockade can be achieved both by a single nucleotide modification or only by several consecutively linked nucleotide modifications (eg, as a sequence fragment consisting of modified nucleotides). For example, at least two, especially at least five, especially at least ten such nucleotide modifications can be attached adjacent to each other in the controller oligonucleotide.

Controller oligonucleotides can have a uniform type of nucleotide modification or include at least two different types of nucleotide modifications.

The position of such a nucleotide modification in the controller oligonucleotide is specifically to prevent the polymerase from extending the 3'end of the first oligonucleotide primer attached to the controller oligonucleotide.

In certain embodiments, such nucleotide modifications are located in the second region of the controller oligonucleotide. In certain embodiments, such nucleotide modifications are located in the third region of the controller oligonucleotide. In certain embodiments, such nucleotide modifications are located in the second and third regions of the controller oligonucleotide.

For example, at least 20%, especially at least 50%, of the location of the second region of the controller oligonucleotide consists of such nucleotide modifications.

For example, the third region of the controller oligonucleotide consists of at least 20%, especially at least 50%, especially at least 90% of the positions of such nucleotide modifications. In certain embodiments, the entire third region comprises a nucleotide modification that prevents the polymerase from using a controller oligonucleotide as a template to extend the primer attached to such region. In certain embodiments, the entire third and second regions contain such nucleotide modifications. In certain embodiments, the entire first, second and third regions contain such nucleotide modifications. Therefore, this controller oligonucleotide can consist entirely of such nucleotide modifications. Such modified controller oligonucleotides can be used, for example, in multiplex analyzes where additional oligonucleotide primers are used. This is to prevent unintended primer extension reactions with one or more controller oligonucleotides.

The sequences of these nucleotide-modified nucleobases meet the requirements for the sequences of the respective regions.

The rest are, for example, natural nucleotides or nucleotide modifications that do not interfere with polymerase function at all or very slightly, such as DNA nucleotides, PTO nucleotides, LNA nucleotides, RNA nucleotides. Here, further modifications such as 2-amino-adenosine, 2-aminopurine, 5-methyl-cytosine, inosin, 5-nitroindole, 7-deaza-adenosine, 7-deaza-guanosine, 5-propyl-cytosine, 5 Base modifications such as -propyl-uridine, or non-nucleotide modifications such as dyes, or MGB modifications can be used, for example, to adjust the binding strength of individual regions of the controller oligonucleotide. The individual nucleotide monomers can be attached to each other via conventional 5'-3'bonds or via 5'-2'bonds.

The segment of the controller oligonucleotide that contains the nucleotide modification that prevents the extension of the 3'end of the first oligonucleotide primer bound to the controller oligonucleotide by the polymerase is called the "second blocking unit". The length of the segment can include 1 to 50 nucleotide modifications, especially 4 to 30 nucleotide modifications. The segment can be placed on the controller oligonucleotide, for example, such that the 3'end of the bound first oligonucleotide primer is in this segment. Therefore, this segment can extend to regions 2 and 3.

In certain embodiments, no linker or spacer structure, such as C3, C6, HEG linkers, is used, in particular to prevent extension of the 3'end of the first oligonucleotide primer bound to the controller oligonucleotide.

In certain embodiments, the controller oligonucleotide comprises at least one component of the detection system (eg, a fluorescent reporter or fluorescent quencher or donor fluorescent dye molecule) in its third region. In certain embodiments, the location of this component is at the 5'end of the controller oligonucleotide. In another embodiment, this component is located in the internal sequence segment of the third region. Here, the distance to the 5'end of the controller oligonucleotide can be 2-50 nucleotides, especially 2-20 nucleotides, especially 2-10 nucleotides. If the oligonucleotide probe and controller oligonucleotide bind simultaneously to the same first primer extension product, the two components of the detection system will be in close proximity (eg, the fluorescence reporter of the oligonucleotide probe and the controller oligonucleotide). Between donor fluorescent dye molecules).

In addition to regions 1, 2, and 3, the controller oligonucleotide can also include additional sequence segments flanking the above regions, for example, in the 5'or 3'segment of the controller oligonucleotide. Such sequence elements can be used, for example, for additional functions such as interaction with the probe, binding to the solid phase. Such regions do not particularly interfere with the functions of regions 1-3. The length of these flanking sequences can be, for example, 1 to 50 nucleotides. In addition, the controller oligonucleotide can include at least one element for immobilization to the solid phase, such as a biotin residue. In addition, the controller oligonucleotide can include at least one element for detection, eg, a fluorescent dye.

In the presence of controller oligonucleotides, newly synthesized sequences are tested taking into account their sequence contents by interaction with the controller oligonucleotides.
• The template strand is replaced with the newly synthesized strand when the correct sequence content of the controller oligonucleotide strand substitution occurs. Thereby, the primer binding site is converted into a single-stranded state, which can be used for new interactions with the primer and thus for further synthesis. In this way, the system of both primer extension products is activated. Therefore, the controller oligonucleotide has an activating effect on this system.
• If it does not match the given sequence content of the controller oligonucleotide, the strand substitution of the template strand of the newly synthesized strand is affected. Chain substitution and / or separation is quantitatively reduced or completely discontinued. Therefore, the primer binding site is never or less frequently converted to a single-stranded state. Therefore, there are no or few primer binding sites available for new interactions with primers. Therefore, the systems of both primer extension products become less frequent or no active state is achieved.

The efficiency of double-strand opening of the newly synthesized primer extension product after each single synthesis step affects the possible yields obtained in subsequent cycles: fewer free / single-strand primer binding sites. If provided to the amplified nucleic acid strand, the number of newly synthesized strands of the nucleic acid strand amplified in this step will be reduced. In other words: the yield of the synthetic cycle is proportional to the amount of primer binding sites available for interaction with the corresponding complementary primers. In summary, the control loop can be realized in this way.

The control loop corresponds to real-time control (real-time / online) of the synthesized fragment, and sequence control is performed in the reaction mixture while amplification is taking place. The sequence control is performed according to a given pattern, and the oligonucleotide system can distinguish between the "correct" and "wrong" states without external intervention (due to the strand cleavage effect of the controller oligonucleotide). In the correct state, sequence synthesis continues, and in the wrong state, synthesis slows down or is completely prevented. The difference obtained in the yield of "correct" and "wrong" sequences after each step affects the overall amplification including several such steps.

In exponential amplification, the dependencies are exponential, so that even the slightest difference in effectiveness in one single synthetic cycle due to sequence differences causes a significant delay in the overall amplification time. Or the detectable amplification in a given time frame is completely lost.

This effect of real-time control of newly synthesized nucleic acid chains is associated with the use of controller oligonucleotides, so the effect of controller oligonucleotides in the context of amplification is far beyond the region containing the length of the oligonucleotide primer. Extend beyond.

Reaction Conditions for Chain Substitution Reactions Substitution of the second primer extension product from binding to the first primer extension product by sequence-dependent chain substitution with a controller oligonucleotide forms individual steps in amplification. The reaction conditions during the steps are adjusted accordingly. The reaction temperature and reaction time are selected so that the reaction can occur successfully.

In certain embodiments, chain substitution with the controller oligonucleotide is carried out from binding to the first primer extension product to elimination / dissociation of the second primer extension product. Such dissociation of the 3'segment of the first primer extension product in the complementary portion of the second primer extension product can occur spontaneously during temperature-dependent / temperature-related separation of both primer extension products. Such dissociation has a favorable effect on the kinetics of the amplification reaction and can be influenced by the choice of reaction conditions, such as temperature conditions. Therefore, the temperature conditions are selected so that successful strand substitution by complementary binding of the controller oligonucleotide favors the dissociation of the second primer extension product from the 3'segment of the first primer extension product.

Although the embodiment does not represent "classical" PCR, the thermal evoked dissociation of the 3'segment is well below the temperature of the classical PCR, which is well below the reaction that induces general strand separation at> 90 ° C. Proceed in range.

In a further specific embodiment, the strand substitution with the controller oligonucleotide is such that the 3'segment of the second primer extension product (P2.1-Ext) is complementary to the first primer extension product (P1.1-Ext). Proceeding from binding to separation / dissociation, the 3'segment of this second primer extension product (P2.1-Ext) is at least one segment complementary to the first primer and the first primer extension product (P2.1-Ext). It contains one segment complementary to P1.1-Ext), which is formed only during enzyme synthesis. As a result, the complex (C1.1 / P1.) Containing the first primer extension product (P1.1-Ext), the second primer extension product (P2.1-Ext) and the controller oligonucleotide (C1.1). It results in the formation of 1-Ext / P2.1-Ext) (FIG. 2B). In such complexes, the 3'segment of the second primer extension product is at least temporarily in single-stranded form, as the controller oligonucleotide can replace the binding to the first primer extension product. Exists. The 3'segment of the first primer extension product (P1.1-Ext) is present in such a complex hybridized to the second primer extension product (P2.1-Ext) (FIG. 2B).

Due to the partially open primer binding site of the first oligonucleotide primer (3'segment of the second primer extension product), the new oligonucleotide primer is still a complex under reaction conditions (P2.1-). It can bind to the primer binding site of this single-stranded sequence segment of Ext) and thus initiate the synthesis of a new first primer extension product (P1.2-Ext) by the polymerase. As a general rule, the 3'segment of P2.1-Ext is not permanently single-stranded and behaves competitively with the controller oligonucleotide, thus binding to P1.1-Ext causes single-stranded and double-stranded. This reaction slows down and proceeds because it appears alternately.

Continuing the synthesis of this newly initiated P1.2-Ext using a second primer extension product (P2.1-Ext) as a template is followed by a polymerase-related strand substitution complex (C1.1 / P1.1). -Ext / P2.1-Ext) dissociation can also be achieved. Here, controller oligonucleotides, temperature-dependent double-strand destabilization, and polymerase strand substitution work synergistically and complementarily. This causes the 3'segment of the first primer extension product (P1.1-Ext) to dissociate from the complementary portion of the second primer extension product (P2.1-Ext).

Such dissociation has a positive effect on the kinetics of the amplification reaction, depending on the choice of reaction conditions, eg temperature conditions. Can be affected. The involvement of polymerase-mediated synthesis-dependent chain substitutions in the dissociation of P1.1-Ext and P2.1-Ext has a favorable effect on chain separation.

The temperature in the step includes, for example, a range of 15 ° C. to 75 ° C., particularly 30 ° C. to 70 ° C., particularly 50 ° C. to 70 ° C.

With a given length of the first region of the controller oligonucleotide and the second region of the first oligonucleotide primer (eg, including the range of 3-25 nucleotide monomers, especially 5-15 nucleotide monomers), generally , The chain substitution reaction can be started successfully. When the controller oligonucleotide is completely complementary to each portion of the first primer extension product, the controller oligonucleotide binds to the first primer extension product except for the 3'segment of the first primer extension product. The second primer extension product can be substituted. Therefore, the second primer extension product remains connected to the 3'segment of the first primer extension product. The strength of the connection can be affected by temperature. When the critical temperature is reached, this connection breaks and both primer extension products can dissociate. The shorter the arrangement of the 3'segments, the more unstable the connection and the lower the temperature at which spontaneous dissociation occurs.

Spontaneous dissociation can be achieved, for example, in a temperature range that is an approximate melting temperature. In certain embodiments, the temperature of the chain replacement step with the controller oligonucleotide is the 3'segment of the first primer extension product that is not bound by the controller oligonucleotide, and the second oligonucleotide primer or the second primer extension product, respectively. Approximate melting temperature (Tm ± 3 ° C.) of the complex containing.

In certain embodiments, the temperature of the chain replacement step with the controller oligonucleotide is the 3'segment of the first primer extension product and the second oligonucleotide primer or the second primer extension product, respectively, which are not bound by the controller oligonucleotide. It is approximately the melting temperature (Tm ± 5 ° C.) of the containing complex.

In certain embodiments, the temperature of the chain replacement step with the controller oligonucleotide is the 3'segment of the first primer extension product that is not bound by the controller oligonucleotide, and the second oligonucleotide primer or the second primer extension product, respectively. Exceeds the melting temperature of the complex containing. Such temperatures include a temperature range of about Tm + 5 ° C. to Tm + 20 ° C., and better, Tm + 5 ° C. to Tm + 10 ° C. By using a higher temperature, the equilibrium in the reaction step can be shifted towards dissociation. This can have a positive effect on the kinetics of the reaction. Using temperatures that are too low in the chain replacement step with controller oligonucleotides can lead to a significant slowdown in amplification.

In certain embodiments, the first primer extension product is not bound by the controller oligonucleotide and comprises a 3'segment containing 4-6, better 7-8, particularly about 18 nucleotides in sequence length. In the embodiment, spontaneous dissociation is usually already achieved in the temperature range between 40 ° C and 65 ° C. Higher temperatures also lead to dissociation.

In certain embodiments, the first primer extension product is not bound by a controller oligonucleotide and comprises a 3'segment containing a sequence length of 15 to about 25 nucleotides. In this embodiment, spontaneous dissociation is usually already achieved at temperatures between 50 ° C and 70 ° C. Higher temperatures also lead to dissociation.

In certain embodiments, the first primer extension product is not bound by a controller oligonucleotide and comprises a 3'-segment containing a sequence length of 20 to about 40 nucleotides. In this embodiment, spontaneous dissociation is usually already achieved in the temperature range between 50 ° C and 75 ° C. Higher temperatures also lead to dissociation.

The composition of the 3'segment of the first primer extension product and optionally additional melting temperature-influenced oligonucleotide modifications (MGB, etc.) or reaction conditions (TPAC, betaine, etc.) can influence temperature selection. Is. Therefore, the corresponding adjustments can be made.

In certain embodiments, all steps of amplification proceed under stringent conditions that prevent or slow down the formation of non-specific products / by-products. Such conditions exceed, for example, higher temperatures, such as 50 ° C.

In certain embodiments, the individual steps of strand substitution with the controller oligonucleotide proceed at the same temperature as the synthesis of the first and second primer extension products. In a further embodiment, the individual steps of chain substitution with the controller oligonucleotide proceed at a temperature different from the temperature of each synthesis of the first and second primer extension products. In a further embodiment, the synthesis of the first primer extension product and the strand substitution with the controller oligonucleotide proceed at the same temperature. In a further embodiment, the synthesis of the second primer extension product and the strand substitution with the controller oligonucleotide proceed at the same temperature.

The concentration of the controller oligonucleotide comprises the range of 0.01 μmol / l to 50 μmol / l, better the range of 0.1 μmol / l to 20 μmol / l, preferably the range of 0.1 μmol / l to 10 μmol / l.

Specific Embodiments of the Second Oligonucleotide Primer (Primer 2):
Oligonucleotides with 3'segments can bind to essentially complementary sequences within the amplified nucleic acid or equivalent and initiate a specific second primer extension reaction (FIGS. 14, 27). ~ 29, 55, 68 ~ 71). Thus, this second oligonucleotide primer can bind to the 3'segment of the first specific primer extension product of the first oligonucleotide primer and initiate polymerase-dependent synthesis of the second primer extension product. be able to. In certain embodiments, each second oligonucleotide primer is specific for one nucleic acid to be amplified.

The second oligonucleotide primer should be copyable during reverse synthesis and also functions as the template itself during the synthesis of the first primer extension product.

The length of the second oligonucleotide primer can be between 15 and 100 nucleotides, particularly between 20 and 60 nucleotides, particularly preferably between 30 and 50 nucleotides. In particular, the nucleotide building blocks are linked to each other via a common 5'-3'phosphodiester or phosphothioester bond. Such oligonucleotide primers can be chemically synthesized in a desired manner.

In certain embodiments, the second oligonucleotide primer can contain nucleotide monomers that do not or have a slight effect on the function of the polymerase, and these are present, for example:
-Natural nucleotides (dA, dT, dC, dG, etc.) or their modifications without base pair modification
• Modified nucleotides, 2-amino-dA, 2-thio-dT, or other nucleotide modifiers with different base pairs (eg, universal base pairs such as inosin 5-nitroindole).

In certain embodiments, the 3'-OH end of this region is unmodified and has a functional 3'-OH group that is recognized by the polymerase and can be extended depending on the template. In a further specific embodiment, the 3'segment of the second primer contains at least one phosphorothioate compound, whereby the 3'end of the primer cannot be degraded by the 3'exonuclease activity of the polymerase.

The second oligonucleotide primer can be used in several separate steps. First, it exerts a primer function in amplification. As a result, the primer extension reaction is carried out using the first primer extension product as a template. In certain embodiments, the second oligonucleotide primer can use the starting nucleic acid chain as a template at the initiation of the amplification reaction. In certain embodiments, the second oligonucleotide primer can be used to design / provide the starting nucleic acid strand.

Within the amplification, the second primer functions as an initiator in the synthesis of the second primer extension product, using the first primer extension product as a template. The 3'segment of the second primer contains sequences that are primarily complementary to the first primer extension product. Enzymatic extension of the second oligonucleotide primer using the first primer extension product as a template results in the formation of the second primer extension product. Such synthesis is generally performed in parallel with the substitution of the controller oligonucleotide from binding to the first primer extension product. The substitution is primarily accomplished by the polymerase and can be partially performed by a second oligonucleotide primer. Such a second extension product comprises a target sequence or a segment thereof. In the process of synthesizing the second primer extension product, the sequence of the copyable portion of the first oligonucleotide primer is recognized by the polymerase as a template, and each complementary sequence is synthesized. The sequence is in the 3'segment of the second primer extension product and contains the primer binding site of the first oligonucleotide primer. The synthesis of the second primer extension product is carried out up to the stop position of the first oligonucleotide primer. Immediately after the synthesis of the second primer extension product, this product binds to the first primer extension product to form a double-stranded complex. The second primer extension product is sequence-specifically substituted from the complex by a controller oligonucleotide. After successful strand substitution with the controller oligonucleotide, the second primer extension product itself can then serve as a template for the synthesis of the first primer extension product.

In addition, the second oligonucleotide primer can function as an initiator for the synthesis of the second primer extension product starting from the starting nucleic acid strand at the start of amplification. In certain embodiments, the sequence of the second primer is completely complementary to the corresponding sequence segment of the starting nucleic acid strand. In certain embodiments, the sequence of the second oligonucleotide primer is only partially complementary to the corresponding sequence segment of the starting nucleic acid strand. However, the difference complementarity does not prevent the second oligonucleotide primer from initiating a predominantly sequence-specific primer extension reaction. Each difference in the complementarity of the second oligonucleotide primer to each position of the starting nucleic acid chain is particularly second, as the 3'segment allows predominantly complementary base pairing and initiation of synthesis. Is in the 5'segment of the oligonucleotide primer. For the initiation of synthesis, for example, the first 4-10 positions of the 3'segment need to be completely complementary to the template (starting nucleic acid strand). The remaining nucleotide positions can differ from perfect complementarity. Therefore, the degree of perfect complementarity in the 5'segment may range from 10% to 100%, especially between 30% and 100% of the base composition. Depending on the length of the second oligonucleotide primer, these differences from the perfect complementarity of the 5'segment include positions of 1-40, especially 1-20 nucleotides. In certain embodiments, the second oligonucleotide primer binds to the starting nucleic acid strand only in its 3'segment, but not in its 5'segment. The length of such a 3'segment of the second oligonucleotide primer, which is completely complementary to the starting nucleic acid strand, includes a range between 6-40 nucleotides, especially 6-30 nucleotides, especially 6-20 nucleotides. .. The length of the corresponding 5'segment of the second oligonucleotide primer, which is non-complementary to the starting nucleic acid strand, comprises a range between 5-60 nucleotides, in particular 10-40 nucleotides. Therefore, the second oligonucleotide primer can initiate the synthesis of the starting nucleic acid chain. In subsequent synthesis of the first primer extension product, the first primer extension product sequence segment of the second oligonucleotide primer is copied by the polymerase, whereby in subsequent synthesis cycles, fully complementary primer binding. Sites are formed within the first primer extension product that binds the second oligonucleotide and become available in subsequent synthetic cycles.

In certain embodiments, the second oligonucleotide primer can be used in the preparation of the starting nucleic acid chain. Here, such a second oligonucleotide primer binds primarily / preferably sequence-specificly to a nucleic acid (eg, single-stranded genomic DNA or RNA containing the target sequence or its equivalent) and is present in the polymerase. The template-dependent primer extension reaction can be initiated below. The binding position is selected such that the primer extension product contains the desired target sequence. Extension of the second oligonucleotide primer results in a strand with a sequence complementary to the template. Such chains can be separated by a template (eg, by heat or alkali) and thus converted into a single chain form. Such a single-stranded nucleic acid strand can function as a starting nucleic acid strand at the start of amplification. In the 5'segment such initiation nucleic acid strand comprises a sequence portion of a second oligonucleotide primer, which further comprises a target sequence or equivalent thereof and a primer binding site for the first oligonucleotide primer. Further steps are described in the section "Initiation of Nucleic Acid Chains".

In certain embodiments, the second oligonucleotide primer binds complementaryly and sequence-specifically to the sequence segment of the target sequence, at least in its 3'segment, to initiate / support a normal primer extension reaction by the polymerase. Includes an array part that can be used. The length of such sequence segments includes the range of 6-40 nucleotides, especially 8-30 nucleotides, especially 10-25 nucleotides.

In certain embodiments, the second oligonucleotide primer in the 3'and 5'segments comprises a copyable sequence segment that is copied by the polymerase in the synthesis of the first primer extension product. Therefore, all sequence segments of the second primer are copied by the polymerase. As a result, a primer binding site is formed in the 3'segment of the first primer extension product.

In certain embodiments, the second oligonucleotide primer, which has a copyable portion in its length, corresponds to the 3'segment of the first primer extension product that is not bound by the controller oligonucleotide. In a complex comprising a second oligonucleotide primer and a first primer extension product, the 3'end of such a second oligonucleotide primer flanks the controller oligonucleotide that binds to the first primer extension product. .. Such primer extension is performed using the first primer extension product as a template. In such primer extension, substitution of the controller oligonucleotide from binding to the first primer extension product is performed by polymerase-dependent chain substitution.

In certain embodiments, the second oligonucleotide primer with its copyable sequence portion is shorter than the 3'segment of the first primer extension product that is not bound by the controller oligonucleotide. In a complex containing a second oligonucleotide primer and a first primer extension product, the first primer extension is between the 3'end of such primer and the controller oligonucleotide bound to the first primer extension product. There is a single chain site of the product. Such primer extension is performed using the first primer extension product as a template. In such primer extension, substitution of the controller oligonucleotide from binding to the first primer extension product is performed by polymerase-dependent chain substitution.

In certain embodiments, the second oligonucleotide primer with its copyable portion is longer than the 3'segment of the first primer extension product that is not bound by the controller oligonucleotide. In the complex of the second oligonucleotide primer and the first primer extension product, the 3'segment of the second primer and the 5'segment of the controller oligonucleotide compete for binding to the first primer extension product. Binding of the 3'segment of the second primer to the extension product of the first primer, which is required to initiate synthesis, occurs at the same time as the partial substitution of the 5'segment of the controller oligonucleotide.

Substitution of such primers occurs by using the first primer extension product as a template after initiation of synthesis by the polymerase. In such primer extension, substitution of the controller oligonucleotide from binding to the first primer extension product is performed by polymerase-dependent chain substitution. The sequence length of the 3'segment of the second oligonucleotide primer that replaces the 5'segment of the controller oligonucleotide includes the following range: 1-50 nucleotides, especially 3-30 nucleotides, especially 5-20 nucleotides. It is advantageous in some embodiments, for example, to use a longer second oligonucleotide primer that exceeds the length of the 3'segment of the first primer extension product. Such embodiments include, for example, a first primer extension product having a 3'segment that is 5-40 nucleotides in length, particularly 10-30 nucleotides and is not bound by a controller oligonucleotide. In particular, a longer second oligonucleotide primer with a shorter 3'segment provides improved sequence specificity at the start of synthesis.

The strength of the second oligonucleotide primer to its primer binding site depends on the length of the primer. In general, longer second oligonucleotide primers can be used at higher reaction temperatures.

In particular, the sequences of the first and second oligonucleotide primers and controller oligonucleotides are adapted to each other so that side reactions, such as primer dimer formation, are minimized. Thus, for example, the sequences of the first and second oligonucleotide primers are compatible with each other so that both oligonucleotide primers cannot initiate the amplification reaction in the absence of the appropriate template and / or target sequence and / or starting nucleic acid strand, respectively. Will be done. This can be achieved, for example, by the second oligonucleotide primer not containing the primer binding site of the first oligonucleotide primer and the first oligonucleotide primer not containing the primer binding site of the second oligonucleotide primer. .. Furthermore, it should be avoided that the primer sequence contains a self-complementary structure (self-complementary) in which it is extended.

The synthesis of the second primer extension product is a primer extension reaction, forming the individual steps of the first amplification. The reaction conditions during this step are adjusted accordingly. The reaction temperature and reaction time are selected so that the reaction can occur successfully. Each preferred temperature in this step depends on the strength of the polymerase used and each second oligonucleotide primer bound to its primer binding site, eg, 15 ° C. to 75 ° C., especially 20 to 65 ° C., especially 25. Includes the range of ° C to 65 ° C. The concentration of the second oligonucleotide primer includes a range of 0.01 μmol / l to 50 μmol / l, particularly 0.1 μmol / l to 20 μmol / l, particularly 0.1 μmol / l to 10 μmol / l.

In certain embodiments, all steps of amplification proceed under stringent conditions that prevent or slow down the formation of non-specific products / by-products. Such conditions exceed, for example, higher temperatures, such as 50 ° C.

When two or more specific nucleic acid chains are amplified in one batch, in certain embodiments, each sequence-specific oligonucleotide primer is preferably used for amplification of the corresponding target sequence. Will be done.

In certain embodiments, the synthesis of the first and second primer extension products proceeds at the same temperature. In certain embodiments, the synthesis of the first and second primer extension products proceeds at different temperatures. In certain embodiments, the synthesis of the second primer extension product and the strand substitution with the controller oligonucleotide proceed at the same temperature. In a further embodiment, the synthesis of the second primer extension product and the strand substitution with the controller oligonucleotide proceed at different temperatures.

Polymerase:
Preferably, a template-dependent polymerase capable of strand substitution is used.

In one embodiment, a large fragment of Bst polymerase or a modification thereof (eg, Bst2.0 DNA polymerase) can be used. In addition, Klenow fragment, Vent exo minus polymerase, Deepent exo minus DNA polymerase, large fragment of Bsu DNA polymerase, large fragment of Bsm DNA polymerase can be used. Vent exo minus polymerase, Deepent exo minus DNA (manufactured by NEB) and PyroPhage polymerase manufactured by Lucien are thermostable enzymes having strand substitution activity.

In one embodiment, a polymerase that is inactive at room temperature, the so-called hot start polymerase or warm start polymerase, is used. BST 2.0 Polymerase Warm Start (manufactured by NEB) is an example of this.

Specific Embodiment of the Third Oligonucleotide Primer (Component of Second Amplification System)
In one embodiment, the third oligonucleotide primer is identical to the first oligonucleotide primer in the first amplification system.

In certain embodiments, the third oligonucleotide primer is not identical to the first oligonucleotide primer in the first amplification system.

The length of the third oligonucleotide primer can be between 15-100 nucleotides, especially 20-60 nucleotides, especially 30-50 nucleotides. For example, the level of CG is between 20-80%, especially 30-79%. Nucleotide building blocks are particularly interconnected via conventional 5'-3'phosphodiester or phosphothioester bonds. Such oligonucleotide primers can be chemically synthesized in the desired form.

In certain embodiments, the third oligonucleotide primer can include a nucleotide monomer that has little or no effect on the function of the polymerase, such as:
-Natural nucleotides (dA, dT, dC, dG, etc.) or their modified base pairs unchanged-Modified nucleotides, nuclease-resistant phosphorothioate compound (PTO) modified products, LNA modified products, 2-amino-dA, 2- Thio-dT, or other nucleotide modifiers with different base pairs (eg, universal base pairs such as inosin 5-nitroindole).

In certain embodiments, the 3'-OH end of the third oligonucleotide primer has a functional 3'-OH group that is unmodified, is recognized by the polymerase and can be extended in a template-dependent manner. In a further specific embodiment, the 3'segment of the third oligonucleotide primer contains at least one phosphorothioate compound, so degradation of the 3'end of the primer by the 3'exonuclease activity of the polymerase cannot occur.

In certain embodiments, the 3'end of the third oligonucleotide primer is blocked. For example, it has a 3'-phosphate group or a C3 linker. In the embodiment, the 3'segment of the third oligonucleotide primer is not activated until the second amplification using, for example, the 3'-5'exonuclease activity of the second polymerase.

In particular, the third oligonucleotide primer does not contain any sequence segment that can complementarily bind to the first segment of the controller oligonucleotide.

The third oligonucleotide primer (FIGS. 15-31) can be arranged differently with respect to the first amplification fragment 1.1 (first amplification product 1.1 containing the target sequence).

To allow the second amplification reaction to be initiated using the first amplification fragment 1.1 as a template (starting nucleic acid chain 2.1), the third and fourth primers are provided by the first amplification fragment. It needs to bind within a pre-determined sequence and be extended by the polymerase.

In certain embodiments, the third oligonucleotide primer is capable of binding to one strand of the first amplified fragment 1.1. In particular, the third oligonucleotide primer can bind to the second primer extension product and initiate primer extension using the appropriate polymerase and reaction conditions.

In certain embodiments, this binding of the third oligonucleotide primer preferably occurs in the 3'segment of the second primer extension product.

The third oligonucleotide primer preferably comprises a sequence segment in a 3'segment that can bind complementaryly or predominantly to the second primer extension product under the reaction conditions used, whereby the polymerase contains a second primer. The synthesis of the primer extension product of 3 can be started.

The length of this segment includes a range of particularly between 6-30 nucleotides, in particular 8-25 nucleotides, in particular 10-20 nucleotides, in particular 10-15 nucleotides. In certain embodiments, this segment is completely complementary to the corresponding segment of the second primer extension product. In certain embodiments, the segment comprises at least one discrepancy with respect to the primer extension product. In particular, the position of this mismatch is not closer than the -4 position, especially closer to -5, especially not closer to -6, especially the 3 of the third oligonucleotide primer, with respect to the 3'end of the third primer. 'No closer than -8 to the end. Such discrepancies weaken the binding of this segment to the controller oligonucleotide, as the third oligonucleotide primer can also bind to the controller oligonucleotide in this 3'segment. Therefore, when choosing the length and number of mismatches, consider that the 3'segment should bind to the second primer extension product and initiate the primer extension reaction, but at the same time the reaction conditions used by the segment. Consider below that it does not irreversibly bind to the controller oligonucleotide and therefore inactivates it.

Therefore, position and length selection considers interactions between the third oligonucleotide primer and controller, as well as the second primer extension product.

The position of binding of the segment to the second primer extension product can be selected in a variety of ways (FIGS. 27-31, 57).

For example, the third oligonucleotide primer can bind to the same sequence segment as the first oligonucleotide primer in a predominantly complementary manner. The 3'end position of the third oligonucleotide primer corresponds to the 3'end position of the first oligonucleotide primer. Thus, the third oligonucleotide primer can at least partially (using its 3'segment) bind to a portion of the target sequence in a complementary manner. Therefore, the 3'end of the third primer is located within the second blocking unit of the controller oligonucleotide and cannot be extended by the polymerase using the controller as a template (FIG. 20).

In certain embodiments, the third oligonucleotide primer binds predominantly complementary to the second primer extension product, the 3'end of which is shifted relative to the 3'end of the first oligonucleotide primer. To. In certain embodiments, the 3'end of the third oligonucleotide primer is shifted with at least one nucleotide in the 3'direction of the second primer extension product. Therefore, the segment of the second primer extension product to which the third oligonucleotide primer can bind predominantly complementarily is shifted in the 3'direction (FIG. 19).

In certain embodiments, the 3'end of the third oligonucleotide primer is shifted by at least one nucleotide in the 5'direction of the second primer extension product. Therefore, the segment of the second primer extension product to which the third oligonucleotide primer can bind predominantly complementarily is shifted in the 5'direction (FIG. 21).

In certain embodiments, the binding position of the third oligonucleotide primer extends the second primer so that the third oligonucleotide primer does not overlap the binding position of the first oligonucleotide primer in the first amplification system. The product is shifted in the 5'direction. Therefore, the segment of the second primer extension product to which the third oligonucleotide primer can bind mainly complementarily is shifted in the 5'direction with respect to the binding position of the first oligonucleotide primer (FIG. 22). ..

Thus, the third oligonucleotide primer contains a sequence segment that can complementarily bind to the controller oligonucleotide. The segment of the controller oligonucleotide that can complementarily bind to the third oligonucleotide primer is called the fourth segment of the controller oligonucleotide. To prevent unwanted primer extension of the third oligonucleotide primer in the controller, the position of the controller oligonucleotide located at least around the 3'end of the third oligonucleotide primer is modified. For example, this can be done in a similar manner to the modification of the second blocking unit. The controller segment containing such modifications (preventing extension of the third oligonucleotide primer) is referred to as the fourth blocking unit (FIGS. 19-22). Its position is determined by the possible binding position of the third oligonucleotide primer in the controller.

The third oligonucleotide primer needs to be copyable during the reverse synthesis and also functions as the template itself for the synthesis of the fourth primer extension product.

In certain embodiments, the third oligonucleotide primer comprises at least one sequence segment in its 3'region, which is capable of predominantly complementary binding to the first target sequence. This segment can predominantly complementarily bind to the second primer extension product (including the corresponding segment of the target sequence) and the polymerase can carry out a primer extension reaction.

The third oligonucleotide primer, in one embodiment, comprises at least one segment in the 5'segment that is not complementary to the target sequence. For example, the segment may contain 1-60 nucleotides and may be used for other purposes such as bar coding, cloning, immobilization, probe binding and the like. The sequence composition of this 5'segment is adjusted so as not to interfere with the second amplification.

The third oligonucleotide primer may include, for example, a fluorescent dye (eg, FAM, Cy5, etc.), a fluorescent quencher (eg, BHQ1, BHQ2, etc.), an affinity marker (eg, biotin, digoxigenin, etc.). In one embodiment, the third oligonucleotide primer may include a linker (eg, C3 or HEG linker) so that its 5'segment is not copied by the polymerase.

In certain embodiments, the third oligonucleotide primer is immobilized on the solid phase prior to the second amplification reaction. Therefore, primer extension of this third oligonucleotide primer results in immobilization of the entire third primer extension product.

The concentration of the third oligonucleotide primer used may range from 0.01 μmol / l to about 10 μmol / l, particularly from 0.1 μmol / l to about 2 μmol / l.

Specific Embodiment of the Fourth Oligonucleotide Primer (Component of Second Amplification System)
In one embodiment, the fourth oligonucleotide primer is identical to the second oligonucleotide primer in the first amplification system.

In certain embodiments, the fourth oligonucleotide primer is not identical to the second oligonucleotide primer in the first amplification system.

The length of the fourth oligonucleotide primer can be between 15-100 nucleotides, especially 20-60 nucleotides, especially 30-50 nucleotides. For example, the level of CG is between 20-80%, especially 30-79%. Nucleotide building blocks are particularly interconnected via conventional 5'-3'phosphodiester or phosphothioester bonds. Such oligonucleotide primers can be chemically synthesized in the desired form.

In certain embodiments, the fourth oligonucleotide primer can include nucleotide monomers that have little or no effect on the function of the polymerase, such as:
-Natural nucleotides (dA, dT, dC, dG, etc.) or their modified base pairs unchanged-Modified nucleotides, nuclease-resistant phosphorothioate compound (PTO) modified products, LNA modified products, 2-amino-dA, 2- Thio-dT, or other nucleotide modifiers with different base pairs (eg, universal base pairs such as inosin 5-nitroindole).
In certain embodiments, the 3'-OH end of the fourth oligonucleotide primer has a functional 3'-OH group that is unmodified, is recognized by the polymerase and can be extended in a template-dependent manner. In a further specific embodiment, the 3'segment of the fourth oligonucleotide primer contains at least one phosphorothioate compound, so degradation of the 3'end of the primer by the 3'exonuclease activity of the polymerase cannot occur.

In certain embodiments, the 3'end of the fourth oligonucleotide primer is blocked. For example, it has a 3'-phosphate group or a C3 linker. In these embodiments, the activity of the 3'segment of the fourth oligonucleotide primer does not occur until the second amplification using, for example, the 3'-5'exonuclease activity of the second polymerase.

In particular, the fourth oligonucleotide primer does not contain any sequence segment that can complementarily bind to the first segment of the controller oligonucleotide.

The fourth oligonucleotide primer (FIGS. 15-31) can be arranged differently with respect to the first amplification fragment 1.1 (first amplification product 1.1 containing the target sequence).

To initiate a second amplification reaction using the first amplification fragment 1.1 as a template (starting nucleic acid chain 2.1), the third and fourth primers are pre-determined by the first amplification fragment. It needs to be bound within the sequence and extended by the polymerase.

In certain embodiments, the fourth oligonucleotide primer is capable of binding to one strand of the first amplified fragment 1.1. Preferably, the fourth oligonucleotide primer can bind to the first primer extension product and initiate primer extension using the appropriate polymerase and reaction conditions.

In certain embodiments, this binding of the fourth oligonucleotide primer preferably occurs in the 3'segment of the first primer extension product.

The fourth oligonucleotide primer preferably comprises a sequence segment in a 3'segment that can bind complementaryly or predominantly to the first primer extension product under the reaction conditions used, whereby the polymerase contains the first primer. The synthesis of the primer extension product of 4 can be started.

The length of the segment includes in particular a range of 6-40 nucleotides, in particular 8-30 nucleotides, in particular 10-25 nucleotides, in particular 10-20 nucleotides. In certain embodiments, this segment is completely complementary to the corresponding segment of the first primer extension product. In certain embodiments, the segment comprises at least one discrepancy with respect to the first primer extension product. In particular, this mismatched position is no closer than the -4 position, especially no closer than -5, especially no closer than -6, especially with respect to the 3'end of the fourth primer, especially the fourth oligonucleotide primer. No closer than the -8 position with respect to the 3'end of.

The position of binding of the segment to the first primer extension product can be selected in various ways (FIGS. 23-31, 57).

For example, the fourth oligonucleotide primer can bind to the same sequence segment predominantly complementary to the second oligonucleotide primer. The 3'end position of the fourth oligonucleotide primer corresponds to the 3'end position of the second oligonucleotide primer. Therefore, the fourth oligonucleotide primer contains, at least in part (using its 3'segment), part of the target sequence.

In certain embodiments, the fourth oligonucleotide primer binds predominantly complementary to the first primer extension product, its 3'end being shifted relative to the 3'end of the second oligonucleotide primer. To. In certain embodiments, the 3'end of the fourth oligonucleotide primer is shifted with at least one nucleotide in the 3'direction of the first primer extension product. Therefore, the segment of the first primer extension product to which the fourth oligonucleotide primer can bind predominantly complementarily is shifted in the 3'direction (FIG. 1).

In certain embodiments, the 3'end of the fourth oligonucleotide primer is shifted with at least one nucleotide in the 5'direction of the first primer extension product. Therefore, the segment of the first primer extension product to which the fourth oligonucleotide primer can bind predominantly complementarily is shifted in the 5'direction.

In particular, the fourth oligonucleotide primer does not contain any sequence segment that can complementarily bind to the controller oligonucleotide.

The fourth oligonucleotide primer must be copyable during reverse synthesis and itself also serves as a template for the third primer extension product during synthesis.

In certain embodiments, the fourth oligonucleotide primer comprises a portion of the first target sequence and thus comprises at least one sequence segment in the 3'segment that can bind to a strand complementary to the target sequence. .. The segment can predominantly complementarily bind to the first primer extension product (including the corresponding segment of this target sequence) and the polymerase can carry out a primer extension reaction.

In certain embodiments, the fourth oligonucleotide primer comprises at least one sequence segment in the 5'segment that does not include the sequence segment of the first target sequence. For example, the segment may contain 1-60 nucleotides and may be used for other purposes such as bar coding, cloning, immobilization, probe binding and the like. The sequence composition of this 5'segment is adjusted so as not to interfere with the second amplification.

The fourth oligonucleotide primer may include further modifications, such as fluorescent dyes (eg, FAM, Cy5, etc.), fluorescent quenchers (eg, BHQ1, BHQ2, etc.), affinity markers (eg, biotin, digoxigenin, etc.). .. In one embodiment, the fourth oligonucleotide primer may include a linker (eg, C3 or HEG linker), which leaves the 5'segment uncopied by the polymerase.

In certain embodiments, the fourth oligonucleotide primer is immobilized on the solid phase prior to the second amplification reaction. Therefore, such primer extension of the fourth oligonucleotide primer results in immobilization of the entire fourth primer extension product.

The concentration of the fourth oligonucleotide primer used may range from 0.01 μmol / l to about 10 μmol / l, especially from 0.1 μmol / l to about 2 μmol / l.

Second amplification system polymerase:
Many polymerases are known for performing PCR.

In certain embodiments, a thermostable template-dependent DNA polymerase with 5'-3'exonuclease activity is used.

In certain embodiments, Taq polymerase and / or a formulation and / or modified form thereof (eg, Ampli-Taq) is used to perform the second amplification.

In certain embodiments, a thermostable template-dependent DNA polymerase with strand substitution activity is used. For example, Vent Exo-minus (manufactured by NEB), PyroPhage polymerase (manufactured by Lucigene), SD polymerase (manufactured by Bioron).

In certain embodiments, a thermostable template-dependent DNA polymerase with 3'-5 proofreading activity is used. For example, Vent exo plus, Deep Vent exo plus (manufactured by NEB), Pfu polymerase (Jena Biosciences), Phusion polymerase (NEB).

In certain embodiments, for example, a thermostable template-dependent DNA polymerase that is conjugated to another protein is used to increase the process activity of the polymerase. For example, Phusion Polymerase (NEB).

Oligonucleotide primers containing additional sequence segments:
The above structures of the first primer and the second primer can be regarded as the so-called "basic structure of primer" and "minimum structure of primer", respectively.

Such a basic structure of an oligonucleotide having a primer function (for example, a first oligonucleotide primer, a second oligonucleotide primer, optionally a third oligonucleotide primer, optionally a fourth oligonucleotide primer, etc.) may be used. Includes sequence segments that are advantageous for performing amplification procedures, such as the first and second segments of the first primer.

Such basic structure of oligonucleotide primers can be extended by additional sequence segments. Such additional sequence segments contain structures that are not required to perform the amplification procedure, but may be useful for other tasks.

Such additional sequence segments can optionally be introduced into oligonucleotide primers and used for further function or reaction. In this way, primer extension products synthesized by polymerases (eg, starting with first and / or second primers) can be linked to such sequence segments. This allows the integration of such additional sequence segments and primer extension products into the molecular structure. Such integration can be advantageous in certain embodiments. Various uses of oligonucleotide primer sequences with additional sequence segments are known to those of skill in the art.

Several functions are known to those of skill in the art supported by additional sequence segments of oligonucleotide primers.

For example, the introduction of additional structures can be used as a means of mediating intermolecular or intramolecular bonds. Some examples of such structures are known to those of skill in the art. For example, the probe can be designed according to such a principle of intramolecular binding, for example in the Scorpion-primer. For example, sequence segments can still be used to bind additional oligonucleotides. Sequence-specific intermolecular bonds can be achieved by using stringent conditions. Such interactions can be used, for example, to bind the amplification product to a solid phase by complementary binding to an immobilized oligonucleotide.

Another example is the introduction of so-called adapter sequences and / or the use of additional segments for the unique coding or sequence-specific labeling of primers and resulting primer extension products (so-called primer barcoding). It is used, for example, to tune the NGS library (Stahberg et al., Nucleic Acids Res, June 20, 2016, 44 (11): e105). In sequence analysis of primer extension products, such labeling can be used for later sequence assignment.

Further examples provide the use of additional sequence segments to introduce specific sequences with specific protein bindings, such as restriction enzymes.

Another example provides the use of additional segments to introduce spacer sequences that are not intended to bind to specific interaction partners, but rather increase the distance between adjacent sequences.

Such additional sequences can either be placed in the copyable portion of the primer or added to the non-copyable portion of the oligonucleotide primer. Several factors play a role in deciding whether to copy an array segment. For example, the arrangement of sequence segments in each oligonucleotide, the nucleotide modifications used (eg, C3, HEG, 2'-Ome, etc.) determine whether the sequence segments are used as templates during the process step. be able to.

In one embodiment, an additional sequence segment is introduced into the copyable region of the primer, eg, in the 5'segment of the copyable portion of the second primer, thereby, for example, during the process of synthesizing the target sequence. When the primer sequence is read in, additional sequence segments are also read by the polymerase. The length of such additional sequence segments includes the range of 3-50 nucleotides. The composition of these sequence segments allows in this type of embodiment synthesis by polymerases, thus the segments serve as templates for polymerase-dependent synthesis. In such segments, for example, natural nucleotides such as dA, dG, dC, dT are used.

In a further embodiment, additional sequence segments can be placed, for example, at the 5'end of oligonucleotide primers that should not be copied during the synthesis of specific amplified fragments containing the target sequence. This involves placing, for example, one or more modifications or chemical groups that prevent the polymerase from synthesizing complementary strands, such as segments containing 4-10 nucleotides containing HEG, C3, 2'-Ome modifications. Can be achieved by. Such modifications can be placed, for example, at the 5'end of the copyable portion of the second primer and can interfere with continued synthesis. For example, a HEG group can be introduced at the 5'end of the copyable segment of the second primer, and then an additional sequence segment can be introduced.

In addition, additional sequence segments can be placed at the 5'end of the second region of the first primer. Such localization of additional sequence segments interferes with the synthesis of complementary strands during normal synthesis of specific amplification products containing the target sequence.

The length of such additional sequence segments includes the range of 3-50 nucleotides. The base composition is, for example, modification of a naturally occurring nucleobase (A, G, C, T, U, inosine) or nucleotide at different positions (eg, 2-aminoadenine, iso-guanine, iso-cytosine, 5-propargyl). It can contain bases such as -uridine, 5-propargyl-cytosine, or (in sugar phosphate backbones such as LNA, 2'-Ome, 2'-halogen). In one embodiment, the first primer and additional sequence segments are combined to form an oligonucleotide primer. In certain embodiments, a second primer and additional sequence segments are combined to form an oligonucleotide.

In such additional sequence segments, oligonucleotides are designed in such a way that they do not interfere with the amplification of the target sequence. This is achieved, for example, by avoiding or reducing inhibitory interactions with the structures of primers or controllers that are essential to the process. In certain embodiments, the additional structure is capable of forming double-stranded segments complementary to other primer regions under selected reaction conditions. However, especially such double-stranded segments do not interfere with the specific amplification of the target sequence. In certain embodiments, such additional sequence segments do not interact with or bind to the first or second primer region of the first primer. In certain embodiments, such additional segments do not interact with the controller oligonucleotide. In certain embodiments, such additional sequence segments do not interact with other primers in the reaction. In certain embodiments, such additional sequence segments do not interact with P1.1-Ext or P2.1-Ext or other amplified fragments containing the target sequence. In certain embodiments, such additional sequence segments provide a stable double-stranded segment with the first or second region of the first primer that completely prevents the first or second region from functioning. Does not form under reaction conditions.

In certain embodiments, such additional segments do not interact with or bind to the second primer. In certain embodiments, such additional sequence segments do not specifically interact with the 3'segment of the second primer.

In certain embodiments, the first primer comprises an additional sequence segment of the first primer (additional sequence variant P1) at the 5'end of the second region. The segment optionally comprises a sequence of 10 to 50 nucleotides that does not interfere with the procedure for amplifying the target sequence (eg, does not form secondary structure with primers). In addition, the segment optionally comprises a sequence of approximately 5-15 nucleotides of the copyable first segment of the first primer. The additional sequence variant P1 contains native nucleotides as monomers (A, C, G, T) and is capable of functioning as a polymerase template.

In certain embodiments, the second primer comprises an additional segment of the second primer (additional sequence variant P2) at its 5'end. Optionally, the segment comprises a sequence of 10 to 50 nucleotides that does not interfere with the procedure for amplifying the target sequence (eg, does not form secondary structure with the primer). In addition, the segment optionally comprises a sequence of approximately 5-15 nucleotides in the copyable region of the second primer. The additional sequence variant P2 contains native nucleotides as monomers (A, C, G, T) and is capable of functioning as a polymerase template.

An oligonucleotide containing such a first primer and an additional sequence variant P1 or an oligonucleotide containing a second primer and an additional sequence variant P2 is an oligonucleotide containing only the first primer or a second primer. It was observed that it was less susceptible to side reactions than oligonucleotides containing only. In certain embodiments, for example, the formation and / or amplification of non-specific primer dimer structures can be delayed. Therefore, in such side reactions, the formation of by-products that do not contain the target sequence can be optionally reduced or delayed. In this way, for example, early consumption of primers can be reduced or delayed. The use of such oligonucleotides is such that, for example, a primer dimer containing a first primer (PD P1) or a primer dimer containing a second primer (PD P2) is produced by a side reaction and is being reacted. It is advantageous when it leads to early consumption of the primer. The use of primers with such additional structure (the first primer with the additional sequence variant P1 and / or the second primer with the additional sequence variant P2) causes a non-specific reaction in the amplification reaction. When observed, it is advantageous in certain embodiments. Such side reactions are facilitated by several factors, including:
-Long reaction time (for example, in the range of 1 hour to 100 hours)
-Primers are used in high concentrations (eg, in the range between 1 μmol / l and 1 mmol / l).
• High concentrations of polymerase are used (eg, in the range greater than 10 units / 10 μl)
Multiple reactions (eg, amplification of more than 10 different target sequences in one reaction approach).
High concentrations of complex nucleic acid strands in the reaction mixture (eg, concentrations greater than 1 μg hg DNA in 50 μl)
Factors alone or in combination with other factors can promote side reactions.

In general, side reactions (eg, non-specific primer-dimer formation) are such that by optimizing the reaction components and / or reaction conditions, for example, reducing the concentration of individual components, shortening the reaction time, primer sequences. This can be prevented by selecting more stringendo reaction conditions for the sequence design of. The additional sequence segments described in the advantageous embodiments (oligonucleotides containing a first primer and an additional sequence variant P1 or oligonucleotides containing a second primer and an additional sequence variant P2) are specific. Shows additional potential to delay side reactions.

In the examples, oligonucleotide primers with additional sequence segments are shown. In the embodiment, additional sequence segments are used that do not participate in the specific amplification of the target sequence and contribute to the delay of side reactions. Therefore, an oligonucleotide containing a first primer and an additional sequence variant P1 and an oligonucleotide containing a second primer and an additional sequence variant P2 are used.

Those skilled in the art will appreciate that in addition to the primer structure in which the oligonucleotide favors the specific amplification of the target sequence (this structure is also referred to as the "basic structure" or "minimal structure"), additional sequence segments (eg, for example). It will be recognized that additional sequence variants P1 or additional sequence variants P2) may also be included. Such additional sequence segments can provide a variety of other advantages or useful properties or functions.

Specific Embodiments of the Detection System The detection system comprises at least one fluorescent reporter (reporter) and at least one oligonucleotide probe capable of hybridizing to at least one of the primer extension products formed during amplification. In addition, the detection system can include a fluorescent quencher (called a quencher) compatible with the fluorescent reporter, whereby the quencher reduces or signals the fluorescent signal of the fluorescent reporter under certain circumstances. The strength can be reduced. In addition, the detection system can include a donor fluorescent dye molecule that is compatible with the fluorescent reporter, whereby the donor fluorescent dye molecule can enable the fluorescent signal of the fluorescent reporter by energy transfer under certain circumstances. it can. In addition, the detection system can include controller oligonucleotides, such controller oligonucleotides that include either a fluorescent reporter or donor fluorochrome molecule or a fluorescent quencher.

The arrangement of individual elements (fluorescent reporter, fluorescent quencher, donor fluorochrome molecule) on the oligonucleotide probe and / or controller oligonucleotide from the fluorescent reporter in the presence of primer extension products, each forming a complementary complex. Changes in the fluorescent signal need to be brought about.

Those skilled in the art understand a number of reporter systems developed over the last two decades in the real-time PCR segment. These are specifically cleaved using probes with self-complementary segments, such as molecular beacons, probes based on exonuclease degradation (so-called Taqman probes, which use the 5'-3'nuclease activity of Taq polymerase. A probe-based signal with two oligonucleotides labeled with a FRET pair to generate a signal and capable of binding to a single strand, a primer-based probe (eg, a LUX primer, which is self-complementary). Includes "Scorpion-primers" (having segments complementary to the strands synthesized with the primers), etc., which change the intensity of the signal when the structure is developed during inverse synthesis). Some probes can be used as endpoint measurements between signal acquisitions (eg, molecular beacons). Some probes are used to detect PCR kinetics, such as the 5'-3'nuclease probe. Many different arrangements of probes and fluorescent reporters, fluorescent quenchers, and donor fluorochrome molecules are known in the literature. Many fluorescent reporter quenchers and fluorescent reporter-donor fluorescent dye molecules (also called donor-acceptor pairs or FRET pairs) are also known ("Fluorescent Energy Transfer Nucleic Acid Probes", edited by Didenko, 2006, eg, Chapters 1 and 2; Products. Publication "Fluorescence Molecular Probes", published by Gene Link Inc). Most probes have been developed for PCR-based methods and use specific arrangements of primers, probes, and polymerases used, such as 3'-exonuclease (FEN).

In general, such probes can bind more or less specifically to DNA fragments generated during amplification, and signals can signal to such binding (eg, nuclease degradation or adjacent segments) alone or in combination with other events. It changes as a result of the binding of another oligonucleotide. This change can be detected and quantified.

Therefore, by using PCR in the second amplification, such known techniques and probes can also be used. The sequence of the oligonucleotide probe is adapted to the amplification product produced during the reaction.

The choice of probe also depends, for example, on whether a particular variant of the target sequence needs to be detected by a target sequence-specific probe, or whether only primer consumption is recorded (as a sign of amplification). Therefore, different probe formats can be selected depending on the job.

Therefore, the details of such adaptation for some embodiments will be described below.

Non-uniform form The components of both amplification systems are separated at the beginning of the first amplification, so that the influence of oligonucleotide probes or PCR primers on the first amplification reaction cannot occur. Conversely, after the completion of the first amplification, the aliquot is transferred from the first amplification to the second amplification. The resulting diluent of the reaction components of the first amplification system is advantageous for the use of probe structures known for PCR reactions. For example, in the presence of a controller oligonucleotide at a concentration of less than 100 nmol / l, particularly less than 10 nmol / l, particularly less than 1 nmol / l, particularly less than 0.01 nmol / l, it binds to the complementary segment of the primer extension fragment formed. Since the probe is largely unaffected under PCR conditions, probe-based detection methods known to those of skill in the art can be used.

Oligonucleotide probes can bind to one of the primer extension products formed (eg, third and fourth primer extension products), and this binding event uses a variety of techniques (eg, Taqman probe and Taq polymerase). , Or a "molecular beacon" can be detected by "binding to a complementary segment of the corresponding primer extension product).

Uniform Form In uniform form, the components of both amplification systems are in the same batch at the beginning of the first amplification, which allows them to interact with other components and intervene in a particular process. In particular, the presence of effective concentrations of controller oligonucleotides (eg, between 0.01 μmol / l-10 μmol / l) and oligonucleotide probes (eg, between 0.01 μmol / l-10 μmol / l) is between these components. Causes the interaction of substeps or affects the outcome of these steps. For this reason, the following aspects need to be considered in particular:
The presence of the controller oligonucleotide can affect the binding of the oligonucleotide probe to the primer extension product formed. This is the case, for example, when the probe and controller contain significant overlap in a segment complementary to the primer extension product (eg, the third segment of the controller and the oligonucleotide probe are the first and / or third primers. Therefore, it is advantageous to limit the degree of such duplication (which can partially hybridize to the same segment of the elongation product). For example, the length of the controller and probe segments can be adjusted to minimize duplication. Preferably, the sequence segments do not overlap when complementarily bound to the formed primer extension product.
• Oligonucleotide probes can bind and influence controller oligonucleotides as needed when strand substitutions occur. This can occur, for example, when the controller oligonucleotide and the oligonucleotide probe each contain a segment, which results in the formation of a double-stranded complex between the controller and the probe. Therefore, it is advantageous to limit the degree of such complementary segments. For example, the length of the controller and probe segments can be adjusted to reduce complementarity as much as possible. Preferably, the controller and probe do not contain complementary sequence segments.

For this reason, it is advantageous to design oligonucleotide probes in uniform form, for example, to selectively hybridize to the 3'segment of the first or third primer extension product. These segments do not hybridize to controller oligonucleotides.

Another essential aspect of the invention is the possible change of the polymerase when switching from the first amplification to the second amplification: the first polymerase can be inactivated and the second polymerase can be activated. .. This allows, for example, the use of a 5'-3 nuclease-sensitive probe ("Taqman probe") during the second amplification: for the first amplification, for example, a large fragment of Bst polymerase is preferred. This fragment cannot cut the Taqman probe. At the start of the second amplification, a large fragment of Bst polymerase is inactivated and Taq polymerase (eg, used as a hot start polymerase) is activated. This allows the use of 5'-3'nuclease sensitive probes.

Depending on the group selected between the fluorescent reporter and / or the donor fluorochrome molecule and / or the fluorescent quencher, this change can lead to an increase or decrease in the signal intensity of the fluorescent reporter. This change can be detected during or after the reaction by known appropriate means (eg, with a real-time PCR device such as StepOne-PCR or Lightcycler or Rotorgene, with reference to the manufacturer's instructions). By detecting signal changes, it is possible to draw conclusions about the course of the reaction, eg, signal amplitude, kinetics, time or concentration dependence of signal appearance. When multiple target sequences are used, multiplex analysis can be appropriately encoded by different spectral properties so that multiple reactions can be observed in parallel.

The present invention describes various embodiments of oligonucleotide probes that are particularly advantageous in detecting the progress of a reaction.

The oligonucleotide probe is preferably an oligonucleotide composed of DNA nucleotides. In embodiments other than DNA, the oligonucleotide is composed of other nucleotide monomers, such as RNA or nucleotide modifiers (eg, having glycophosphate backbone modifications such as PTO, LNA, 2'-O-Me). obtain. In another embodiment, the oligonucleotide sample is a mixed polymer containing both DNA and non-DNA elements (eg, RNA, PTO LNA). The oligonucleotide probe may include other oligonucleotide modifications, such as linkers or spacers (C3, HEG, debasement monomers, such as THF modifications, etc.).

The base composition of the oligonucleotide probe preferably contains a base capable of forming a complementary bond with a naturally occurring nucleobase (A, C, T, G) under hybridization conditions. In a further embodiment, the probe also comprises a modification comprising, for example, a universal base (eg, inosin, 5-nitro-indole). The probe can include additional modifications that affect the binding behavior of the oligonucleotide, such as the MGB modification.

The length of the oligonucleotide probe is preferably between 8-80 nucleotides, especially 12-80 nucleotides, especially 12-50 nucleotides, especially 12-35 nucleotides. Oligonucleotide probes usually contain segments that can complementarily bind to at least one of the formed products. In one embodiment, the oligonucleotide probe comprises at least one additional segment that cannot complementarily bind to one of the formed primer extension products.

The probe can be arranged differently in relation to the other components of the amplification system. Thereby, certain embodiments are preferred.

In one embodiment, the oligonucleotide probe is essentially complementary to at least one of the primer extension products formed (P1.1-Ext, P2.1-Ext, P3.1-Ext, P4.1-Ext). Sequence segment, which can hybridize to at least one of the formed primer extension products under appropriate conditions (hybridization conditions). In a further embodiment, the sequence segment is complementary to the first and / or third primer extension products. In a further embodiment, this sequence segment is complementary to the first and / or third primer extension products, and the oligonucleotide probe is a 3'segment of each primer extension product that is not complementaryly bound by the controller oligonucleotide. Can be complementary to. In a further embodiment, this sequence segment is complementary to the second and / or fourth primer extension products. In a further embodiment, this sequence segment of the oligonucleotide probe comprises a length ranging from 10 nucleotides to 50 nucleotides, particularly between 15-40, especially 15-30 nucleotides. In a further embodiment, this sequence segment of the oligonucleotide probe does not contain a sequence region that is essentially complementary to the controller oligonucleotide. In a further embodiment, the segment of the oligonucleotide probe sequence comprises a sequence region that is essentially complementary to the controller oligonucleotide, and the length of the segment is less than 20 nucleotides, particularly less than 15 nucleotides, particularly less than 10 nucleotides. Especially less than 5 nucleotides.

In a further embodiment, this sequence segment of the oligonucleotide probe does not contain a sequence region that is essentially complementary to one of the oligonucleotide primers. In a further embodiment, said segment of the oligonucleotide probe sequence comprises a sequence region that is essentially complementary to one of the oligonucleotide primers, and the length of the segment is less than 20 nucleotides, particularly less than 15 nucleotides, particularly 10 Less than nucleotides, especially less than 5 nucleotides.

In a further embodiment, this sequence segment of the oligonucleotide probe does not contain a sequence region that is essentially identical to the sequence of the third region of the controller oligonucleotide. In a further embodiment, the region of the oligonucleotide probe sequence comprises a sequence region that is essentially identical to the sequence of the third region of the controller oligonucleotide, and the segment length is less than 20 nucleotides, particularly 15 nucleotides. Less than, especially less than 10 nucleotides, especially less than 5 nucleotides.

In one embodiment, the controller oligonucleotide comprises one of the following components (fluorescent reporter and / or fluorescent quencher and / or donor fluorescent dye molecule), at least one of these components being the first of the controller oligonucleotide. It is located in the area of 3.

In one embodiment, the oligonucleotide probe is at least partially cleaved by a 5'-3'nuclease. In one embodiment, the controller oligonucleotide is cleaved by a 5'-3'nuclease at least in its 5'segment (third region of the controller).

In a further embodiment, the oligonucleotide probe comprises a sequence segment that is essentially complementary to the target sequence or that segment contained in one of the amplification products, said segment length of 5 to 50 nucleotides, particularly 10 to 10 nucleotides. It ranges from 40 nucleotides, especially between 15 and 30 nucleotides.

In a further embodiment, the oligonucleotide probe does not contain a sequence segment that is essentially complementary to the target sequence or its complementary strand. For example:

In one embodiment, the 3'end of the oligonucleotide probe is modified, eg, by a fluorescent dye molecule or quencher or donor, or another modification that prevents the polymerase from using the oligonucleotide as a primer (eg, phosphate residue). Blocked by groups or C3 linkers or dideoxynucleotide modifications).

In a further embodiment, the 3'end of the oligonucleotide probe is free and can complementarily bind to the first primer extension product to initiate synthesis by the polymerase. This allows the oligonucleotide probe to be extended like a primer, resulting in the formation of a probe extension product.

In one embodiment, the sequence composition of the probe corresponds to the sequence composition of the primer.

The location of the fluorescent reporter, fluorescent quencher or donor fluorescent dye molecule can vary depending on the embodiment of the oligonucleotide probe. The element can be covalently attached to either one of the respective ends of the oligonucleotide probe or to any of the central regions. Many such modifications are known to those of skill in the art, for example, dT for binding the FAM reporter to the 3'or 5'end of the nucleotide, or for binding the probe in the central or inner sequence segment of the probe. Use of -BHQ1 or dT-FAM or dT-TMR modifications. Such modified oligonucleotide probes are available from commercial suppliers (eg, Sigma-Aldrich, Eurofins, IDT, Eurogentec, Thermo Fisher Scientific).

For example, an oligonucleotide probe comprises at least one sequence segment capable of binding essentially complementaryly to a third or fourth primer extension product formed during amplification under the appropriate reaction conditions of the detection step. In this process, the oligonucleotide probe is complementarily attached, for example, to the single-stranded 3'segment of the synthesized third primer extension product or the 5'segment of the synthesized fourth primer extension product. Such probes do not contain any sequence segment that is identical to or complementary to the controller. Therefore, a complex containing a primer extension product and an oligonucleotide probe is produced. Binding of the oligonucleotide probe and controller oligonucleotide to the synthesized third primer extension product is preferably sequence-specific. The length of this sequence segment, which is complementary to the 3'segment of the first primer extension product, is, for example, 8-80 nucleotides, especially 12-80 nucleotides, especially 12-50 nucleotides, especially 12-35 nucleotides, especially 15-35. The range is between 25 nucleotides.

A detection system containing at least one fluorescent reporter bound to either the oligonucleotide probe or the controller oligonucleotide may alter the signal generation or signal intensity of the fluorescent reporter depending on the binding of the oligonucleotide probe to the complementary sequence. it can. Depending on the embodiment of the detection system, this change can result in the generation or increase or decrease of signals.

During amplification, the third and fourth primer extension products are separated such that the three segments of the third primer extension product and the corresponding fragments of the fourth primer extension product are present in a single strand form. The oligonucleotide probe can bind to the 3'segment of the third primer extension product or the 5'segment of the fourth primer extension product only during or after the reaction.

The binding is sequence-specific in nature, but it can also be resistant to differences from full complementarity.

Binding of oligonucleotide probes preferably does not interfere with amplification. The concentration of the probe and its length are adjusted so that a sufficient amount of primer can initiate the synthesis of primer extension products.

As the reaction proceeds, a sufficient amount of primer extension product is formed and the oligonucleotide probe can also bind well to result in a detectable signal change.

A suitable detection system that includes at least one fluorescent reporter, in one embodiment, further comprises at least one fluorescent quencher that matches the fluorescent reporter. This allows the fluorescent quencher (also known as the quencher) to function as a contact quencher or a FRET quencher. Suitable examples are from the literature ("Fluorescent Energy Nucleic Acid Probes" edited by Didenko, 2006, eg Chapters 1 and 2; Product Description "Flurescent Molecular Probes", published by GeneLink. For example, the fluorescence reporter can be fluorescein (FAM) and a suitable fluorescence quencher can be BHQ-1 or BHQ-2 or TAMRA. In one embodiment, the guanosine nucleobase can act as a quencher (eg, in combination with FAM as a reporter). When the fluorescence reporter is excited at the appropriate wavelength of light, a fluorescence signal is emitted in the absence of the quencher. However, if the fluorescent reporter is in close proximity to a suitable quencher, the intensity of the fluorescent reporter will be reduced. As the distance / separation between the fluorescent reporter and the quencher increases, the signal intensity increases.

Further suitable detection systems that include at least one fluorescent reporter further include, in one embodiment, at least one donor fluorescent dye molecule (also referred to as a donor) that matches the fluorescent reporter so that a FRET pair is formed. Suitable examples are from the literature ("Fluorescent Energy Nucleic Acid Probes" edited by Didenko, 2006, eg Chapters 1 and 2; Product Description "Flurescent Molecular Probes", published by GeneLink. A FRET pair usually includes a donor and an acceptor (usually the reporter acts as an acceptor). For example, the fluorescent reporter can be tetramethylrodamine (TAMRA), the appropriate counterpart of the FRET pair can be FAM (donor), another example can be FAM (donor) and Cy3 (as acceptor or reporter). .. In spatial proximity, excitation of a donor (such as FAM) causes energy to be transferred to a reporter (TAMRA or Cy3), so that the reporter itself emits electromagnetic radiation (detectable optical or fluorescent signal). You will be able to release energy as. This fluorescent signal of the reporter can be detected by appropriate means. As the spatial separation between the reporter and the donor fluorochrome increases, the signal gradually diminishes and is no longer measurable and detectable from distances usually greater than 50 nucleotides (measured as double-stranded 50 nucleotides).

Proper placement of individual elements of the detection system on oligonucleotide probes and / or controller oligonucleotides to detect binding events of these components to the first primer extension product by increasing or decreasing signals. It is possible to do.

Such detection can be performed either during amplification (eg, as online detection), at appropriate time intervals, or only at the end of the reaction.

The fluorescent signal is acquired in one detection step. In the detection step of this method, it is necessary to confirm whether the oligonucleotide probe is complementarily bound to the 3'segment of the first primer extension product (P1-Ext). Therefore, in the detection step, the reaction temperature is adjusted so that the probe can bind to the 3'segment of the first primer extension product in an essentially complementary manner. The temperature can correspond to one of the temperatures during amplification, or can represent a step separate from the temperature step of amplification.

During the steps, the reaction can be excited by a light source that uses light of the appropriate wavelength. Depending on the design of the detection system, the wavelength is adapted to the absorption spectrum of the fluorescent reporter or donor fluorochrome molecule. A signal from the fluorescent reporter can be expected if the oligonucleotide probe can bind to the 3'end of the first primer extension product synthesized. The signal has a unique spectrum of wavelengths and can be detected and quantified by an appropriate detection system. Current real-time PCR devices typically include both a light source for excitation and a detection system for detecting the fluorescence of the reporter, and a temperature control container for the reaction batch. Real-time PCR devices such as StepOne, LightCycler, and RotorGene are good examples.

This detection can be used to quantify one or more starting nucleic acid strands present in the reaction. In addition, this detection can be used to detect the availability of the starting nucleic acid chain at the onset of the reaction. In addition, the detection system can be used in combination with an internal amplification control.

In a further embodiment, the two or more nucleic acid chains can be specifically amplified in the amplification batch. For example, a combination of a specific first primer and / or a specific controller oligonucleotide and / or a specific second primer can be used. For example, the target sequence and internal amplification control are amplified in one batch. Therefore, it is advantageous to detect individual nucleic acid chains separately and independently during their amplification.

In one embodiment, a specific detection system is used in each case, whereby monitoring of nucleic acid chain amplification is performed by the specific detection system in each case. Specific signals from the fluorescent reporter can be detected at the same time. Preferably, the spectral properties of the fluorescence reporter differ to the extent that they can be achieved by detecting each fluorescence signal at a characteristic wavelength. For example, two or three or four fluorescent reporters can be used. Each specific wavelength of the fluorescence signal is preferably greater than about 10 nm, particularly greater than about 20 nm, particularly greater than about 30 nm, as measured by the maximum intensity (fluorescence peak) of the fluorescence spectrum. Such combinations are known. For example, a combination containing FAM and / or Cy3 and / or Cy5 or FAM and / or HEX and / or ROX is appropriate. Each quencher is preferably specifically selected for each fluorescence reporter so that signal reduction by the quencher has high efficiency. For example, a combination of FAM / BHQ-1 and HEX / BHQ-2 or FAM / BHQ-1 and Cy5 / BHQ-2 is used.

In a further embodiment, a detection system can be used that allows monitoring of amplification of groups of different nucleic acid chains. The group can include two or more nucleic acid chains that are amplified. The components of the detection system are adjusted accordingly. In one embodiment, such a group of different nucleic acid chains to be amplified is, for example, at least one homogeneous specific to the oligonucleotide probe used for complementary binding of the oligonucleotide probe under the reaction conditions of the detection step. Contains array segments. In a further embodiment, such a group of different nucleic acid chains to be amplified comprises, for example, at least one sequence segment for predominantly complementary binding of the oligonucleotide probe, the sequence composition of this sequence segment. Different within this group. These differences may include 1-10 nucleotides, preferably 1-3 nucleotides.

Further embodiments and embodiments are found in the following items that describe the invention without limitation:
Item 1: A method for amplifying nucleic acid.
a) A sample containing a first nucleic acid polymer comprising a first target sequence M [and a (reverse) complementary sequence M'], where M has 5'-sequence segments MPL, MS and MPR. The sample, which is contained in the nearest sequence in the 3'direction, is added to the next component in the first amplification step:
b) First template-dependent nucleic acid polymerases, especially DNA polymerases, and substrates for template-dependent nucleic acid polymerases (particularly ribonucleoside triphosphates or deoxyribonucleoside triphosphates) and appropriate cofactors;
c) First left oligonucleotide primer PL1 that is (essentially) identical to MPL;
d) The first right oligonucleotide primer PR1, which contains the sequence segment PCR and PMR in the 5'-3'direction in the immediate vicinity, where the PMR has a [hybridizing] sequence complementary to the MPR. However, the first right oligonucleotide primer PR1, which cannot bind to M [or the sequence immediately following MPR with respect to sequence M of 3', and here PR1 (particularly the PCR site), is the sequence segment PCR. Containing a modified nucleotide building block such that PCR cannot serve as a template for activating the first template-dependent nucleic acid polymerase; and e) Sequence segments CSR, CPR and CCR in the 5'-3'direction. The most recent contiguous inclusion of the controller oligonucleotide CR, the CSR is identical to the segment of MS at 5'of the MPR [and is read first at the initiation of the polymerase of PR, ie the CSR is of primer PR1. Can bind to polymer products], CPR is complementary to PMR (and is identical to MPR), and CCR is complementary to PCR, said controller oligonucleotide CR,
And here, the CR comprises a nucleotide building block modified to the CSR so that the CSR cannot function as a template for activating the template-dependent nucleic acid polymerase;
A method of amplifying the nucleic acid in contact with.
Item 2: In addition to the sequence region PCR and PMR, a first primer comprising a synthetic region PSR essentially complementary to the target sequence M in the 5'-3'direction in the region adjacent to the MPR at 5'. An extension product PR1'is obtained, and in addition to the sequence region MPL, a second primer extension product PL1' is obtained that contains a synthetic region PSL that is essentially identical to the target sequence M in the adjacent region at 3'.
And
-The reaction conditions of any of the first amplification steps are selected and / or-the length and melting temperature of PCR and MS, if applicable, are selected.
As a result, PR1'can form a double strand with M, [PL1'can form a double strand with M'], [PR1'can form a double strand with PL1'], and PR1'is A double strand can be formed with C, and the formation of the double strand of PR1'and C takes precedence over the formation of the double strand of PR1'with M (at least in the MPR region).
The method for amplifying the nucleic acid according to item 1.
Item 3:
-The reaction conditions of any of the first amplification steps are selected and / or-appropriately, the length and melting temperature of the PCR and the positions of MS and / or MPL and MPR are selected.
As a result, in the absence of the controller oligonucleotide CR, the first primer extension product PR1'does not separate from the second primer extension product PL1'.
The method for amplifying the nucleic acid according to item 2.
Item 4: In the second amplification step, the sample is divided into the following components:
a) The second left oligonucleotide primer PL2, which is the same as the first secondary primer binding region MPL2,
b) Second right oligonucleotide primer PR2, which is complementary to region MPR2,
Here, MPL2 and MPR2 are included in M, and the 3'end [at least 20 positions] of MPL2 is located at 5'at the 5'end of MPR2 (in particular, MPL2 is identical to MPL and / or MPR2. Is the same as MPR),
Contact,
The method for amplifying the nucleic acid according to any one of items 1 to 3.
Item 5: The method of item 4, wherein PL2 and / or PR2 [directly] contain the sequence portion PCL2 or PCR2 in 5'of the sequence portion that is identical to MPL2 or complementary to MPR2, respectively.
Item 6: A second template-dependent nucleic acid polymerase, particularly a DNA polymerase, and optionally a template-dependent nucleic acid polymerase substrate (particularly ribonucleoside triphosphate or deoxyribonucleoside triphosphate and suitable cofactors) is the second. The method according to any one of items 1 to 5, wherein the sample is brought into contact with the sample in the amplification step.
Item 7: The item according to any one of items 1 to 6, wherein the modified nucleotide building block comprises a 2'-O-alkylribonucleoside building block, particularly a 2'-O-methylribonucleoside building block. Method.
Item 8: The method according to any one of items 1 to 7, wherein the first amplification step is performed essentially at an isothermal temperature.
Item 9: The method of any one of items 1-8, wherein the MS has a length of 20 to 200 nucleotides.
Item 10: The method of any one of items 1-9, wherein PR1 has a length in the range of 15 to 100 nucleotides.
Item 11: The PCR has a length in the range of 5 nucleotides to 85 nucleotides, in particular the PCR has a length between 50% and 300% of the sequence segment PMR, items 1-10. The method according to any one of the above.
Item 12: The method of any one of items 1-11, wherein the CR (controller oligonucleotide) has a length in the range of 20 to 100 nucleotides.
Item 13: The method of any one of items 1-12, wherein the first amplification and the second amplification are performed in the same reaction batch.
Item 14: The method according to any one of items 1 to 13, wherein the second template-dependent nucleic acid polymerase is a thermostable polymerase, particularly a thermostable DNA polymerase.
Item 15: Items 1-14, wherein the first template-dependent nucleic acid polymerase is thermally inactivated, particularly the first template-dependent nucleic acid polymerase is a mesophilic polymerase. The method according to any one of the above.
Item 16: A second template-dependent nucleic acid polymerase, a second right oligonucleotide primer PR2 and / or a second left oligonucleotide primer PL2 can be activated and / or a controller oligo. The method according to any one of items 1 to 15, wherein the nucleotide CR can be inactivated.
Item 17: The method according to any one of items 1 to 16, wherein the components of the second amplification step come into contact with the sample after the first amplification step has been performed.
Item 18: In any one of items 1 to 17, wherein in CSR and CPR the CR comprises a sequence region capable of specifically binding the sequence to the second right oligonucleotide primer PR2. The method described.
Item 19: Any of items 1-18, wherein the selected reaction condition, in particular the reaction condition of the first amplification step, comprises a reaction temperature in the range of 25 ° C. to 80 ° C., particularly 50-70 ° C. The method described in item 1.
Item 20: A kit for carrying out the method according to any one of items 1 to 19.
The first right oligonucleotide primer PR1, which contains the sequence segment PCR and PMR in the most recent sequence in the −5'-3'direction, where PMR is eukaryotic or pathogenic bacteria, especially mammals, in particular. The primer binding site MPR of the genome sequence M of the human target sequence can be bound, and in the 5'-3'direction, M contains the sequence segments MPL, MS and MPR in the immediate vicinity, and the sequence segment PCR is performed. A nucleotide that cannot bind to a sequence located directly at 3'of the MPR, and that PR1 (particularly the PCR site) cannot function as a template for PCR to activate the first template-dependent nucleic acid polymerase. The first left oligonucleotide primer PL1 which is (essentially) identical to the first right oligonucleotide primer PR1; and-MPL, which comprises a building block.
A controller oligonucleotide CR containing the sequence segments CSR, CPR and CCR in the most recent sequence in the −5'-3'direction, the CSR of which is identical to the segment of MS at 5'of the MPR [and of the PR. It is read first at the initiation of the polymerase, so the CSR can bind to the polymer product of primer PR1], the CPR is complementary to the PMR (and is identical to the MPR), and the CCR is complementary to the PCR. Here, in the CSR, the CR comprises the nucleotide building block modified in such a way that the CSR cannot function as a template for activating a template-dependent nucleic acid polymerase;
-The second left oligonucleotide primer PL2, which is the same as the first secondary primer binding region MPL2, and-the second right oligonucleotide primer PR2, which is complementary to the region MPR2.
Here, MPL2 and MPR2 are contained in M, and the 3'end [at least 20 positions] of MPL2 is located at 5'at the 5'end of MPR2 (in particular, MPL2 is identical to MPL and / or MPR2 is the same as MPR), said second right oligonucleotide primer PR2,
The kit comprising.
Item 21: A second template-dependent nucleic acid polymerase, in particular DNA polymerase, and optionally a DNA polymerase substrate (particularly ribonucleoside triphosphate or deoxyribonucleoside triphosphate) and suitable cofactors, in particular medium temperature template-dependent polymerase. The kit of item 20, which further comprises a medium temperature template-dependent polymerase, in particular which has no 5'-3'exonuclease activity.
Item 22: A second template-dependent nucleic acid polymerase, especially a DNA polymerase, and optionally a template-dependent nucleic acid polymerase substrate (particularly ribonucleoside triphosphate or deoxyribonucleoside triphosphate), and suitable cofactors, particularly thermophilic. The kit according to any one of items 20 and 21, further comprising a template-dependent polymerase, particularly a thermophilic template-dependent polymerase having 5'-3'exonuclease activity.
Item 23: A method of amplifying nucleic acid, the next step:
a) The first amplification step, the next step:
− The step of hybridizing the first oligonucleotide primer (P1.1) to the amplified nucleic acid containing the target sequence, the first oligonucleotide primer (P1.1) is the following region:
A first region that can be sequence-specifically bound to the region of the nucleic acid to be amplified, the region of the nucleic acid to be amplified that contains at least the 5'end of the target sequence or is located in the 5'direction of the target sequence. , Said first region;
A second region adjacent to the 5'end of the first region or connected via a linker, the second region can be bound by a controller oligonucleotide and under selected reaction conditions. The step, comprising a second region, which remains essentially uncopied by the polymerase used in.
-Obtain a first primer extension product (P1.1-Ext) containing a synthetic region that is essentially complementary to the amplified nucleic acid or target sequence in addition to the first oligonucleotide primer (P1.1). The step of extension of the first oligonucleotide primer (P1.1) by the first polymerase for the purpose, wherein the first primer extension product and the amplified nucleic acid are present as double strands;
− The step of binding the controller oligonucleotide (C1.1) to the first primer extension product (P1.1-Ext), the controller oligonucleotide (C1.1) is the following region:
A first region capable of binding to a second region (overhang) of the first primer extension product (P1.1-Ext),
A second region that is essentially complementary to the first region of the first oligonucleotide primer (P1.1), and a synthetic region of the first segment of the primer extension product (P1.1-Ext). A third region that is essentially complementary to at least a portion of; and where the controller oligonucleotide (C1.1) does not serve as a template for primer extension of the first oligonucleotide primer (P1.1). And the first controller oligonucleotide (C1.1) binds to the first and second regions of the first primer extension product (P1.1-Ext), and at the same time, the first and second regions. Complementarily to replace the amplified region of nucleic acid;
Including the above steps,
-The step of hybridizing the second oligonucleotide primer (P2.1) to the first primer extension product, where the second primer (P2.1) is complementary to at least the 5'end of the target sequence. , Or a region that is sequence-specifically capable of binding to the synthetic region of the first primer extension product (P1.1-Ext) located in the 3'direction thereof.
-In addition to the second oligonucleotide primer (P2.1), obtain a second primer extension product (P2.1-Ext) containing a synthetic region that is essentially identical to the nucleic acid or target sequence to be amplified. This is a step of extension of the second oligonucleotide primer (P2.1) by the first polymerase for the purpose, and is a first primer extension product (P1.1-Ext) and a second primer extension product (P2.1). To form the first double-stranded amplification product, said step;
The first amplification step, and (b) the second amplification step, comprising:
-A step of hybridizing the third oligonucleotide primer (P3.2) to the second primer extension product (P1.1-Ext) of the first amplification product, which is a step of hybridizing the third oligonucleotide primer (P3.2). ) Includes at least the 5'end of the target sequence, or is located in the 5'direction thereof, the first region capable of sequence-specifically binding to the segment of the second primer extension product (P1.1-Ext). Including, said step;
-In addition to the third oligonucleotide primer (P3.2), a second primer extension product (P2.1-Ext) or a third primer extension product containing a synthetic region that is essentially complementary to the target sequence. A step of extension of a third oligonucleotide primer (P3.2) by a second polymerase to obtain (P3.2-Ext), the second primer extension product (P2.1) and the third. The step in which the primer extension product (P3.1) is present as a double strand;
-A step of hybridizing the fourth oligonucleotide primer (P4.2) to the first primer extension product (P1.1-Ext), where the fourth oligonucleotide primer (P4.2) is of the target sequence. Includes a first region capable of sequence-specific binding to the synthetic region of the first primer extension product (P1.1-Ext), which is complementary to at least the 5'end or located in the 3'direction thereof. Said step;
-In addition to the fourth oligonucleotide primer, a fourth primer extension product containing a synthetic region that is essentially complementary to the first primer extension product (P1.1-Ext) or is identical to the target sequence. A step of extension of the fourth oligonucleotide primer (P4.2) by the second polymerase to obtain (P4.2-Ext), the first primer extension product (P1.1-Ext) and the first. The 4-primer extension product (P4.1-Ext) is present as a double strand, said step;
-Double strands from the first primer extension product (P1.1-Ext) and the fourth primer extension product (P4.2-Ext), and the second primer extension product (P2.1) and the third The step of separating the double strand from the primer extension product (P3.1);
-Hybrid the third oligonucleotide primer (P3.2) to the fourth primer extension product (P4.2-Ext) and extend the third oligonucleotide primer (P3.2) with the second polymerase. Steps; and-The step of hybridizing the fourth oligonucleotide primer to the third primer extension product (P3.2-Ext) and extending the fourth oligonucleotide primer (P4.2) with the second polymerase. ,
The second amplification step, which comprises:
The method described above.
Item 24: The first amplification step is the copy number of the first amplification product in the range of 10-100,000,000,000, especially 100-1,000,000,000,000, especially 1000-100,000,000. 23. The method of item 23, characterized in that it is repeated until it exists.
Item 25: A third oligonucleotide primer and / or a fourth oligonucleotide primer having a second region adjacent to the 5'end of the first region or connected via a linker, second 23 or 24, wherein the region of is not complementary to the first primer extension product (P1.1-Ext) or the second primer extension product (P2.1-Ext). Method.
Item 26: Double-strand separation of the first primer extension product (P1.1-Ext) and the fourth primer extension product (P4.2-Ext), and the second primer extension product (P2.1) and Any of items 23-25, characterized in that the double-stranded separation of the third primer extension product (P3.1) is carried out by thermal denaturation, in particular at temperatures in the range of 85 ° C to 105 ° C. The method described in paragraph 1.
Item 27: The method according to any one of items 23 to 26, wherein the first polymerase and the second polymerase are the same.
Item 28:
-The first oligonucleotide primer (P1.1) and the third oligonucleotide primer (P3.2) are essentially the same; and / or-the second oligonucleotide primer (P2.1) and The third oligonucleotide primer (P4.2) is essentially the same.
23. The method according to any one of items 23 to 27.
Item 29: The method of any one of items 23-28, wherein the first amplification and the second amplification are performed in the same reaction batch.
Item 30: A second polymerase, a third oligonucleotide primer (P3.1) and / or a fourth oligonucleotide primer (P4.1) can be activated and / or a controller oligonucleotide. 29. The method of item 29, wherein (C1.1) can be deactivated.
Item 31: The method of any one of items 23-28, wherein the first amplification is performed in a first reaction batch and the second amplification is performed in a second reaction batch. ..
Item 32: The method of item 31, wherein an aliquot of the first reaction batch or a complete first reaction batch is added to the second reaction batch.
Item 33: Items 23-32, wherein the controller oligonucleotide (C1.1) comprises a fourth region capable of sequence-specific binding to a third oligonucleotide primer (P3.2). The method according to any one of the above.
Item 34: The method of any one of items 23-33, wherein the first amplification is performed essentially at an isothermal temperature.
Item 35: The first oligonucleotide primer (P1.1) contains one or more modifications in the second region, particularly immediately after the first region of the first oligonucleotide primer, thereby the second. The method according to any one of items 23 to 24, which comprises stopping the first polymerase in the region of.
Item 36: A kit for carrying out the method according to any one of items 23 to 35.
− The first oligonucleotide primer (P1.1), which is the following region:
A first region that can be sequence-specifically bound to the region of the nucleic acid to be amplified, and the region of the nucleic acid to be amplified contains at least the 5'end of the target sequence, or itself in the 5'direction of the target sequence. The first area to be arranged;
A second region that is close to the 5'end of the first region or is connected via a linker, the second region can be bound by the first controller oligonucleotide and is selected. The second region, which remains essentially uncopied by the polymerase used for amplification under reaction conditions;
Here, the first oligonucleotide primer (P1.1) can be extended by a polymerase to a first primer extension product containing a synthetic region in addition to the first oligonucleotide primer (P1.1). Is;
The first oligonucleotide primer (P1.1) having
-A second oligonucleotide primer (P2.1), the second oligonucleotide primer (P2.1), which is at least complementary to the 5'end of the target sequence, or is located in the 3'direction thereof. A second primer extension product that contains a region capable of sequence-specific binding to the synthetic segment of the first primer extension product (P1.1-Ext) and that contains the synthetic segment in addition to the oligonucleotide primer (P2.1). The second oligonucleotide primer (P2.1), which can be extended to (P2.1-Ext) by a polymerase,
− Controller oligonucleotide (C1.1) in the following regions:
A first region capable of binding to a second region of the first primer extension product (P1.1-Ext),
A second region that is essentially complementary to the first region of the first oligonucleotide primer (P1.1), and a synthetic region of the first region of the primer extension product (P1.1-Ext). A third region that is essentially complementary to at least part of the
Including
Here, the controller oligonucleotide (C1.1) does not function as a template for primer extension of the first oligonucleotide primer (P1.1), and the controller oligonucleotide (C1.1) is the first primer. The controller oligonucleotide (C1.) Which binds to the first and second regions of the extension product (P1.1-Ext) and at the same time replaces the region of the nucleic acid which is complementarily amplified in the first and second regions. 1);
-A third oligonucleotide primer (P3.2), wherein the third oligonucleotide primer (P3.2) contains at least the 5'end of the target sequence or is located in the 5'direction thereof. A third primer extension product that is capable of sequence-specific binding to the region of the primer extension product (P1.1-Ext) and contains a synthetic region in addition to the third oligonucleotide primer (P3.2). The third oligonucleotide primer (P3.2); and the-fourth oligonucleotide primer (P4.2), which comprises a first region in (P3.2-Ext) that can be extended by a polymerase. The fourth oligonucleotide primer (P4.2) is at least complementary to the 5'end of the target sequence, or the synthesis of the first primer extension product (P1-Ext) located in the 3'direction thereof. It is capable of sequence-specific binding to a region and can be extended by a polymerase to a fourth primer extension product (P4.2-Ext) containing a synthetic region in addition to an oligonucleotide primer (P4.2). The fourth oligonucleotide primer (P4.2), which comprises region 1.
The kit comprising.
Item 37: Use of the kit according to item 36 for performing the method according to any one of items 23-35.
Item 38: A method of amplifying nucleic acid, the next step:
a) The first amplification step, the next step:
− Steps of providing the starting nucleic acid 1.1 containing the target sequence,
-A step of hybridizing the first oligonucleotide primer (P1.1) to an amplifying nucleic acid having a target sequence (either the starting nucleic acid 1.1 or P2.1-Ext), the first oligonucleotide primer. (P1.1) is the following area:
The first region (P1.1.1), which can be sequence-specifically bound to the region of nucleic acid to be amplified (P2.1E1),
A second region (P1.1.2) adjacent to the 5'end of the first region or connected via a linker, the second region being bound by a controller oligonucleotide. The second region (P1.1.2), which can and remains essentially uncopied by the polymerase used under the reaction conditions of choice;
In addition to the above-mentioned step-first oligonucleotide primer (P1.1), which comprises a synthetic segment (P1.1E1 to P1.1E4) that is essentially complementary to the nucleic acid or target sequence to be amplified. It is a step of extension of the first oligonucleotide primer (P1.1) by the first polymerase to obtain the first primer extension product (P1.1-Ext), and is amplified with the first primer extension product. The nucleic acid is present as a double strand, said step;
-The step of binding the controller oligonucleotide (C1.2) to the first primer extension product (P1.1-Ext), where the controller oligonucleotide (C1.2) is in the following region;
The first region (C1.2.1), which can bind to the second region (overhang) of the first primer extension product (P1.1E6),
The second region (C1.2.2), which is complementary to the first region of the first oligonucleotide primer (P1.1.1), and the first primer extension product (P1.1E4). It comprises a third region (C1.2.3); which is essentially complementary to at least a portion of the synthetic region, and where the controller oligonucleotide (C1.1) is the first oligonucleotide primer (P1). It does not function as a template for primer extension in 1), and the first controller oligonucleotide (C1.1) binds to the first primer extension products (P1.1E6, P1.1E5, P1.1E4) at the same time. , The step of substituting the complementary regions (P2.1E1, P2.1E2) of the nucleic acid amplified in the region;
-The step of hybridizing the second oligonucleotide primer (P1.1) to the first primer extension product, the second oligonucleotide primer (P2.1) is the first primer extension product (P1.1E1). The above step, which includes a region (P2.1.1) capable of sequence-specific binding to the synthetic region of).
-A second primer extension product (P2.1E4 to P2.1E1) containing a synthetic region (P2.1E4 to P2.1E1) that is essentially identical to the nucleic acid or target sequence to be amplified in addition to the second oligonucleotide primer (P2.1). A step of extension of the second oligonucleotide primer (P2.1) by the first polymerase to obtain P2.1-Ext), the first primer extension product (P1.1-Ext) and the second. The first amplification step, wherein the primer extension product (P2.1) forms the first double-stranded amplification product; and-until the desired amount of the first amplification product is synthesized. Steps that need to be repeated,
The first amplification step, comprising:
b) The second amplification step, the next step:
− A step of hybridizing a third oligonucleotide primer (P3.2) to a second and / or fourth primer extension product (P1.1-Ext or P4.1-Ext), the third oligonucleotide. The primer (P3.2) is a first region (P3.1E1) capable of specifically binding a sequence to a region of a second primer extension product (P2.1E1) and a fourth primer extension product (P4.1E2). The step, including 1).
-From a synthetic region (P3.1E5 to P3.1E2 or P3.1E5) that is essentially complementary to the second primer extension product (P2.1-Ext) or target sequence in addition to the third primer extension product. A step of extension of a third oligonucleotide primer (P3.2) by a second polymerase to obtain a third oligonucleotide primer extension product (P3.2-Ext) with P3.1E1). The step 2 in which the primer extension product (P2.1) and the third primer extension product (P3.1) are present as double strands;
− A step of hybridizing a fourth oligonucleotide primer (P4.2) to a first primer extension product (P1.1-Ext) and / or a third primer extension product (P3.1-Ext). The fourth oligonucleotide primer (P4.2) is a first segment capable of sequence-specific binding to the synthetic region of the first primer extension product (P1.1E1) and the third primer extension product (P3.1E2). The step, which comprises (P4.1.1).
-From the synthetic region (from P4.1E5) that is essentially complementary to the first primer extension product (P1.1-Ext) or essentially identical to the target sequence, in addition to the fourth oligonucleotide primer. A fourth oligonucleotide primer (P4.2) with a second polymerase to obtain a fourth primer extension product (P4.2-Ext) containing P4.1E2 or P4.1E5 to P4.1E1). The step of extension, wherein the first primer extension product (P1.1-Ext) and the fourth primer extension product (P4.1-Ext) are present as double strands;
-Separate the double strands of the first primer extension product (P1.1-Ext) and the fourth primer extension product (P4.2-Ext), and the second primer extension product (P2.1-Ext). And the double strand of the third primer extension product (P3.1-Ext) are separated, and the fourth primer extension product (P4.1-Ext) and the third primer extension product (P3.1-Ext). Steps to separate the double strands of
-Hybrid the third oligonucleotide primer (P3.2) to the fourth primer extension product (P4.2-Ext) and extend the third oligonucleotide primer (P3.2) with the second polymerase. Steps; and-The step of hybridizing the fourth oligonucleotide primer to the third primer extension product (P3.2-Ext) and extending the fourth oligonucleotide primer (P4.2) with the second polymerase. ,
The second amplification step, comprising:
The method described above.
Item 39: The first amplification step is a copy number of the first amplification product in the range of 10-100,000,000,000, especially 100-1,000,000,000,000, especially 1000-100,000,000. 38. The method of item 38, characterized in that it is repeated until it is present.
Item 40: A third oligonucleotide primer and / or a fourth oligonucleotide primer forms a second region (P3.1.2 or P4.1.2, respectively) adjacent to the 5'end of the first region. Item 38, wherein the second region cannot complementarily bind to the first primer extension product (P1.1-Ext) or the second primer extension product (P2.1-Ext). Or the method according to 39.
Item 41: Double-strand separation of the first primer extension product (P1.1-Ext) and the fourth primer extension product (P4.2-Ext), and the second primer extension product (P2.1-Ext). ) And the third primer extension product (P3.1-Ext), and the fourth primer extension product (P4.1-Ext) and the third primer extension product (P3.1-Ext). 38. The method of any one of items 38-40, characterized in that the double-stranded separation of is achieved, in particular, by thermal denaturation at a temperature in the range of 85 ° C. to 105 ° C.
Item 42: The method according to any one of items 38 to 40, wherein the first polymerase and the second polymerase are the same.
Item 43:
The first oligonucleotide primer (P1.1) and the third oligonucleotide primer (P3.2) are essentially the same; and / or the second oligonucleotide primer (P2.1) and The fourth oligonucleotide primer (P4.2) is essentially the same.
38. The method according to any one of items 38 to 42.
Item 44: The method of any one of items 38-40, wherein the first amplification and the second amplification are performed in the same reaction batch.
Item 45: A second polymerase, a third oligonucleotide primer (P3.1) and / or a fourth oligonucleotide primer (P4.1) can be activated and / or a controller oligonucleotide. 44. The method of item 44, wherein (C1.1) can be deactivated.
Item 46: Any of items 38-45, wherein the fluorescent dye-labeled first oligonucleotide probe can complementarily bind to the third primer extension product and the fluorescent dye signal is detected. The method described in item 1.
Item 47: Any of items 38-46, wherein the fluorescent dye-labeled first oligonucleotide probe can complementarily bind to the fourth primer extension product and the fluorescent dye signal is detected. The method described in item 1.

Materials and methods:
Reagents were purchased from the following vendors:

Unmodified and modified oligonucleotides (Eurofins MWG, Eurogentec, Biomers, Trilink Technologies, IBA Solutions for Life Sciences); Polymerase NEB (New England Biolabs; Other chemicals: Sigma-Aldrich; Plastic products: Polymerase

Solution 1 (amplification reaction solution 1):
Monopotassium glutamate, 50 mmol / l, pH 8.0; magnesium acetate, 10 mmol / l; dNTP (dATP, dCTP, dTTP, dGTP), 200 μmol / l, respectively; polymerase (Bst2.0 WarmStart, 120.000 U / ml NEB), 12 Unit / 10 μl; TritonX-100, 0.1% (v / v); EDTA, 0.1 mmol / l; TPAC (tetrapropylammonium chloride), 50 mmol / l, pH 8.0; EvaGreen dye (dye is manufacturer Used in a 1:50 dilution according to the instructions in).

Solution 2 (amplification reaction solution 2):
1x Isothermal Buffers (New England Biolabs); In a single concentration, this buffer contains: 20 mM Tris-HCl; 10 mM (NH4) 2SO4; 50 mM KCl; 2 mM 00754; 0.1% Tween® 20 At pH 8.8 and 25 ° C.); dNTP (dATP, dCTP, dUTP, dGTP), 200 μmol / l each;
EvaGreen dye (dye used at 1:50 dilution according to manufacturer's instructions)

Solution 4 (Amplification Reaction Solution 4); 1x Isothermal Buffers (New England Biolabs) See above.
All concentrations are the final concentration in the reaction. Deviations from the standard reaction are shown accordingly.

The melting temperature (Tm) of the components involved is measured at a concentration of 1 μmol / l of each component in the solution 1. Deviation parameters are given in each case.

General information about the reaction (first amplification)
The Bst 2.0 polymerase warm start was initiated by heating the reaction solution to the reaction temperature, as the temperature sensitive oligonucleotide (according to the manufacturer's specifications) significantly suppressed its function at low temperatures. Polymerase becomes more and more active at temperatures above 45 ° C. At a temperature of 65 ° C., no difference could be detected between polymerase Bst 2.0 and Bst 2.0 warm start. Polymerase Bst 2.0 warm start was used to prevent the formation of large amounts of by-products (such as primer dimers) during the preparation phase of the reaction. The deviation due to this is shown concretely.

The reaction was stopped by heating the reaction solution above 80 ° C. (eg, 95 ° C. for 10 minutes). At this temperature, polymerase Bst 2.0 is irreversibly denatured and the results of the synthetic reaction cannot be modified later.

The reaction was performed on a thermostat equipped with a fluorescence measuring device. For this purpose, a commercially available real-time PCR device, StepOne Plus (Biosystems, supplied by Thermofischer) was used. The standard reaction volume was 10 μl. The deviation due to this is shown.

Both end point determination and kinetic observations were made. In determining the endpoint, the signal was recorded, for example, by a dye bound to nucleic acid, eg, by TMR (tetramethylrhodamine, also called TAMRA) or FAM (fluorescein). Wavelengths for excitation and measurement of fluorescent signals of FAM and TMR are stored as default settings in the StepOne Plus real-time PCR device. Also, an insert dye (EvaGreen) was used for end point measurements (eg, melting curve measurements). EvaGreen is an insert dye, an analog of the frequently used dye Sybrgreen, but with slightly lower polymerase inhibition. The wavelengths for excitation and measurement of the fluorescence signals of SYbrGreen and EvaGreen are the same and are stored in the StepOne Plus real-time PCR device as a factory setting. Fluorescence can be detected continuously, i.e. "online" or "in real time" by a built-in detector. Since the polymerase synthesizes double strands during its synthesis, this technique could be used for kinetic measurements of reactions (real-time monitoring). Due to specific interference between the color channels of the StepOne Plus device, an increase in basal signal intensity was sometimes observed in measurements using TMR-labeled primers at concentrations above 1 μmol / L (eg, 10 μmol / L). It was observed that the TMR signal of the Sybr-Green channel resulted in an increase in basal signal intensity. The increased basal value described above was taken into account in the calculation.

Kinetic observations of the reaction process were recorded regularly using fluorescent signals from fluorescein (FAM-TAMRA fret pair) or insert dye (EvaGreen). The time dependence of the signal was recorded (real-time signal acquisition using the StepOne plus PCR device). The increase in signal during the reaction compared to the control reaction was interpreted according to the structure of the batch. For example, the increase in signal when using the Evagreen dye was interpreted as indicating an increase in the amount of double-stranded nucleic acid strands in the reaction and thus as a result of synthesis by DNA polymerase.

For some reactions, melting curve measurements were performed after the reaction. From such measurements, it is possible to conclude, for example, that there are double strands that can absorb the inserted dye and thus significantly amplify the signal intensity of the dye. As the temperature rises, the proportion of double strands decreases and the signal intensity also decreases. The signal depends on the length of the nucleic acid chain and the sequence composition. The technique is well known to those of skill in the art.

When using melting curve analysis in connection with reactions containing significant amounts of modified nucleic acid strands (eg, controller oligonucleotides or primers), the signal from the EvaGreen dye is modified, for example, with type B of DNA. It was found that the nucleic acid chain can behave differently from the A type. For example, in type B of a double-stranded nucleic acid strand (usually assumed in the classical DNA section), it is presumed to have a morphology similar to type A (eg, due to some 2'-O-Me modifications of nucleotides). Higher signal intensity than double-stranded nucleobases having the same sequence of nucleobases that can be observed was observed. This observation was taken into account when using insert dyes.

If necessary, the reaction was analyzed by capillary electrophoresis and the length of the formed fragments was compared to the standard. As a preparation for capillary electrophoresis, buffer the reaction mixture (Tris-HCl, 20 mmol / l, pH 8.0, and EDTA, 20 mmol / l, to the extent that the concentration of the labeled nucleic acid is about 20 nmol / l. , PH 8.0). Capillary electrophoresis was performed by GATC-Biotech (Constance, Germany) as a contract service. According to this supplier, capillary electrophoresis was performed on the ABI 3730 capillary sequencer at about 50 ° C. and constant voltage (about 10 kV) under standard conditions for sanger sequencing using a POP7 gel matrix. The conditions used resulted in double-stranded denaturation, which resulted in the single-stranded form being separated from the nucleic acid strand by capillary electrophoresis. Electrophoresis is a standard technique in gene analysis. Today, automated capillary electrophoresis is commonly used for sanger sequencing. Fluorescent signals are continuously recorded during capillary electrophoresis (usually using a virtual filter), resulting in an electrophoresis map in which signal intensity correlates with the duration of electrophoresis. For short fragments, eg, unused primers, earlier signal peaks are observed, and for longer fragments, there is a signal time shift proportional to the length of the elongated segment. The length of the elongated fragment can be measured using a control of known length. This technique is known to those of skill in the art and is also used as a standard for fragment length polymorphisms.

Unless otherwise defined for a particular sequence, both uppercase and lowercase agct and AGCT indicate a deoxyribonucleotide building block of DNA, or a corresponding ribonucleotide or base analog. In some examples, the uracil nucleobase was used in the modified sequence segment, thereby using the 2'-OMe modification as the sugar (ribose with 2'-O-methyl modification). Other 2'-O-alkyl modifications are also possible. Batches with dUTP (particularly the first amplification reaction) produce amplified fragments containing dUMP (as protection against contamination). However, in general, the uracil base can be used with 2'-deoxyribose as well as ribose. Those skilled in the art will be associated with each sequence segment of the sequences used herein from new facts to determine at what point a classical DNA backbone, RNA, or base analog can be effectively applied. Apply as an overall teaching.

Example 1 (FIG. 36)
Use of Human Genome DNA as a Source for Target Sequences This example demonstrates the use of human genomic DNA (hgDNA) as a source for target sequences. The sequence segment of the factor V Leiden gene (Homo sapiens coagulation factor V (F5), mRNA, hereinafter referred to as the FVL gene) was selected as the target sequence.

Only the first amplification was performed. This example shows that a controller-dependent first amplification can be performed. In this example, the second amplification was not performed.

Target sequence:

The binding sequence of the first oligonucleotide primer is underlined. The second oligonucleotide primer binds to the inverse complementary sequence of the double underlined sequence at its 3'segment.

A first primer, a second primer, and a controller oligonucleotide were designed and synthesized for variants of the FVL mutation in the gene.

First oligonucleotide primer: P1F5-200-AE2053

The segments used as primers in the reaction are underlined.

A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

This oligonucleotide contains the following modifications:
The synthesis of the second primer extension product was stopped using the 1 = C3 linker.

The primer [CUCU GAUGCUC] segment in square brackets contained a 2'-O-Me modification and served as a second primer region that bound to the first region of the controller oligonucleotide.
A = (2'-O-methyladenosine); G = (2'-O-methylguanosine); C = (2'-O-methylcytosine); U = (2'-O-methyluridine)

This oligonucleotide primer contains the first region (positions 1-12 from the 3'end), the second region (C3 linker, and positions 13-24 from the 3'end), and additional sequence variant P1. Includes segments (positions 25-57 from the 3'end). The first region and the second region are necessary for performing specific amplification and can be outlined as "basic structure of first primer" or "minimum structure of first primer". The additional sequence variant P1 provides an example of additional segments that can be incorporated into the first oligonucleotide primer. Positions 1-12 serve as templates for the synthesis of the second primer extension product. The C3 modification and the second region prevent the continuation of synthesis at positions 25-57 during the synthesis of the second primer extension product.

Primer 2: P2G3-5270-7063

The segments used as primers in the reaction are underlined.

This oligonucleotide contains the following modifications:
6 = HEG linker A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

This oligonucleotide primer contains a copyable region and a non-copyable region. The copyable regions are (positions 1 to 13 from the 3'end that can complementarily bind to the sequence of the FVL gene in hgDNA and the first primer that does not complementarily bind to the sequence of the FVL gene, but during amplification. 14-35 positions) that can bind to the elongation product). The copyable region can be summarized as the "basic structure of the second primer" or the "minimum structure of the second primer".

The non-copyable region (positions 36-70 from the 3'end that does not complementarily bind to the FVL gene sequence) is separated from the copyable region by HEG modification, thereby synthesizing the first primer extension product. Prevents continuation of synthesis at positions 36-70 of. The non-copyable segment is an example of an additional sequence variant P2 that can be incorporated into a second oligonucleotide primer.

Ａ＝２’−デオキシアデノシン；Ｃ＝２’−デオキシシトシン；Ｇ＝２’−デオキシグアノシン；Ｔ＝２’−デオキシチミジン（チミジン） The following controller oligonucleotides were used: AD-F5-1001-503

A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

The 5'segment of oligonucleotides in square brackets [UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC] contained a 2'-O-Me nucleotide modification:
Modifications: A = (2'-O-methyladenosine); G = (2'-O-methylguanosine); C = (2'-O-methylcytosine); U = (2'-O-methyluridine)
X = 3'-phosphate group that blocks possible elongation by the polymerase.

Nucleotides and nucleotide modifications are linked to each other by phosphodiester bonds. The 3'end of the controller oligonucleotide is blocked with a phosphate group to prevent possible elongation by the polymerase.

The first primer contains, in its first region, a sequence capable of specifically binding to the sequence of the Factor V Leiden gene in genomic DNA, so that synthesis can be initiated by the polymerase. The second region of the first primer contains a sequence that does not specifically hybridize to the sequence of the FVL gene. In addition, the first primer contains an additional sequence segment linked to the 5'end of the second region (additional sequence variant P1). The segment is not involved in the specific amplification of the Factor V Leiden segment. The function of the segment is mainly seen in the delay of side reactions.

The second primer contains in its 3'segment a segment that can specifically bind to genomic DNA and, as a result, initiate synthesis by the polymerase. The 5'segment of the second primer contains a sequence that does not specifically hybridize to the sequence of the FVL gene. You can copy this sequence segment during the back synthesis. The second primer contains a controller oligonucleotide, a first primer or an additional sequence segment that cannot specifically hybridize to the second primer. The segment is located at the 5'end of the second primer and is separated from the 5'end of the primer by a HEG linker (additional sequence variant P2). The segment is not involved in specific amplification. The function of the segment is mainly due to the delay of side reactions.

The controller oligonucleotide is constructed to exactly match the sequence of the Factor V Leiden mutation resulting in the FVL gene. The controller oligonucleotide contains the first, second and third regions.

The WHO standard for FVL mutations was used as genomic DNA. Prior to use in the reaction, the DNA was denatured by heating (95 ° C. for 5 minutes) to transfer from the double-stranded state to the single-stranded state. Using this single-stranded hgDNA, the starting nucleic acid strand was first created by primer extension. Subsequently, exponential amplification was performed starting from this starting nucleic acid strand. The specificity of amplification was determined by melting curve analysis and sanger sequencing with sequencing primers.

All reactions were carried out in amplified solution 1.
The dNTPs used included dATP, dGTP, dCTP, dUTP (instead of dTTP).
Bst2.0 warm start polymerase manufactured by NEB was used as a polymerase.

The starting nucleic acid strand was created as follows:
Approximately 50,000 haploid genomic equivalents (HGE), 150 ng hg DNA, second primer (0.5 μmol / l) and Bst-2.0 warm start polymerase (approximately 1 unit), and dNTP (approximately 250 μmol / l). ) And in hybridization conditions (amplified solution 1, temperature about 60 ° C.), contacted in a reaction volume of 50 μl and incubated for about 10 minutes. During this stage, the second primer is extended and the genomic DNA acts as a template. The result is a primer extension product that can be used as the starting nucleic acid. After completion of this reaction, the reaction mixture was heated to 95 ° C. for about 10 minutes to separate the starting nucleic acid chain. The reaction mixture was frozen and used as a source of starting nucleic acid chains as needed.

Specific amplification of the target sequence of the FVL gene was performed using 5 μl of the reaction mixture with the starting nucleic acid strand (corresponding to about 5000 HGE).

Other reaction components (first primer, second primer, controller oligonucleotide, Eva-Green dye, polymerase Bst. 2.0 warm start, dNTP) were added to obtain a reaction termination volume of about 10 μl. The final concentrations of the components are as follows: first primer: 5 μmol / l, second primer: 2 μmol / l, controller oligonucleotide: 1 μmol / l, Eva-Green dye (1:50), polymerase Bst. 2.0 warm start (about 8 units), dNTP: about 250 μmol / l.

No hgDNA was added to the control batch.

The reaction was performed on a Step-One Plus device (Thermorphisher Scientific).

The reaction temperature was initially changed by periodic changes (30 cycles) between 65 ° C. (5 minutes, including detection steps) and 55 ° C. (1 minute), then at 65 ° C. for 1 hour (2 minutes). (Detection step for each) It was kept constant. After the reaction process, signal detection of EvaGreen dye was performed. After completion of the reaction, the reaction mixture was first brought to 95 ° C. for 10 minutes and then the melting curve of the formed product was measured.

First, the starting nucleic acid strand was established (FIG. 50), followed by exponential amplification using controller oligonucleotides by extending the first and second primers.

The result of amplification is the deposition of amplified fragments. The amplification detection result is shown in FIG. It can be seen that the visible increase in fluorescence occurs about 2 hours after the reaction (36A). Subsequent melting curve analysis showed a specific melting curve with a Tm of 84 ° C. (FIG. 36B).

Sequence check (Fig. 36C):
Sequence validation was performed using sequencing primers:
5'-AAG CTCGACAAAAAGAAC TCAG AG TGTG CTCGAC AC tacctgtattcc ttgcc 3'(SEQ ID NO: 005)
A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

To verify the sequence, the reaction mixture (after measuring the melting curve) was diluted with water (about 1: 10 to about 1: 100) and the resulting aliquot was mixed with sequencing primers (concentration of 2 μmol / l). Add in). The mixture was shipped by a commercial sequencing company (GATC-Biotec) and was sequenced using Sanger sequencing as a contracted sequencing method. The resulting electrophoretic map was checked for matching with the FVL sequence gene. As a result of the reaction, the sequence of the FVL gene was identified.

Example 2 (FIG. 37):
Selective amplification reaction of sequence variants of the target sequence.
In this example, the effect of sequence changes on the amplification in the template was investigated. When the first oligonucleotide primer is extended, it has sequences complementary to the template and thus forms complementary strands containing these sequence mutations. The purpose was to evaluate the effect of such discrepancies between the resulting first primer extension product and the controller oligonucleotide on amplification. The location of the discrepancy is in the 3'direction of the first primer, which is assessed by the controller's oligonucleotide rather than the primer.

Therefore, discrimination between individual sequence variants of the target sequence is performed by controller oligonucleotides that use uniform first and second primers.

Only the first amplification was performed. This example shows that the discrimination between two target sequence variants can be used to perform a controller-dependent first amplification. In this example, no second amplification was performed.

Ａ＝２’−デオキシアデノシン；Ｃ＝２’−デオキシシトシン；Ｇ＝２’−デオキシグアノシン；Ｔ＝２’−デオキシチミジン（チミジン） The following mold was used:
Template with sequence composition that yields a first primer extension product that perfectly matches the controller oligonucleotide: M2SF5-M001-200

A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

The binding sequence of the first oligonucleotide primer is underlined. The second oligonucleotide primer binds to the inverse complement of the double underlined sequence (35 positions at the 5'end).

Ａ＝２’−デオキシアデノシン；Ｃ＝２’−デオキシシトシン；Ｇ＝２’−デオキシグアノシン；Ｔ＝２’−デオキシチミジン（チミジン） A template having a sequence composition that results in a first primer extension product that forms a mismatch with the controller oligonucleotide at a single base position (bold):

A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

The binding sequence of the first oligonucleotide primer is underlined. The second oligonucleotide primer binds to the inverse complement of the double underlined sequence.

The following mold was used.
First oligonucleotide primer:
P1F5-200-AE2053

The segments used as primers in the reaction are underlined.
A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

This oligonucleotide contains the following modifications:
The synthesis of the second primer extension product was stopped using the 1 = C3 linker.
The primer segment [CUCU GAUGCUC] contained a 2'-O-Me modification and served as a second primer region that binds to the first region of the controller oligonucleotide.
A = (2'-O-methyladenosine); G = (2'-O-methylguanosine); C = (2'-O-methylcytosine); U = (2'-O-methyluridine)

Primer 2: P2G3-5270-7063

The segments used as primers in the reaction are underlined.
This oligonucleotide contains the following modifications:
6 = HEG linker A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

Ａ＝２’−デオキシ−アデノシン；Ｃ＝２’−デオキシ−シトシン；Ｇ＝２’−デオキシ−グアノシン；Ｔ＝２’−デオキシ−チミジン（チミジン）
角括弧のオリゴヌクレオチドの５’セグメント［ＵＡＡＵＣＵＧＵＡＡ ＧＡＧＣＡＧＡＵＣＣ ＣＵＧＧＡＣＡＧＧＣ ＡＡ ＧＧＡＡＵＡＣ］は、２’Ｏ−Ｍｅヌクレオチド修飾を含んだ： The following controller oligonucleotides were used:
AD-F5-1001-503

A = 2'-deoxy-adenosin; C = 2'-deoxy-cytosine; G = 2'-deoxy-guanosine; T = 2'-deoxy-thymidine (thymidine)
The 5'segment of oligonucleotides in square brackets [UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC] contained a 2'O-Me nucleotide modification:

Qualification:
A = (2'-O-methyladenosine); G = (2'-O-methylguanosine); C = (2'-O-methylcytosine); U = (2'-O-methyluridine)
X = 3'-phosphate group that blocks possible elongation by the polymerase.

Nucleotides and nucleotide modifications are linked to each other by phosphodiester bonds. The 3'end of the controller oligonucleotide is blocked with a phosphate group to prevent possible elongation by the polymerase.

4 batches were prepared:
Batch 1, mold M2SF5-M001-200 as the starting nucleic acid strand (exact match situation) is at a concentration of 300 fmol / l (about 2x10 corresponding to ⁶ copies / batch).

Batch 2, mold M2SF5-M001-200 as the starting nucleic acid strand (the status of exact) contains a concentration of 300Amol / l (corresponding to about 2x10 ³ copies / Batch).

Batch 3 forms a control because it does not contain a template.

Batch 4 is started as a nucleic acid strand template M2SF5-WT01-200 (single mismatch situation) is at a concentration of 300 pmol / l (approximately 2x10 ⁹ copies per batch).

Primer 1 was used at 5 μmol / l, controller oligonucleotide was used at 2 μmol / l, and primer 2 was used at 1 μmol / l.

Further reaction conditions are as follows: Amplified solution 2.

To mimic the presence of genomic DNA in the assay, 100 ng of freshly denatured fish DNA (salmon DNA) was added per reaction.

The thermal reaction condition was a cyclically changing temperature change, with a 2-minute interval at 55 ° C followed by a 5-minute interval at 65 ° C. Amplification was monitored over 100 cycles. The detection of the EvaGreen fluorescent signal was performed at 65 ° C.

Successful amplification was measured by an increase in EvaGreen fluorescence signal over time.

Temperature changes and real-time monitoring were performed using Thermo Fisher's StepPne Plus real-time PCR device.

FIG. 37 shows a typical curve of the EvaGreen signal. The Y-axis shows the increase in fluorescence signal (Delta Rn), and the X-axis shows the reaction time (as the number of cycles). Arrows indicate individual reaction preparations. The marked positions belong to the following batch:

Arrow 1: 300 fmol / l of exact match template (approximately 2x10 ⁶ copies / Batch)

Arrow 2: 300 fmol / l exact match template (approx. 2x10 ³ copies / batch)

Arrow 3: No mold

Arrow 4: 300 pmol / l mismatch template (approximately 2x10 ⁹ copies per batch) of.

An increase in the fluorescence signal can be confirmed in both the exact match and the mismatched mutations of the template, and the signal of the mismatched mutation (4) appears later, despite a 100-fold excess. It can be seen that the mismatched amplification signal is significantly delayed compared to the exact match amplification signal. A delay of about 15 cycles is observed for a single discrepancy. The time delay (= number of cycles) is an immediate measurement of identification in amplification. Further quantification of discrimination in amplification can be obtained by comparing with the template concentration series under exact coincidence amplification.

When using an exact match template, complementary strands of primer extension products are synthesized. This extension is complementary to both the exact match template and the controller oligonucleotide used.

In contrast, when a mismatched sequence is used, the synthesis of the first segment of the primer extension product results in the production of a complementary strand of the extension product that is completely complementary to the mismatched template, thereby causing the oligonucleotide controller. It differs from the complementarity with the third region. This difference occurs in the 5'-chain segment of the extension product, which should allow the chain substitution process to proceed by reacting with the controller oligonucleotide. As shown in this example, the mismatch interferes with the strand substitution by the controller oligonucleotide.

Controlled reactions using the exact match template (arrows 1 and 2) showed concentration dependence of amplification. As the concentration decreased, the reaction time was longer for the synthesis to be sufficient to amplify the nucleic acid at which the signal was amplified above baseline levels.

This result indicates the importance of the base composition of the controller oligonucleotide: the difference from complementarity between the controller oligonucleotide and the primer extension product can slow or even interrupt amplification.

In this example, the sequence ends of the exact and mismatched templates were exactly the same, so the likelihood of binding of both oligonucleotide primers was the same, but both reactions were completely different. It was found: If there was perfect complementarity between the controller oligonucleotide and the 5'segment of the extension product of the first oligonucleotide primer, the amplification was as planned. Interruption of strand substitution due to sequence differences (in this case due to mismatch) resulted in suppression of amplification.

Example 3 (FIGS. 38-42):
Use of Two Amplifications: First Amplification, Subsequent Second Amplification In this example, the amplification product of the first amplification (first amplification) is followed by classical PCR (second amplification). It is shown that it can be used as a starting nucleic acid chain. Therefore, a total of two separate amplification reactions were performed: first amplification first, then second amplification.

First Amplification First, the first amplification is performed using the single-stranded nucleic acid strand containing the target sequence (here, as the single-stranded starting nucleic acid strand 1.1) using the first amplification system. I went. In the reaction conditions used (temperatures 55 ° C and 65 ° C), the double strands formed during the reaction spontaneously separate, including the first primer extension product and the second primer extension product in the absence of the controller oligonucleotide. Is not allowed to occur.

The first amplification system contained the following components:
-First oligonucleotide primer,
-Second oligonucleotide primer,
・ Controller oligonucleotide and ・ Polymerase with strand substitution properties (BST2.0 warm start polymerase),
-And the substrates required for the polymerase (dNTPs: dATP, dCTP, dGTP, dTTP) and the appropriate buffer system (reaction was performed with isothermal buffer 1x (NEB)).

Reaction progression was detected by EvaGreen at 65 ° C.

In the concentration ratio of the first oligonucleotide primer (5 μmol / l) to the controller oligonucleotide (2 μmol / l) used, the first primer is in excess relative to the controller oligonucleotide. The melting temperature of the complex containing the first oligonucleotide primer and the controller oligonucleotide was about 63 ° C. (Tm) and was measured with the same reaction solution.

Two temperature ranges were used in the first amplification reaction:
55 ° C (2 minutes) and 65 ° C (2 minutes). At the 55 ° C. temperature step, the controller oligonucleotide was fully complexed with the first oligonucleotide primer. This prevented the controller oligonucleotide from binding to the first primer extension product. In the second temperature step (65 ° C.), the complex was capable of at least partially dissociating from the complex, resulting in the availability of free single-stranded controller oligonucleotides in batches. Such free controller oligonucleotides can either form a complex with a new first oligonucleotide primer or anneal to a first primer extension product. Contact with the first primer extension product begins with the complementary binding of the sequence segment of the first region of the controller oligonucleotide to the corresponding sequence segment of the second region of the primer, which is not copied by the polymerase.

The individual synthetic and chain separation methods essentially consist of the following processes:
The first oligonucleotide primer is primarily capable of specifically binding to the 3'segment of the provided nucleic acid strand (starting nucleic acid strand 1.1) and can be extended by the polymerase (this reaction is predominantly carried out at 55 ° C.). .. Synthesis proceeds to the end of the template strand (5'region of the starting nucleic acid strand). This resulted in a first primer extension product. The controller oligonucleotide can bind to the first primer extension product by complementary base pairing and replace the template strand (here the first starting nucleic acid strand) by complementary base pairing (this reaction is predominantly 65). Performed at ° C). At 65 ° C., the 3'segment of the first primer extension product (not bound by the controller) was separated from the complex with the template strand mainly due to temperature (Tm of the 3'segment was 65 ° C. Was less than). Therefore, the 3'segment of the first primer extension product became single strand. Therefore, the first primer extension product was present in combination with the controller oligonucleotide.

The second oligonucleotide primer was thus capable of binding to said segment of the first primer extension product and initiated the synthesis of the second primer extension product by the polymerase (this step was 55 ° C. and 65 ° C.). Can be run at ° C). At the same time as the synthesis was performed, the controller oligonucleotide was co-substituted from binding to the first primer extension product. The polymerase used in the first primer extension reaction (Bst2.0 warm start) has chain substitution properties. Synthesis of the second primer extension product was carried out up to the first region of the first primer copied by the polymerase and including the first region. Synthesis of the second primer extension product was stopped by the C3 linker of the first primer extension product, so that the second region of the first primer extension product was not copied by the polymerase. As a result, a second primer extension product forming a double strand with the first primer extension product was obtained (Tm of this double strand = the first amplification product was about 79 ° C.). Repeated heating of the reaction mixture to 65 ° C. resulted in the recombination of the controller oligonucleotide to the first primer extension product, thereby releasing the second primer extension product from binding to the first primer extension product. ..

As a result of the release of the second primer extension product, this product can more function as a template, the second primer extension product complementing the first region of the first primer in the 3'segment. Sequences, thus new oligonucleotide primers can be attached and synthesis can be initiated by the polymerase.

As a result, repeated synthesis and chain separation processes were performed, allowing both synthesized primer extension products to serve as templates in subsequent cycles.

The first amplification product could be synthesized starting from the starting nucleic acid strand (1.1) using the provided primers and with the help of the provided controller oligonucleotide until the desired copy number was achieved. (Here, the concentration of the first amplification product 1.1 is about 0.8-1 μmol / l towards the end of the first amplification, which is about 1,000,000 copies in a 10 μl reaction batch. Corresponds to). The number of copies was estimated from the concentration for illustration.

The reaction was stopped by heating to 95 ° C. for 10 minutes to denature the first polymerase. Next, a short fusion analysis was performed to confirm specific amplification. The reaction batch was frozen and used in a second amplification as needed.

Second amplification (PCR)
After completion of the first amplification, a portion of the first reaction batch was added to the second amplification. The first amplified fragment (1.1) synthesized during the first amplification served as the second starting nucleic acid chain (2.1) of the second amplification.

Several dilution steps of the first completed batch were added to the second amplification reaction to give one dilution series.

It should be noted that the first amplified fragment (1.1) formed was not purified, only diluted. Therefore, all unused components of the first amplification system, albeit in diluted form, were also present in the second amplification step.

Standard PCR (second amplification) was performed using PCR reaction conditions (three temperature ranges of 55 ° C. (1 minute) and 72 ° C. (3 minutes) and 95 ° C. (20 seconds)).

The second amplification system contains the following components, respectively:
-Third oligonucleotide primer,
・ Fourth oligonucleotide primer,
-Polymerase that is a thermostable polymerase (Taq polymerase)
And substrates for polymerases (dNTPs: dATP, dCTP, dGTP, dTTP) and suitable buffer systems.

In the PCR, the general concentrations of the individual components were used: PCR primers with a final concentration of 0.5 μmol / l each, and Taq polymerase at a concentration (1,100 dilution, equivalent to about 1 unit / reaction) and a concentration of about. 200 μmol / l dNTP (A, G, C, T). This reaction was performed in isothermal buffer 1x (NEB).

The second amplification was performed using this first amplification fragment as the starting nucleic acid chain (2.1) and using the second amplification system. The progress of the synthesis was detected by EvaGreen at 72 ° C. NTC and the starting nucleic acid chain 1.1, which can also be used as templates in the second amplification system, were used as control reactions.

The number of cycles required for the second amplification until the specific signal increased was measured and compared to the reference value. A comparison of the number of cycles required for detectable amplification concluded the successful use of the first amplified fragment (1.1) in PCR.

For more information, I did the following:
The first amplification product of the first amplification reaction based on the controller oligonucleotide was diluted and amplified as a template (input) under common PCR conditions. In this example, subsequent PCR detections are performed using the insert dye EvaGreen. By comparing with a number of different control approaches, we have shown that subsequent PCR signals are generated only if the controller oligonucleotide-based amplification reaction is successful. For this reason, the subsequent conventional PCR is also referred to as detection PCR.

The amplification product of the amplification reaction based on the controller oligonucleotide was essentially produced as described in Example 2.

反応でプライマーとして使用されるセグメントには下線が引かれている。
Ａ＝２’−デオキシアデノシン；Ｃ＝２’−デオキシシトシン；Ｇ＝２’−デオキシグアノシン；Ｔ＝２’−デオキシチミジン（チミジン）
このオリゴヌクレオチドは以下の修飾を含む：
１＝Ｃ３リンカーを使用して、第２のプライマー伸長産物の合成を停止した。
２＝２’−デオキシイノシン The following oligonucleotide primers were used:
First oligonucleotide primer: P1F5-200-AE205

The segments used as primers in the reaction are underlined.
A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)
This oligonucleotide contains the following modifications:
The synthesis of the second primer extension product was stopped using the 1 = C3 linker.
2 = 2'-deoxyinosine

反応でプライマーとして使用されるセグメントには下線が引かれている。
このオリゴヌクレオチドは以下の修飾を含む：
６＝ＨＥＧリンカー
Ａ＝２’−デオキシアデノシン；Ｃ＝２’−デオキシシトシン；Ｇ＝２’−デオキシグアノシン；Ｔ＝２’−デオキシチミジン（チミジン）
以下のコントローラーオリゴヌクレオチド（配列番号４）を使用した：
ＡＤ−Ｆ５−１００１−５０３

Ａ＝２’−デオキシアデノシン；Ｃ＝２’−デオキシシトシン；Ｇ＝２’−デオキシグアノシン；Ｔ＝２’−デオキシチミジン（チミジン） The primer segment [CUCU GAUGCUC] contained a 2'-O-Me modification and served as a second primer region that binds to the first region of the controller oligonucleotide.
A = (2'-O-methyladenosine); G = (2'-O-methylguanosine); C = (2'-O-methylcytosine); U = (2'-O-methyluridine)
Second oligonucleotide primer: P2G3-5270-7063

The segments used as primers in the reaction are underlined.
This oligonucleotide contains the following modifications:
6 = HEG linker A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)
The following controller oligonucleotide (SEQ ID NO: 4) was used:
AD-F5-1001-503

A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

The 5'segment of oligonucleotides in square brackets [UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC] contained a 2'-O-Me nucleotide modification:
A = (2'-O-methyladenosine); G = (2'-O-methylguanosine); C = (2'-O-methylcytosine); U = (2'-O-methyluridine)
X = 3'-phosphate group that blocks possible elongation by polymerase

Nucleotides and nucleotide modifications are linked to each other by phosphodiester bonds. The 3'end of the controller oligonucleotide is blocked with a phosphate group to prevent possible elongation by the polymerase.

Starting nucleic acid strand (1.1) with exact match with the controller.
M2SF5-M001-200
5'GCT CATA CTACAATGTCA CT TA CTGTAA GAGCAGATCC CTGGACAGGC AA GGAATACAGGTA AAAAA 3'(SEQ ID NO: 006)
The starting nucleic acid chain was used at a concentration of about 1 pM.

As a result, the amplified fragment can be expected to produce a second primer extension product with the following sequence:
5'GCT CATA CTACAATGTCA CT TA CTGTAA GAGCAGATCC CTGGACAGGC AA GGAATACAGGTA 3'(SEQ ID NO: 10)
(The sequence segment of the second primer that cannot be copied is not shown here)

PCR was performed with three different primer pairs. This primer covers different sequence regions of the amplification product. For better understanding, each primer pair is also marked on the primer binding site of the template chain (= amplification product).

What was used for PCR:
PCR primer pair 1: consisting of a third oligonucleotide primer SeqP1F5 300-35x (SEQ ID NO: 8) and a fourth oligonucleotide primer P2-4401-601 TMR (SEQ ID NO: 9).

Third oligonucleotide primer SeqP1F5 300-35x
5'ACGAAGCTCGCAAGACACTCAGAGTGTGCTGACTACACTGTATTCC 3'(SEQ ID NO: 8)
Fourth Oligonucleotide Primer P2-4401-601
TMR 5'GCTCATACTACAAATGTCACTTAC 3'(SEQ ID NO: 9)
It has A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine) :.
TMR = tetramethylrhodamine (P2-4401-601 TMR) connected to the 5'end of this chain

The two primers bind to the template strand in the following manner (here, shown as the second primer extension product of the first amplification):

In a regulatory approach, the primers of this primer pair can also bind to the starting nucleic acid chain 1.1 as follows:
M2SF5-M001-200 (SEQ ID NO: 006):

The binding sequence of the first oligonucleotide primer is underlined.

The second oligonucleotide primer binds to the inverse complement of the double underlined sequence.

PCR primer pair 2: consisting of third oligonucleotide primer P3F5D-600-203 (SEQ ID NO: XY) and fourth oligonucleotide primer P2-4401-601 TMR:

Third oligonucleotide primer:
P3F5D-600-203
-5'TMR GTACCGAAGC TCGCAGGAAC TCAGAGTGTG GAGAGGACGA AAATGTCCAG GGA 3'(SEQ ID NO: 11)

Fourth Oligonucleotide Primer P2-4401-601
TMR TMR-5'GCTCATACTACAAATGTCACTTAC 3'(SEQ ID NO: 9)
It has A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine) :.

Qualification:
TMR = tetramethylrhodamine connected to the 5'end of the chain (P3F5D-600-203) and P2-4401-601 TMR

The two primers bind to the template strand in the following manner (here, shown as the second primer extension product of the first amplification):

In a controlled batch, the primers of this primer pair can also bind to the starting nucleic acid chain 1.1 as follows:

第１のオリゴヌクレオチドプライマーの結合配列には下線が引かれている。
第２のオリゴヌクレオチドプライマーは、二重下線付きの配列の逆相補に結合する。 M2SF5-M001-200

The binding sequence of the first oligonucleotide primer is underlined.
The second oligonucleotide primer binds to the inverse complement of the double underlined sequence.

PCR Primer Pair 3: Consisting of Third Oligonucleotide Primer SeqP1F5-35-X02 and Fourth Oligonucleotide Primer P2-4401-601 TMR:
Third oligonucleotide primer SeqP1F5-35-X02
5'AAGCTCGACAAAAAGAACTCAGAGTGTGTCCGACACTACCTGTATTTCCTTG 3'(SEQ ID NO: 012)

Fourth Oligonucleotide Primer P2-4401-601 TMR
TMR-5'GCTCATACTACAAATGTCACTTAC 3'(SEQ ID NO: 009)
It has A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine) :.
TMR = tetramethylrhodamine connected to the 5'end of this chain (P2-4401-601 TMR)

The two primers bind to the template strand in the following manner (here, shown as the second primer extension product of the first amplification):

第１のオリゴヌクレオチドプライマーの結合配列には下線が引かれている。
第２のオリゴヌクレオチドプライマーは、二重下線付きの配列の逆相補に結合する。 In a controlled batch, the primers of this primer pair can also bind to the starting nucleic acid chain 1.1 as follows:
M2SF5-M001-200:

The binding sequence of the first oligonucleotide primer is underlined.
The second oligonucleotide primer binds to the inverse complement of the double underlined sequence.

Nucleotides and nucleotide modifications are linked to each other by phosphodiester bonds.

Taq polymerase (NEB, catalog number M0273S) was used in PCR. A 1: 100 polymerase dilution was used for each reaction.

Amplification products from the controller oligonucleotide-based amplification reaction (first amplification) were used for PCR at a dilution of 1: 5,000 to 1: 500,000,000.

As a control, a reaction mixture containing no template for amplification was also investigated (NTC = untemplated control = negative control).

In addition, a further regulatory approach was used to determine if the controller oligonucleotide functions as a template for PCR primers. To this end, the controller oligonucleotide (SEQ ID NO: 4 = referred to herein as "controller 503") previously used during controller oligonucleotide-based amplification was used by PCR at concentrations of 1 μmol / l to 1 pmol / l. Provided for amplification of.

In a further control, a defined amount of template M2SF5-M001-200 was provided for amplification in PCR (positive control). The control demonstrated the function of the PCR-primer pair (under ideal conditions). In addition, samples could be quantified after the first amplification by comparing the received Ct values with the standard dilution series of positive controls. The template M2SF5-M001-200 was tested at a concentration of 1 nmol / l to 1 fmol / l.

The third and fourth oligonucleotide primers were used at a concentration of 0.5 μmol / l, respectively.

Further reaction conditions are as follows:
1x Isothermal Buffer (New England Biolabs); At a single concentration, the buffer contains: 20 mM Tris-HCl; 10 mM (NH4) 2SO4; 50 mM KCl; 2 mM sulfonyl4; 0.1% Tween® 20; pH 8.8 at 25 ° C.); dNTPs (dATP, dCTP, dUTP, dGTP), 200 μmol / l each;
EvaGreen dye (dye used at 1:50 dilution according to manufacturer's instructions)

The thermal reaction conditions were selected as follows:
20 seconds 95 ° C, activate the system 30 cycles, each with the following temperature-time profile o 1 minute 55 ° C
o 3 minutes 72 ° C
o 20 seconds 95 ° C
・ 72 ° C for 10 minutes for standardization of amplification products
・ Melting curve analysis

Amplification was typically monitored over 30 cycles, ie 30x (1 minute 55 ° C. + 3 minutes 72 ° C. + 20 seconds 95 ° C.) = about 30x260 seconds = 130 minutes. Successful amplification was measured by an increase in EvaGreen fluorescence signal over time.

Analysis of Detected PCR The following techniques were used to analyze the detected PCR and evaluate the resulting amplification products.
-Fluorescent signal of the inserted dye (EvaGreen) -Melting curve analysis of the obtained amplification product When analyzing PCR, only the data obtained by diluting Taq polymerase at 1: 100 will be described.

Control experiments with the template (M2SF5-M001-200) confirmed that all three selected PCR primer pairs were able to produce specific PCR products under the PCR conditions used. It was also confirmed that all three primer pairs failed to form a product starting with the controller oligonucleotide. This was the expected result as all the "third" primers used bind to the modified portion of the controller oligonucleotide.

FIG. 38A shows the typical time course of the EvaGreen fluorescent signal obtained during amplification of a primer-to-one diluted controller oligonucleotide amplification approach. The Y-axis shows the fluorescence signal of the EvaGreen dye and the X-axis shows the reaction time (as the number of cycles). Arrows indicate different amplification approaches. Arrows indicate the next batch:

Arrow 6 indicates a negative control batch that does not generate an amplification signal during the experiment because it does not contain a template.

Depending on the selected dilution of the controller oligonucleotide amplification batch, a time-delayed amplification signal is observed. For PCR primer pair one, dilutions from 1: 5,000 to 1: 50,000,000 are well degradable under selected conditions. A dilution of 1: 50,000,000 produces an amplified signal that is slightly different from the negative control (see arrow 5).

FIG. 38B shows the relevant melting curve analysis of reactions 1-5, revealing that a specific amplification product was formed (Tm = 82.7 ° C.).

FIG. 38B shows the melting curve of the primer-to-one amplification product from FIG. 38A. The Y-axis shows the derivative of the fluorescence signal with respect to temperature, and the X-axis shows the temperature. A uniform peak marked by arrow 1 was detected. The peak at position 1 whose melting point is close to 82.7 ° C belongs to the primer-to-one amplification product. Melting curve analysis shows specific amplification.

FIG. 39A shows a general time curve of the EvaGreen fluorescence signal obtained from amplification of the diluted controller oligonucleotide amplification approach with PCR primer pair 2. The Y-axis is the fluorescence signal of the EvaGreen dye, and the X-axis is the reaction time (as the number of cycles). Arrows indicate different amplification approaches. Arrows indicate the next batch:

Arrow 5 indicates a negative control batch that does not generate an amplification signal during the experiment because it does not contain a template.

Depending on the selected dilution of the controller oligonucleotide amplification batch, a time-delayed amplification signal is observed. For PCR primer pair 2, a dilution of 1: 5,000 to 1: 5,000,000 is fully degradable under selected conditions. A dilution of 1: 5,000,000 produces a striking amplification signal from the negative control (see arrow 4).

FIG. 39B shows the relevant melting curve analysis for reactions 1-4, revealing the formation of specific amplification products.

FIG. 39B shows the melting curve of the PCR primer pair 2 amplification product from FIG. 39A. The Y-axis represents the derivative of the fluorescence signal with respect to temperature, and the X-axis represents temperature. A uniform peak marked by arrow 1 was detected. The peak at position 1 whose melting point is close to 81.9 ° C belongs to the PCR amplification product of primer pair 2. Melting curve analysis shows specific amplification.

FIG. 40A shows a general time curve of the EvaGreen fluorescence signal obtained from amplification of a diluted controller oligonucleotide amplification approach with PCR primer pairs 3. The Y-axis is the fluorescence signal of the EvaGreen dye, and the X-axis is the reaction time (as the number of cycles). Arrows indicate different amplification approaches. Arrows indicate the next batch:

Arrow 7 indicates a negative control batch that does not generate an amplified signal during the experiment because it does not contain a template.

A time-delayed amplification signal is observed, depending on the selected dilution of the controller oligonucleotide amplification approach. For primer pair 3, dilutions from 1: 5,000 to 1: 500,000,000 are well degradable under selected conditions. A dilution of 1: 500,000,000 produces a prominent amplified signal from the negative control (see arrows 6 and 7).

FIG. 40B shows the relevant melting curve analysis for reactions 1-6, revealing the formation of specific amplification products.

FIG. 40B shows the melting curve of the PCR primer vs. 3 amplification product from FIG. 40A. The Y-axis represents the derivative of the fluorescence signal with respect to temperature, and the X-axis represents temperature. A uniform peak marked by arrow 1 was detected. The peak at position 1 whose melting point is close to 81.2 ° C belongs to the PCR amplification product of primer pair 3. Melting curve analysis shows specific amplification.

Analysis of the fluorescent signal during the amplification process (amplification plot) and melting curve analysis at the end of the amplification showed that each of the PCR primer pairs 1-3 used the diluted amplification product obtained from the first amplification reaction as a template. Showed that it can be done.

Depending on the dilution selected, a time-delayed amplification signal is generated, which is common in dilution series. Under the PCR conditions selected here, amplification products diluted 5,000,000 times can be detected. Primer pair 3 is the most sensitive and the amplification product can still be detected at a dilution of 500,000,000 times.

FIG. 41 shows a control experiment using a template (M2SF5-M001-200) used at 1 nmol / l (arrow 1) and 10 pmol / l (arrow 2), and an NTC control (arrow 3).

FIG. 41A shows the results of PCR primer pair 1.
FIG. 41B shows the results of PCR primer pair 2.
FIG. 41C shows the results of PCR primer pair 3.

A comparison of the rise time of the fluorescent signal between the control experiment (template) and the second amplification performed from the first amplification fragment shows that the first amplification can be used as a template in the second amplification. Indicates that it caused.

FIG. 42 shows the process of the first amplification (before PCR).

An increase in signal was seen, which was interpreted as an increase in copy number in the first amplification. Arrow 1 corresponds to the batch having the starting nucleic acid chain 1.1. Arrow 2 = NTC control.

Example 4 (FIGS. 43-47):
Combination of first and second amplifications in the same batch (uniform assay)
In this example, we demonstrate how a controller oligonucleotide-based amplification reaction can be combined with subsequent classical PCR in a homogeneous assay format. Here, we use a highly selective controller oligonucleotide-based amplification reaction in the early stages of amplification and then switch to classical PCR for amplification and detection of amplification products. In contrast to Example 3 (= heterogeneous assay format), the combinations in this example are performed in a homogeneous assay format. All assay components are already available at the start of the reaction. Switching between controller oligonucleotide-based amplification and conventional PCR is done by altering temperature control during the course of the reaction.

First, the first amplification product 1.1 is amplified starting from the single strand initiation nucleic acid strand (1.1) using a 15-30 cycle controller oligonucleotide-based amplification reaction, then 30 Pre-amplification products were detected by further amplification by cycle classical PCR. In this example, the insert dye EvaGreen is used for detection.

The first amplification system contained the following components:
-First oligonucleotide primer,
-Second oligonucleotide primer,
・ Controller oligonucleotide and ・ Polymerase with strand substitution properties (BST2.0 warm start polymerase),
-And the substrates required for the polymerase (dNTPs: dATP, dCTP, dGTP, dTTP) and the appropriate buffer system (reaction was performed with isothermal buffer 1x (NEB)).

The second amplification system contained the following components:
-Third oligonucleotide primer,
・ Fourth oligonucleotide primer,
-Polymerase that is a thermostable polymerase (Taq polymerase)
-And the substrates required for the polymerase (dNTP: dATP, dCTP, dGTP, dTTP) and the appropriate buffer system.

Both amplification systems were available together before the first amplification began.

In this example, a second oligonucleotide primer was used for both amplifications. Therefore, the fourth oligonucleotide primer is the same as the second oligonucleotide primer.

The third oligonucleotide primer was changed.

In this example, the inventors presented four different primers, each of which was used individually for each batch as a "third oligonucleotide primer".

Each third oligonucleotide primer is different from the first oligonucleotide primer. Therefore, each reaction batch is used as three primers, namely a first oligonucleotide primer and a second oligonucleotide primer (as a component of the first amplification system) and a specific primer of the second amplification system. Also contains a third oligonucleotide primer. The role of the fourth primer was taken over by the second primer of the first amplification system, which is also suitable for subsequent PCR reactions. For better understanding, the primer binding sites of each primer combination are marked on the template chain (= amplification product).

The following starting nucleic acid strand (1.1) was used as the single strand template:

第１のオリゴヌクレオチドプライマーの結合配列には下線が引かれている。 A template with a sequence composition that yields a first primer extension product that exactly matches the controller oligonucleotide (SEQ ID NO: 6):
M2SF5-M001-200:

The binding sequence of the first oligonucleotide primer is underlined.

The second oligonucleotide primer binds to the inverse complement of the double underlined sequence. The binding sequence of the third oligonucleotide primer (P3F5D-600-203) is shown in bold italics.

First amplification system:
First oligonucleotide primer: P1F5-200-AE205

The segments used as primers in the reaction are underlined.
A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

This oligonucleotide contains the following modifications:
The synthesis of the second primer extension product was stopped using the 1 = C3 linker.
2 = 2'-deoxyinosine

One segment of the primer [CUCU GAUGCUC] contained a 2'-O-Me modification and served as a second primer region that binds to the first region of the controller oligonucleotide.
A = (2'-O-methyladenosine); G = (2'-O-methylguanosine); C = (2'-O-methylcytosine); U = (2'-O-methyluridine)

Second oligonucleotide primer: P2G3-5270-7063

The segments used as primers in the reaction are underlined.

This oligonucleotide contains the following modifications:
6 = HEG linker A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

Ａ＝２’−デオキシアデノシン；Ｃ＝２’−デオキシシトシン；Ｇ＝２’−デオキシグアノシン；Ｔ＝２’−デオキシチミジン（チミジン） The following controller oligonucleotides were used:
AD-F5-1001-503

A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)

5 the brackets oligonucleotide 'segment [UAAUCUGUAA GAGCAGAUCC CUGGACAGGC A A GGAAUAC] contained a 2'-O-Me nucleotide modifications:
Qualification:
A = (2'-O-methyladenosine); G = (2'-O-methylguanosine); C = (2'-O-methylcytosine); U = (2'-O-methyluridine)
X = 3'phosphate group that blocks possible elongation by polymerase

Nucleotides and nucleotide modifications are linked to each other by phosphodiester bonds. The 3'end of the controller oligonucleotide is blocked with a phosphate group to prevent possible elongation by the polymerase.

The second amplification system specifically comprises a third oligonucleotide primer (P3 primer) from the list below:
-Third oligonucleotide primer: P3F5D-600-201
TMR 5'GTACCGAAGC TCGCAGGAAC TCAGAGTGTG GAGAGGACGA AAA CTGTCCAGGATC 3'(SEQ ID NO: 14)
A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)
Qualification:
TMR (= tetramethylrhodamine) at the 5'position
-Third oligonucleotide primer: P3F5D-600-202
TMR 5'GTACCGAAGC TCGCAGGAAC TCAGAGTGTG GAGAGGACGAAAA CCAGGGAT 3'(SEQ ID NO: 15)
A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)
Qualification:
TMR (= tetramethylrhodamine) at the 5'position
-Third oligonucleotide primer P3F5D-600-203
TMR 5'GTACCGAAGCTCGCAGGGAACTCAGAGTGTGGGAGAGGACGAAAA TGTCCAGGGGA 3'(SEQ ID NO: 11):
A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)
Qualification:
TMR (= tetramethylrhodamine) at the 5'position
-Third oligonucleotide primer: P3F5D-600-204
TMR 5'GTACCGAAGCTCGCAGGGAACTCAGAGTGTGGGAGAGGACGAAAA CTGTCCAG 3'(SEQ ID NO: 16)
A = 2'-deoxyadenosine; C = 2'-deoxycytosine; G = 2'-deoxyguanosine; T = 2'-deoxythymidine (thymidine)
Qualification:
TMR (= tetramethylrhodamine) at the 5'position

Nucleotides and nucleotide modifications are linked together by phosphodiester bonds. This 3'end is blocked with a phosphate group to prevent possible elongation by the polymerase.

The starting nucleic acid chain 1.1 (template) was used at a concentration of 1 pmol / l. Primer 1 was used at 5 μmol / l, controller oligonucleotide was used at 2 μmol / l, primer 2 was used at 1 μmol / l, and primer 3 was used at 0.5 μmol / l. For amplification, two polymerases are present in batch with Taq polymerase (NEB, catalog number M0273S?) At a dilution of 1: 100, and Bst2.0 warm start polymerase (NEB, catalog number M0538S?) At a dilution of 1: 200. ) Existed.

No P3 primer was used in the control reaction. For further regulatory reactions, only BST polymerase was used, not Taq polymerase.

Further reaction conditions are as follows:
1x Isothermal Buffer (New England Biolabs); At a single concentration, the buffer contains: 20 mM Tris-HCl; 10 mM (NH4) 2SO4; 50 mM KCl; 2 mM sulfonyl4; 0.1 % Tween® 20; pH 8.8 at 25 ° C.); dNTPs (dATP, dCTP, dUTP, dGTP), 200 μmol / l each;
EvaGreen dye (dye used at 1:50 dilution according to manufacturer's instructions)
The thermal reaction conditions were selected according to the following profile:
-To activate the system at 65 ° C for 2 minutes-15 or 30 cycles at the next temperature-time profile (amplification reaction based on controller oligonucleotide)
o 2 minutes 55 ° C
o 2 minutes 65 ° C
20 seconds 95 ° C, to activate the system 30 cycles (PCR) with the following temperature-time profile
o 1 minute 55 ° C
o 3 minutes 72 ° C
o 20 seconds 95 ° C
・ 10 minutes 72 ℃, for standardization of amplification products ・ Melting curve analysis

Total amplification was typically performed over 45-60 cycles, i.e.
First amplification: (15 or 30) x (2 min 55 ° C + 2 min 65 ° C) = (15-30) x 4 min = 60-120 min Second amplification (PCR): 30 x (1 min 55 ° C + 3 min) 72 ° C. + 20 seconds 95 ° C.) = 30 x 260 seconds = 130 minutes (monitored). This corresponds to a total time of 190-250 minutes.

Successful amplification could be measured by an increase in EvaGreen fluorescence signal over time.

Analysis of the combination of amplification reaction based on controller oligonucleotide and detection PCR.
The resulting amplification products were analyzed and evaluated using the following techniques:
-Fluorescent signal of the inserted dye (EvaGreen) -Melting curve analysis of the obtained amplification product

The first partial amplification (by 15 or 30 cycles) results in enrichment of the nucleic acid strand (first amplified fragment 1.1) and can be used as a template in subsequent PCR. This means that PCR requires fewer cycles to produce a detectable amount of PCR product. By selecting the number of cycles of the two amplification steps, the amplification factor of the total amplification of the two amplification reactions can be adjusted.

By comparing this with several different control approaches, we use the first amplification until a detectable signal is generated (Ct) until a second amplification (PCR). It shows that the number of cycles can be reduced. This reduction in the number of PCR cycles occurred only if the first amplification (amplification reaction based on the controller oligonucleotide) was successful. Next, a second amplification was performed from the first amplification product to the detection. For this reason, subsequent conventional PCR is also referred to as detection PCR.

Furthermore, we have shown that it is possible to combine the first and second amplification systems in one batch, which makes it possible to perform a more uniform assay format.

The results of the individual reaction batches are shown below.
FIG. 43A shows the general course of EvaGreen fluorescence signal during combinatorial amplification of template M2SF5-M001-200 with primer P3F5D-600-201. The Y-axis shows the fluorescence signal of the EvaGreen dye and the X-axis shows the reaction time (as the number of cycles). In cycles 1-15, a controller oligonucleotide-based amplification reaction is used. Conventional PCR (detection PCR) is used from cycle 15 to cycle 45. The switching time (end of cycle 15) is marked with arrow 1.

Arrow 2 indicates an amplification approach in which the template M2SF5-M001-200 is amplified by BST and Taq polymerase starting from a starting concentration of 1 pmol / l.

Arrow 3 indicates the execution of a control containing only BST polymerase. Amplification is omitted because the heat sensitive BST polymerase is denatured after cycle 16 depending on the temperature profile of the detection PCR. Amplification products already formed from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

Arrow 4 indicates another control approach that omits the addition of primer P3F5D-600-201. Exponential amplification in detection PCR is not possible without a third primer, so amplification signals are no longer observed. Amplification products already produced from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

FIG. 43B shows the general course of EvaGreen fluorescence signal during combinatorial amplification of template M2SF5-M001-200 with primer P3F5D-600-202. The Y-axis shows the fluorescence signal of the EvaGreen dye and the X-axis shows the reaction time (as the number of cycles). In cycles 1-15, a controller oligonucleotide-based amplification reaction is used. Conventional PCR (detection PCR) is used from cycle 15 to cycle 45. The switching time (end of cycle 15) is marked with arrow 1.

Arrow 2 indicates an amplification approach in which the template M2SF5-M001-200 is amplified by BST and Taq polymerase starting from a starting concentration of 1 pmol / l.

Arrow 3 indicates the execution of a control containing only BST polymerase. Amplification is omitted because the heat sensitive BST polymerase is denatured after cycle 16 depending on the temperature profile of the detection PCR. Amplification products already formed from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

Arrow 4 indicates another control approach without the addition of primer P3F5D-600-202. Exponential amplification in detection PCR is not possible without a third primer, so amplification signals are no longer observed. Amplification products already produced from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

FIG. 44A shows the general course of EvaGreen fluorescence signal during combinatorial amplification of template M2SF5-M001-200 with primer P3F5D-600-203. The Y-axis shows the fluorescence signal of the EvaGreen dye and the X-axis shows the reaction time (as the number of cycles). In cycles 1-15, a controller oligonucleotide-based amplification reaction is used. Conventional PCR (detection PCR) is used from cycle 15 to cycle 45. The switching time (end of cycle 15) is marked with arrow 1.

Arrow 2 indicates an amplification approach in which the template M2SF5-M001-200 is amplified by BST and Taq polymerase starting from a starting concentration of 1 pmol / l.

Arrow 3 indicates the execution of a control containing only BST polymerase. Amplification is omitted because the heat sensitive BST polymerase is denatured after cycle 16 depending on the temperature profile of the detection PCR. Amplification products already formed from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

Arrow 4 indicates another control approach without the addition of primer P3F5D-600-203. Exponential amplification in detection PCR is not possible without a third primer, so amplification signals are no longer observed. Amplification products already produced from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

FIG. 44B shows the general course of EvaGreen fluorescence signal during combinatorial amplification of template M2SF5-M001-200 with primer P3F5D-600-204. The Y-axis shows the fluorescence signal of the EvaGreen dye and the X-axis shows the reaction time (as the number of cycles). In cycles 1-15, a controller oligonucleotide-based amplification reaction is used. Conventional PCR (detection PCR) is used from cycle 15 to cycle 45. The switching time (end of cycle 15) is marked with arrow 1.

Arrow 2 indicates an amplification approach in which the template M2SF5-M001-200 is amplified by BST and Taq polymerase starting from a starting concentration of 1 pmol / l.

Arrow 3 indicates the execution of a control containing only BST polymerase. Amplification is omitted because the heat sensitive BST polymerase is denatured after cycle 16 depending on the temperature profile of the detection PCR. Amplification products already formed from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

Arrow 4 indicates another control approach without the addition of primer P3F5D-600-204. Exponential amplification in detection PCR is not possible without a third primer, so amplification signals are no longer observed. Amplification products already produced from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

Successful combination of controller oligonucleotide-based amplification reaction in uniform assay format with (conventional) detection PCR with any of the four primer 3 variants (P3F5D-600-201-P3F5D-600-204) You can see that it is doing. The combination of BST and Taq polymerase is important. Equally important is the third primer, without which the amplified signal could not be detected. The variants of Primer 3 act very individually, which can be seen at various points when the amplified signal begins to stand out from baseline. The earlier the amplification signal is detected in the detection PCR, the more effective each primer 3 variant is in the detection of amplification products based on previously generated controller oligonucleotides.

It will be shown below that doubling the number of cycles of the controller oligonucleotide-based amplification reaction in the initial amplification step yields qualitatively very similar results.

FIG. 45A shows the general course of EvaGreen fluorescence signal during combinatorial amplification of template M2SF5-M001-200 with primer P3F5D-600-201. The Y-axis shows the fluorescence signal of the EvaGreen dye and the X-axis shows the reaction time (as the number of cycles). Amplification reactions based on controller oligonucleotides are used in cycles 1-30. Conventional PCR (detection PCR) is used for cycles 31 to 60. The switching time (end of cycle 30) is marked with arrow 1.

Arrow 2 indicates an amplification approach in which the template M2SF5-M001-200 is amplified by BST and Taq polymerase starting from a starting concentration of 1 pmol / l.

Arrow 3 indicates the execution of a control containing only BST polymerase. Amplification is omitted because the heat sensitive BST polymerase is denatured after cycle 16 depending on the temperature profile of the detection PCR. Amplification products already formed from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

Arrow 4 indicates another control approach that omits the addition of primer P3F5D-600-201. Exponential amplification in detection PCR is not possible without a third primer, so amplification signals are no longer observed. Amplification products already produced from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

FIG. 45B shows the general course of EvaGreen fluorescence signal during combinatorial amplification of template M2SF5-M001-200 with primer P3F5D-600-203. The Y-axis shows the fluorescence signal of the EvaGreen dye and the X-axis shows the reaction time (as the number of cycles). Amplification reactions based on controller oligonucleotides are used in cycles 1-30. Conventional PCR (detection PCR) is used for cycles 31 to 60. The switching time (end of cycle 30) is marked with arrow 1.

Arrow 2 indicates an amplification batch in which the template M2SF5-M001-200 is amplified by BST and Taq polymerase starting from a starting concentration of 1 pmol / l.

Arrow 3 indicates the execution of a control containing only BST polymerase. Amplification is omitted because the heat sensitive BST polymerase is denatured after cycle 16 depending on the temperature profile of the detection PCR. Amplification products already formed from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

Arrow 4 indicates another control approach without the addition of primer P3F5D-600-203. Exponential amplification in detection PCR is not possible without a third primer, so amplification signals are no longer observed. Amplification products already produced from the controller oligonucleotide-based amplification reaction remain below the EG detection threshold and remain hidden.

FIG. 46 shows a comparison of the time intervals between combined amplification (including first and second amplification) and PCR alone (second amplification only).
The first amplification was carried out as described above. In the second reaction, a third oligonucleotide primer (P3F5D-600-203) was used. The batch was pipetted and the reaction was carried out as described above. As shown above, controller-dependent amplification was first performed in a combination batch (15 cycles in this case) and then switched to PCR (arrow 1). Time intervals 1 and 2 were compared.

Arrow 2 indicates the course of combined amplification (both Bst and Taq polymerases were present in the batch). Prior to the initiation of the first amplification, the batch contained a starting nucleic acid chain (template) of 1 pmol / l. The increase in fluorescent signal occurs after time interval 1.

Arrow 3 indicates the course of the control batch in which Bst was omitted and as a result the first amplification reaction was "stopped" (all components of the first amplification system are in the batch, but the correct polymerase, in this case). Bst 2.0 was absent). This batch contained a starting nucleic acid chain (template) of 1 pmol / l prior to the initiation of the first amplification. The increase in fluorescent signal occurs after time interval 2. Taq polymerase can also synthesize products from the same template, but PCR alone requires more cycles to do this.

Arrow 4 shows the course of the combination batch without starting nucleic acid chain 1.1 (no template = NTC control).

Overall, increasing the number of amplified fragments 1.1 during the first amplification, which can then serve as the starting nucleic acid chain 2.1 (template) in PCR, results in the production of PCR fragments of the reaction in batch 2. It turns out to be faster overall: the number of PCR cycles required is small until a detectable amount of second amplification product (2.1) is produced. The time interval 1 is shorter than the time interval 2.

FIG. 47 shows a comparison of the time intervals between combined amplification (including first and second amplification) and PCR alone (second amplification only).
The first amplification was carried out as described above. In the second reaction, a third oligonucleotide primer (P3F5D-600-203) was used. The batch was pipetted and the reaction was carried out as described above. As shown above, controller-dependent amplification was first performed in combination batches (here 30 cycles) and then switched to PCR (arrow 1). Time intervals 3 and 4 were compared.

Arrow 2 indicates the course of combined amplification (both Bst polymerase and Taq polymerase were present in the batch). Prior to the initiation of the first amplification, the batch contained a starting nucleic acid chain (template) of 1 pmol / l. The increase in fluorescent signal occurs after time interval 3.

Arrow 3 indicates the course of the control batch in which Bst was omitted and as a result the first amplification reaction was "stopped" (all components of the first amplification system are in the batch, but the correct polymerase, in this case). Did not have Bst 2.0). This batch contained a starting nucleic acid chain (template) of 1 pmol / l prior to the initiation of the first amplification. The increase in fluorescent signal occurs after time interval 4. Taq polymerase can also synthesize products from the same template, but PCR alone requires more cycles to do this.

Arrow 4 shows the course of the combination batch without starting nucleic acid chain 1.1 (no template = NTC control).

Overall, by increasing the number of amplified fragments 1.1 during the first amplification, which can then serve as the starting nucleic acid chain 2.1 (template) in PCR, PCR fragment production wholes the reaction in batch 2. It turns out that the number of PCR cycles required is small until a detectable amount of the second amplification product (2.1) is produced. The time interval 3 is shorter than the time interval 4.

Example 5 (FIGS. 48-49, 71): Amplification of human FVL target sequence with mutation in the presence of human genomic DNA with wild-type sequence variant This example shows two sequence variants (mutant or wild-type). ) Is included, and the target sequence (coagulation factor V Leiden gene, SNP mutant of FVL gene) is selectively amplified.

The components of the first amplification system (first primer, second primer, controller, polymerase) and the reaction conditions are the nucleic acids to be amplified (both primer extension products (P1.1-Ext and P2.1-Ext)). The amplified fragment 1.1) containing the FVL gene was selected so as to preferably contain a sequence variant having a mutation in the FVL gene. Wild-type sequence variants (WT sequences) should not be significantly amplified during the first amplification operation. The first amplification was initiated from the initiating nucleic acid 1.1 obtained as a primer extension product using a single-stranded human gDNA (template containing the target sequence). Amplification was performed until the desired level of amplified fragment was achieved. The product produced by the first amplification (amplification fragment 1.1) was able to function as a template (starting nucleic acid 2.1) for the second amplification (PCR).

The components of the second amplification system (third primer, fourth primer, polymerase) and reaction conditions (PCR amplification) were selected so that both variants of the target sequence could be amplified. The second amplification (PCR) was performed in a separate step following the first amplification.

Schematic topography of the first amplification system and the second amplification system is shown in FIG. The second amplification system primers were "nested" with respect to the first amplification system primers.

The product obtained with the first amplification (amplified fragment 1.1) and the product obtained with the second amplification (amplified fragment 2.1) were detected and analyzed by different methods. One of the detection methods consists of real-time detection with an insertion dye (Eva-Green dye), and another detection method consists of real-time detection with a sequence-specific oligonucleotide probe (Taqman probe) (sequencing with these probes). Body-specific discrimination (mutants and wild-type mutants)), another method of analysis consisted of sanger sequencing of the amplified fragments obtained by amplification.

Implementation of sequence-specific amplification was achieved in several ways. First, amplification of the target sequence contained mutants (either by the first amplification alone or by a combination of the first and second amplifications). Second, amplification of the target sequence containing one mutant (100 or 10 copies) was achieved in the presence of approximately 30,000 copies of human gDNA with the wild-type mutant. Furthermore, it was shown that under selected conditions, measurable amplification of the target sequence by the wild-type mutant (5000 copies) did not occur.

Materials and methods:
Target sequence:
The following mutants were selected as target sequences with polymorphic loci.
FVL sequence mutant with mutation:

Wild-type sequence mutants:

Both sequences were derived based on the information available from NCBI:
Homo sapiens chromosome 1, GRCh38. p12 primary assembly, SEQ ID NO: NC_000001.11
At coagulation factor V, codon 506 with SNP1691G> A (substitution).

Ingredients in batch for preparation of starting nucleic acid 1.1

１＝Ｃ３
下線の配列は、２’−ＯＭｅ修飾ヌクレオチド（２’−Ｏ−メチルヌクレオチド）からなり、第１のプライマーの第２の領域に相当する。 Human gDNA: WHO standard 04/224 for human gDNA for the FVL gene
-Mutant carrying a mutation in human gDNA (03/260 factor V Leiden homoconjugate) (FVL mutation)
-Wild type (03/254 wild type factor V) (WT sequence) carrying a mutation in human gDNA
Oligonucleotide Primer P1F5G2-2001-203 (SEQ ID NO: 19)

1 = C3
The underlined sequence consists of 2'-OMe modified nucleotides (2'-O-methyl nucleotides) and corresponds to the second region of the first primer.

-Bst 2.0 warm-start DNA polymerase (NEB 120,000 units / ml) (used in the 1: 1000 dilution reaction) (also called "Bst polymerase")

１＝Ｃ３ First Amplification System-First Oligonucleotide Primer P1F5G2-1001-103

1 = C3

２＝ＨＥＧ
Ｘ＝３’−リン酸基 The underlined sequence consists of 2'-OMe modified nucleotides and corresponds to the second region of the first primer.
-Block oligonucleotide: B1-P1F5G2-3501-304

2 = HEG
X = 3'-phosphate group

The underlined sequence in parentheses consists of 2'-OMe modified nucleotides.
Controller oligonucleotide: CF5G2-1002-401 (SEQ ID NO: 22)
5'[Aggacuau couau cougua aga cou cou cou c cou cou cou c cou cou cou cou cou cou cou cou cou cou cou cou cou cou cou cou cou cou cou cou
X = 3'-phosphate group

The sequence in parentheses consists of 2'-OMe modified nucleotides.
Second Oligonucleotide Primer P2-HAF5-081-1051 (SEQ ID NO: 23)
CAcaaTCATCAATACtaataggac 6 ctctggggtatataggacttcttcattgttagtagca
6 = HEG
BST 2.0 warm-start DNA polymerase (NEB 120,000 units / ml), also known as "BST polymerase"

The following concentrations were used for the individual ingredients:
-First oligonucleotide primer 0.3 μmol / l
・ Block oligonucleotide 5 μmol / l
・ Controller oligonucleotide 2 μmol / l
-Second oligonucleotide primer 0.2 μmol / l
The human gDNA used in the reaction at 1: 1000 dilution of BST polymerase (“human male”, manufactured by Promega) was added to the batch of first amplification in the form of a single strand at a concentration of about 100 ng per batch (12 μl). This simulated a test system with a complex genetic background. Human gDNA contains pooled samples (approximately 30,000 copies per batch).

Second amplification system (EvaGreen detection)
-Third oligonucleotide primer P3F5G2-1001-301
5'-CTCGACACTACTTCAGGACAAAATactgtattcc (SEQ ID NO: 24) (used at 0.5 μmol / l)
-Fourth oligonucleotide primer: P4F5G2-1001-401
5'-ctc tgggctata ggactacttc taatatgtaa gagca gat (SEQ ID NO: 25) (used at 0.5 μmol / l)
Taq polymerase hot start (NEB 5,000 units / ml) (used in reaction at 1: 100 dilution)
-Detection using EvaGreen dye (Jena Biosciences Stock 1:50)

The third and fourth primers were designed in a "nested form" with respect to the first and second primers, respectively.

Oligonucleotide probe for detecting FVL system in the second amplification:
Second amplification system (probe detection)
-Third oligonucleotide primer: P3F5G3-1001-3303-4
5'-ACTTCAGGACAAAATactgt att (SEQ ID NO: 26) (used at 0.5 μmol / l)
-Fourth oligonucleotide primer: P4F5G3-1001-3404-2
5'-ATATGTAA GAGCA GAT CCC (SEQ ID NO: 27) (used at 0.5 μmol / l)
Taq polymerase hot start (NEB 5,000 units / ml) (used in reaction at 1: 100 dilution)
・ Detection by oligonucleotide probe

（Ｔｈｅｒｍｏｆｉｓｈｅｒ製）（０，２μｍｏｌ／ｌで使用）
・ ＷＴ特異的検出のためのプローブオリゴヌクレオチド： Ｓ１Ｐ４−ＦＶＬ−１００３−１

（Ｔｈｅｒｍｏｆｉｓｈｅｒ製）（０，６μｍｏｌ／ｌで使用）
識別塩基には下線が引かれている。
ＮＦＱ＝非蛍光クエンチャー（ＢｌａｃｋＨｏｌｅシリーズのクエンチャー）
これらのプローブは、蛍光レポーターと蛍光クエンチャーとを備えた「Ｔａｑｍａｎプローブ」の例である。 The third and fourth primers were designed in a "nested form" with respect to the first and second primers, respectively.
Probe oligonucleotide S1P4-FVL-1007 for specific detection of FVL mutations

(Manufactured by Thermo Fisher) (Used at 0.2 μmol / l)
Probe oligonucleotides for WT-specific detection: S1P4-FVL-1003-1

(Manufactured by Thermo Fisher) (Used at 0.6 μmol / l)
The distinguishing base is underlined.
NFQ = non-fluorescent citrate (BlackHole series citrate)
These probes are examples of "Taqman probes" with fluorescent reporters and fluorescent quenchers.

Buffer solution:
All reactions were performed in the same buffer. 1x buffer component (NEB) 20 mM Tris-HCl
10 mM (NH ₄ ) ₂ SO ₄
50 mM KCl
2 mM sulfonyl ₄
0.1% Tween® 20
PH 8.8 at 25 ° C
-Optionally, Eva-Green dye (Jena Biosciences) (used in the reaction at 1:50 dilution).
-DNTP mix for Bst polymerase: dATP, dCTP, dGTP 200 μmol / L each, dUTP 400 μmol / l
DNTP mix for Taq polymerase: 200 μmol / L each of dATP, dCTP, dGTP, dTTP

Method procedure:
Preparation of Initiating Nucleic Acid for First Amplification First, a primer (P1F5G2-2001-) was added to the initiating nucleic acid 1.1 containing the target sequence starting from the single-stranded human genomic DNA (WHO standard for FVL and WT sequence variants). Synthesized by primer extension (50 ° C. for 10 minutes) with Bst polymerase using 203, inserted at 0.2 μmol / l) and then separated from the template chain by thermal denaturation at 95 ° C. (5 minutes). Generation of starting nucleic acid 1.1 for sequence variants of target sequences with FVL and wild-type mutations was performed in separate batches. Approximately 100,000 copies of human gDNA were used per batch. The primer used for this was similar in structure to the first oligonucleotide primer P1F5G2-1001-103.

After primer extension, starting nucleic acid 1.1 was obtained containing a sequence segment complementary to the target nucleic acid and a primer extension product with an overhang at the 5'end. In the first amplification, such an initiating nucleic acid acts as a template, and the second primer can specifically bind the sequence to such initiating nucleic acid 1.1 and extend with a polymerase. The controller oligonucleotide can bind to the overhang in its first region.

Execution of first amplification and detection:
Starting nucleic acid 1.1 (having FVL mutations and / or WT sequences) was added to amplification batches at various dilutions (100 copies, 10 copies for FVL mutations, and 5000 copies for WT sequences). The reaction was carried out under periodic temperature changes between 50 ° C and 65 ° C. At the above temperatures, natural attenuation, which is a specific amplification problem, did not occur. One cycle included incubation at 50 ° C. (2 minutes) and 65 ° C. (4 minutes). The batches were incubated for different times (up to about 15 hours). A signal from the insert dye Eva Green was detected during the reaction. After completion of the reaction, the melting curve was determined. Amplification resulted in an increase in the amount of double-stranded DNA (amplified fragment 1.1) capable of preserving the insert dye, resulting in an increase in the signal of EvaGreen.

The amplified fragment 1.1 synthesized during this reaction could be used as a template for a second amplification or Sanger sequencing analysis.

Performing a second amplification and detection (EvaGreen):
The product of the first amplification was diluted 1: 6000 before use in the second amplification. The amplified fragment present in the diluted batch served as the starting nucleic acid 2.1 for the PCR reaction. The PCR reaction was performed in 40 cycles. One cycle included: 55 ° C. 1 minute, 68 ° C. 3 minutes, 95 ° C. 20 seconds. Detection was performed using Eva-Green dye. By observing the increase in signal (Ct value), it was possible to estimate the relative amount of template added at the start of the reaction. The amplified fragment 2.1 synthesized in the second amplification could be used for sanger sequencing.

Performing a second amplification and detection (probe oligonucleotide):
The product of the first amplification was diluted 1: 6000 before use in the second amplification. The amplified fragment present in the diluted batch served as the starting nucleic acid 2.1 for the PCR reaction. The PCR reaction was performed in 40 cycles. One cycle included: 57 ° C. 1 minute, 95 ° C. 20 seconds. Detection was performed using a sequence-specific oligonucleotide probe (Taqman probe with Thermo Fisher's fluorescent reporter and MGB). By observing the increase in signal (Ct value), the relative amount of template added at the start of the reaction could be estimated in the same manner as the sequence composition of the relevant sequence segment of the synthetic product.

Results and evaluation:
In the first amplification, an increase in signal (of the EvaGreen dye) was detected at an initial concentration of about 100 and 10 copies per batch in batches starting with starting nucleic acid 1.1 (target sequence with FVL mutant sequence variant). It was possible (FIGS. 48, arrows 1 and 2, A) signal curve during amplification, B) melting curve). No signal increase was detected in batches with an initial concentration of approximately 5000 copies of the target sequence of WT mutants per batch (FIG. 48, arrow 3). This signal was at the level of a batch without starting nucleic acid. After completion of the first amplification, aliquots were taken, diluted and added to the second amplification.

The second amplification was based on the dilution of the batch from the first amplification completed (final dilution 1: 6000). The second amplification used third and fourth primers, which were nested into the first and second primers. The second amplification showed an increase in specific signal in all batches of the first amplification having an amplification fragment derived from the initiating nucleic acid 1.1 with the target sequence of the FVL mutant (FIG. 49). Arrows 2, A) show the progress of the signal during amplification, B) show the melting curve). The increase in signal occurs early in the PCR process (Ct value is about 5 or 8) and indicates a high concentration of amplified fragments from the first amplification. In batches starting with starting nucleic acid 1.1 with the target sequence of the wild-type variant, a signal was observed at the level of the batch without starting nucleic acid (FIG. 49, arrow 4, Ct value about 25) (FIG. 49, Arrow 3, Ct value about 25). A comparison of the time points of signal increase (Ct values) in these batches showed that the amount of amplified fragment with the FVL mutation at the end of the first amplification was significantly higher than the amount of amplified fragment with wild-type DNA. Shown. Given the starting nucleic acid used in the first amplification (10 copies of the FVL sequence variant, vs. 5000 copies of wild-type DNA), amplification of the FVL sequence variant is clearly preferred over the wild-type variant. I was able to observe that. The composition of the amplification products (FVL sequences) of the individual reactions was further confirmed by Sanger analysis and probe-based detection.

This result represents the specific amplification of the target sequence during the first amplification. The amplified fragment synthesized in the first amplification could be used as a template for the second amplification.

The difference between the two variants of the target sequence during the first amplification was made by the influence of a controller oligonucleotide designed to be complementary to the mutated target sequence. Therefore, this controller oligonucleotide contains a mismatch for the wild-type variant of the target sequence at one nucleotide position. The controller oligonucleotide was designed so that the polymorphic locus of the target sequence is located in the third region of the controller oligonucleotide. Therefore, this polymorphic locus was located in the sequence segment of the target sequence, 3'of the first primer and 3'of the second primer. Primers used alone would not have been able to distinguish individual sequence variants at the polymorphic locus of the target sequence.

During the first amplification, selective amplification of products having a sequence (FVL target sequence containing mutations) that was predominantly complementary to the third region of this controller was performed. In this example, a favorable embodiment of the reaction conditions was selected, where on the one hand the primer concentration was lower than the concentration of the controller oligonucleotide and on the other hand periodic temperature changes (between 50 ° C and 65 ° C) were used.

In the oligonucleotide used, the oligonucleotide primer forms an interacting pair with the corresponding controller oligonucleotide (eg, first oligonucleotide primer and first controller oligonucleotide). The interacting pair had a melting temperature of about 63 ° C. under amplification reaction conditions (measured at a concentration of about 1 μmol / l of both components under amplification buffer conditions). The first region of the oligonucleotide primer formed a complex with the complementary sequence portion of the template at a melting temperature of about 50 ° C. (eg, the first segment of the first primer and the second primer extension product (P2. 1-Ext) means that the concentration of both components under buffered conditions of this amplification was measured at about 1 μmol / l).

Depending on the embodiment, primers and controller oligonucleotides can be used in different ratios.

In an advantageous embodiment, a combination of a primer and a controller having a primer concentration higher than that of the controller was used (see, for example, first primer 5 μmol / l and controller 2 μmol / l, Examples 1-3). In the embodiment, excess primers were present, so that the primer extension step at a temperature of 50 ° C. resulted in good yields despite partial binding of the controller oligonucleotide of the oligonucleotide primer.

In a more advantageous embodiment (Example 5), a mixture of a primer having a primer concentration lower than that of the controller oligonucleotide and a corresponding controller oligonucleotide was used (eg, primer 0.5 μmol / l and controller 2 μmol / l). ). Therefore, the controller oligonucleotide was in excess. In the embodiment, lowering the reaction temperature to 50 ° C. resulted in rapid binding of the oligonucleotide primer to the controller oligonucleotide. This reduced the yield of the primer extension step at 50 ° C. and reduced the amplification rate. So-called block oligonucleotides were used to maintain sufficient primer concentration even at low temperatures and thus increase yields in the primer extension step. Such block oligonucleotides competed with oligonucleotide primers for binding to controller oligonucleotides, but could not be extended by the polymerase itself.

By using block oligonucleotides, it was possible to use a combination of oligonucleotide primers and controller oligonucleotides in several concentration ranges and combine them with periodic temperature changes. This improved the reaction rate.

The structure of block oligonucleotides is essentially similar to that of oligonucleotide primers, with the following differences:
-Block oligonucleotides are not extended by polymerase. This is done, for example, by blocking the 3'-end with a modification (eg, 3'-phosphate, 3'-C3, dideoxynucleotide) and / or introducing a terminal mismatch at the 3'-end of the blocked oligonucleotide. Can be achieved by doing.
-Block oligonucleotides may differ in sequence composition from the corresponding primers. The number of sequence mismatches can vary from 1 nucleotide to 20 nucleotides.

When designing a block oligonucleotide, it is advantageous to keep the Tm of the block oligonucleotide in the same range of controller oligonucleotides as the oligonucleotide primer and the Tm of the controller oligonucleotide. As a result, block oligonucleotides and oligonucleotide primers compete for binding to the controller oligonucleotide at similar levels (eg, Tm plus / minus 3 ° C. of the primer-controller complex). By using a block oligonucleotide having a higher concentration than the oligonucleotide primer, the binding ratio can be adversely affected.

Further Examples of Embodiments of the Invention FIG. 1 shows a first amplification (FIG. 1A) (first partial amplification) using components of a first amplification system and a second amplification system, and then a second. The frees and products of amplification, including amplification of (FIG. 1B) (second partial amplification), are shown schematically. The reaction begins with the starting nucleic acid 1.1, which comprises the target sequence and is used as a template for initiating the first partial amplification.

The components of the first amplification system are particularly: first oligonucleotide primer (P1.1), second oligonucleotide primer (P2.1), controller oligonucleotide (C1.1) and first polymerase (Pol1). .1), dNTP.
During the first partial amplification (A1.1), the specific amplification of the first amplification fragment 1.1 (first primer extension product P1.1-Ext and second primer extension product P2.1-Ext). Including) is performed. The primer extension product P1.1-Ext is realized as a template-dependent extension of P1.1 by a polymerase under the reaction conditions of the first partial amplification. The primer extension product 2.1-Ext results from the template-dependent extension of P2.1 under the reaction conditions of the first partial amplification. Both primers P1.1 and P2.1 include a sequence segment capable of complementarily binding to the target sequence or its complementary sequence segment, so that the primers are placed at both ends of the target sequence.

The amplification product 1.1 formed during the first partial amplification can be used as the starting nucleic acid 2.1 of the second partial amplification (A2.1). During the second partial amplification, the second amplification fragment 2.1 containing the third primer extension product P3.1-Ext and the fourth primer extension product P4.1-Ext was amplified, and the primer extension product P3. 1-Ext is formed as a template-dependent extension of P3.1 and by a polymerase, and the primer extension product 4.1-Ext results from a template-dependent extension of P4.1 under the reaction conditions of the second partial amplification. .. Both primers P3.1 and P4.1 include a sequence segment that can complementarily bind to the target sequence or its complementary sequence segment. P1.1 and P3.1 can complementarily bind controller oligonucleotides in their 3'segment, but due to modification of the controller oligonucleotide they are not extended by the polymerase used.

FIG. 2 schematically shows the free products and products of the first partial amplification. Specific amplification of the first amplification fragment 1.1 during the first partial amplification, including the first primer extension product P1.1-Ext and the second primer extension product P2.1-Ext. .. The primer extension product P1.1-Ext is realized as a template-dependent extension of P1.1, and the primer extension product 2.1-Ext results from a template-dependent extension of P2.1.

The synthetic products (P1.1-Ext and P2.1-Ext) formed during this reaction can form a complex between them and different from the controller oligonucleotide (depending on the concentration ratio and reaction conditions). Specifically, these forms include a complex of P1.1-Ext / C1.1 and / or P1.1-Ext and / or P1.1-Ext / C1.1 / P2.1-Ext. Can be done.

P1-Ext comprises a 3'segment containing a sequence moiety capable of binding essentially complementary to the P4.1 primer under the reaction conditions of the second partial amplification. P2-Ext comprises a 3'segment containing a sequence moiety capable of binding essentially complementary to the P3.1 primer under the reaction conditions of the second partial amplification.

FIG. 3 shows the temperature profile of the two partial amplifications (FIG. 3A) and illustrates the accumulation of products during the first and second partial amplifications (as an increase in copy number in a time-dependent manner). (Fig. 3B).

The first partial amplification section A1.1 is performed under temperature conditions that do not allow non-specific spontaneous chain separation in the absence of specific controller oligonucleotides. In essence, this reaction occurs between temperatures T1 and T2. At lower temperatures (T1), in particular hybridization of at least one primer of the first amplification system and its elongation by the polymerase occurs, and at the second temperature (T2), especially the separation of P1-Ext and P2-Ext. , Which occurs with the involvement of controller oligonucleotides. The reaction proceeds as a periodic change in reaction temperature, the required number of cycles can be selected and the degree of amplification can be affected. Here, a schematic increase to about 10 copies to about 10 ⁸ copies (Figure 3B). Further, after the first partial amplification is completed, a part of the formed product (amplification product 1.1) is transferred as an aliquot to the second partial amplification and functions as a starting nucleic acid chain of the second partial amplification. Here is a schematic description of what you can do. Therefore, if this batch is diluted after the first partial reaction, only a partial amount of the copy formed in the first reaction is transferred to the second reaction. The components of the first amplification system are also diluted, so that their effect on the second amplification system is reduced due to the resulting low concentration.

FIG. 3A more schematically shows the sequence of the second partial amplification (A2.1), the temperature condition used is complementary primer extension in which the separation of the amplification product obtained at temperature (T3) was synthesized. The product is selected to be predominantly non-specific due to the thermal destabilization of the bond between both strands and without the intrinsic involvement of the controller oligonucleotide. The binding and extension of the primer at T1 is the same as in the case of the first partial amplification. The reaction proceeds essentially like PCR, with the required number of PCR cycles performed to produce the required copy number. The reaction involves binding the primers to the template, extending them to the formation of the corresponding primer extension products, and separating the formed chains by temperature. PCR begins with the initial copy number of the first amplification product 1.1 containing a target sequence of about 10 ^5, it amplifies the amplified product 2.1 containing a target sequence to the copy number of about ^{10 10.}

The first partial amplification proceeds under conditions that, with the involvement of the controller oligonucleotide, cause sequence-dependent separation of the synthesized strand. The second partial amplification proceeds as PCR and the strand separation is predominantly sequence non-specific.

FIG. 4 schematically shows the temperature profile of the two partial amplifications (FIG. 4A) and outlines the accumulation of products during the first and second partial amplifications (as an increase in copy number in a time-dependent manner). The figure is shown (FIG. 4B). The reaction proceeds as shown in FIG. 3, but the copy number of the first amplification fragment 1.1 formed by adding the components of the second amplification system to the batch with the first partial amplification The difference is that it constitutes the second partial amplification initiation nucleic acid 2.1 and no significant dilution of the components of the first amplification system occurs.

FIG. 5 shows starting materials and products of amplification including a first partial amplification (FIG. 5A) and a subsequent second partial amplification (FIG. 5B) using the components of the first amplification system and the second amplification system. Is shown schematically. The reaction essentially proceeds as shown in FIG. 1, but during the second partial amplification, primers P3.2 and P4.2 were used, which were P1.1-Ext and P2.1-Ext, respectively. The difference is that the 3'end segment of the is essentially free of complementary sequence segments. Thus, P1.1-Ext and P2.1-Ext products with a 3'end are complementary to primers P3.2 and P4.2, respectively, which are extended by a polymerase (eg, Pol1.1 or Pol2.1). Cannot form a primer. This prevents P1.1-Ext and P2.1-Ext from overstretching when the primers P3.2 and P4.2 are used under the reaction conditions of the first partial amplification. During the second partial amplification, complete P3.1-Ext and P4.1-Ext products are formed during the periodic synthesis process and its propagation. Using such a combination of primers (set 1 containing P1.1 and P2.1 and set 2 containing P3.1 and P4.1), both primer sets are provided in the same reaction batch at the beginning of amplification. It is possible to design a uniform reaction.

FIG. 6 schematically shows the temperature profile of both partial amplifications (FIG. 6A) and is a schematic representation of product accumulation (as copy number increase in a time-dependent manner) during the first and second partial amplifications. (Fig. 6B).

This figure schematically shows the temperature profiles in which both partial amplifications are performed immediately after each other (both primer sets are present in the batch at the start of the reaction). During the first amplification, it is shown schematically that the number of copies of the amplification product 1.1 is increased to about 10 to about 10 ^4. These products contain the target sequence and serve as the starting nucleic acid 2.1 for the second amplification. During the second amplification, the number of the amplified target sequence is further increased to the amplification product 2.2 resulting from the amplification product 1.1 to about 10 ⁴ of approximately ^{10 10.}

FIG. 7A schematically shows the temperature profiles of the two partial amplifications. The series of temperature changes in FIG. 7A correspond to those described in FIG. 3: The first partially amplified segment A1.1 is non-specific spontaneous in the absence of specific controller oligonucleotides. Proceed under temperature conditions that do not allow chain separation. In essence, this reaction occurs between temperatures T1 and T2. At lower temperatures (T1), hybridization of at least one primer of the first amplification system and its elongation by polymerase proceed, especially at the second temperature (T2), especially separation of P1-Ext and P2-Ext. However, it proceeds with the involvement of controller oligonucleotides. In the course of the reaction, amplification of the first amplification product 1.1 occurs, which can be used as the starting nucleic acid 2.1 in the second partial amplification. The reaction proceeds as a periodic change in reaction temperature, the required number of cycles can be selected and the degree of amplification can be affected. FIG. 7A also schematically shows the course of the second partial amplification (A2.1) that proceeds immediately after the first partial amplification, the temperature condition used being that of the amplification product obtained at temperature (T3). Separation is used so that the bond between the two strands of the synthesized complementary primer extension product becomes thermally unstable and is predominantly non-specific with no substantial involvement of the controller oligonucleotide. Will be done. The binding and extension of the primer at T1 is the same as in the case of the first partial amplification. The reaction proceeds essentially like PCR, with the required number of PCR cycles performed to produce the required copy number. The reaction involves binding the primers to the template, extending them to the formation of the corresponding primer extension products, and separating the formed chains by temperature. The first partial amplification is performed under conditions that, with the involvement of the controller oligonucleotide, cause sequence-dependent separation of the synthesized strands. The second partial amplification is performed as PCR and the strand separation is predominantly sequence non-specific.

FIG. 7B shows the first and second amplifications as in FIG. 7A. Both reactions are separated by a further temperature step, here referred to as the "temperature switching step", which involves raising the temperature to about T3 for a period of time. During the steps, for example, inactivation of the polymerase of the first partial amplification and / or activation of the polymerase of the second partial amplification and / or activation of the thermal instability primer can occur. Therefore, such a step also promotes a change in the activity of the individual components of the amplification system.

FIG. 8 schematically shows the temperature profile of both partial amplifications. The sequence of the first partial amplification corresponds to that shown in FIG. 7A. The second partial amplification is divided into two stages. In the first step (A2.1 step 1), a temperature change occurs between T1 and T3. During T1, at least one primer in the second amplification system can bind and extend to the corresponding complementary position of amplification product 1.1. By using a lower temperature, primers with relatively short 3'segments can be used, which can bind complementarily (see Figure 5). Step 1 involves several cycles to form the initial amplification product 2.1, which contains longer complementary segments and thus allows the primers to bind at higher temperatures. This allows higher temperatures to be used for the primer binding and primer extension steps in step 2 (shown here as periodic extension between T2 and T3). For example, raising the temperature can prevent the occurrence of side reactions.

FIG. 8B schematically shows that the temperature is further reduced to temperature T4 during step 1 of A2.1 so that, for example, the primers of set 2 can bind. The use of temperature T4 in step 1 may be due to the use of at least one primer in set 2 containing at least one mismatch for the complementary sequence segment of the first amplification product. The low temperature (T4) is intended to support the initial production of primer extension products starting with P3.1 and / or P4.1. After this initial formation, a fully complementary primer binding site will then be present, allowing the temperature of the step with primer binding and primer extension to be raised (step 2).

FIG. 9A schematically shows the temperature profiles of the two partial amplifications. The first partial amplification proceeds at a uniform temperature, here T2. All necessary steps of the first partial amplification are performed at this temperature. This can be followed, for example, by a temperature rise (temperature switching step) (as described in FIG. 7B). After this, a PCR reaction is performed as a second partial amplification.

The first partial amplification proceeds at a uniform temperature and uses controller oligonucleotides to promote sequence-dependent chain separation.

FIG. 9B schematically shows a further temperature curve of the first partial amplification, including both an isothermal step (at the start) and a subsequent periodic step. Here, the first partial amplification also proceeds at a temperature that promotes sequence-dependent chain separation involving the controller oligonucleotide.

FIG. 10A schematically shows the temperature profiles of the two partial amplifications. The second partial amplification of step 2 involves the use of an additional temperature T5, which is higher than T2. The temperature is between about 68 ° C and 82 ° C. Temperature T5 can be used to weaken the binding of the controller oligonucleotide to P3.1 Ext, thus reducing the effect of this binding on P4.1 elongation, thereby causing P4.1 elongation. Can be more effective.

FIG. 10B schematically shows the temperature profile of both partial amplifications. During the second partial amplification segment, the first production of the second amplification product proceeds in step 1. Primers 3.1 and 4.1, which can bind to complementary positions at higher temperatures (T5) and can be extended by polymerase, can be used to change the temperature of the second stage between T5 and T3. The choice of reaction conditions can affect the formation of intermediates or final products. For example, by using longer P3.1 and P4.1 (eg, 30-40 nucleotides in length) in combination with higher temperatures in the primer binding and primer extension steps in the second partial amplification (eg, T2). And / or (at T5), P3.1-Ext and P4.1-Ext are preferably accumulated.

FIG. 11 schematically shows the temperature profile of both partial amplifications. The transition stage between the two partial amplifications can be designed differently. FIG. 11A shows a continuous rise in temperature towards the end of the first partial amplification. In FIG. 11B, a periodic drop in temperature is shown at the end of the first partial amplification. Therefore, both segments of partial amplification can have partial overlap in the reaction process.

FIGS. 12-18 show the relationships between the individual components of the first amplification system (up to FIG. 14) and the relationships between the components of the first amplification system and the second amplification system. FIG. 15A schematically shows the production of a first amplified fragment (including P1.1-Ext and P2.1-Ext) starting from the starting nucleic acid 1.1 (here schematic as the nucleic acid to be amplified in the reaction). Will be added). FIG. 15B schematically shows the formation of intermediates produced using the first partial amplification primers P3.1 and P4.1 and P1.1-Ext and P2.1-Ext as templates (P3). .1-Ext Part1 and P4.1-Ext Part1). These intermediates are reprimer bound and extended using P3.1-Ext part1 and P4.1-Extpart1 as templates to complete P3.1-Ext and P4.1-Ext in subsequent cycles. Can be converted. The choice of reaction conditions can affect the formation of intermediates or final products. For example, by using longer P3.1 and P4.1 (eg, 30-40 nucleotides in length) in combination with high temperatures in the primer binding step and primer extension step in the second partial amplification (eg, T2). And / or T5) P3.1-Ext and P4.1-Ext are preferably accumulated.

19-26 show the sequences of the controller oligonucleotide (C1.1), the first primer (P1.1) and the third primer (P3.1), and the second primer extension product (P2.1-Ext). An example of segment arrangement is shown schematically. P2.1-Ext contains a sequence complementary to P1.1 in the 3'segment and serves as a template for producing P1.1-Ext in the first partial amplification. In addition, P2.1-Ext contains an additional complementary segment for the 3'segment of P3.1 that binds to P2.1-Ext and is extended by the polymerase, so that P3.1-Ext is the first. It can be produced by partial amplification of 2.

Figures 16-26 summarize examples of controller oligonucleotides and segment placement in P1.1 and P3.1. P3.1 can bind to the controller oligonucleotide in a manner complementary to the 3'segment. This binding occurs in the sequence segment of the controller oligonucleotide, referred to here as the fourth region. Depending on how P3.1 is arranged in detail, this fourth region can have different arrangements with respect to areas 1, 2, or 3. In certain embodiments, the fourth region is located within the second region (FIGS. 19 and 20), and in another embodiment, the fourth region is located at the transition between regions 2 and 3. (FIG. 21), in another embodiment, the fourth region is located in the third region of the controller oligonucleotide (FIG. 22). In particular, the presence of controller oligonucleotides should not lead to the formation of unintended by-products. In particular, primers that bind to the controller oligonucleotide should not result in the polymerase using the controller oligonucleotide to catalyze P1.1 or P3.1 elongation. This is generally achieved by using nucleotide modifications for controller oligonucleotides, for example by using 2'-O-alkyl modifications. Such modifications are particularly used in sequence segments of controller oligonucleotides that contain sequences complementary to at least the 3'segment of each primer. In addition, such modifications can extend beyond the sequence segments complementary to the primers, for example, these sequence segments can be located at about -10 to about plus 10 nucleotides or at the 3'end of each primer. Can include. Sequence segments with such nucleotide modifications are referred to herein as "second blocking units" and include sequence segments complementary to the first primer (P1.1) (FIGS. 1 and 2). Alternatively, it is called a "fourth blocking unit" and contains a sequence segment complementary to a third primer (P3.1) (Fig. 19-22). Such modification of the controller oligonucleotide prevents the polymerase from initiating template-dependent primer extension when the primer binds to the controller oligonucleotide. The composition of the nucleotide modifications may be the same or different in the second and fourth blocking units. The length of each sequence segment that is combined to form a second or fourth blocking unit can also be the same or different for each blocking unit. The position of each blocking unit depends on the position of each possible binding site of the primer on the controller oligonucleotide (FIGS. 19-22). The possible composition of the second blocking unit for the first primer is shown in detail. The components of the fourth blocking unit can be similar to the components of the second blocking unit. In certain embodiments, the third region of the controller oligonucleotide consists entirely of 2'-O-alkyl modifications of the nucleotide. In a further specific embodiment, the third region of the controller oligonucleotide in its 3'segment (eg, 1-20 nucleotide positions adjacent to the second region of the controller oligonucleotide) is the 2'-O-O- of the nucleotides. Completely composed of alkyl modifications. In another particular embodiment, the second region of the controller oligonucleotide consists entirely of 2'-O-alkyl modifications of the nucleotide. In a further specific embodiment, the second region of the controller oligonucleotide and the third region of the controller oligonucleotide (positions 1-20 following the second region) in its 3'segment are completely nucleotides 2'. Consists of —O—alkyl modifications. Therefore, it is not necessary to provide the individual segments with modifications separately, and the complete sequence segment of the controller oligonucleotide can be provided, for example, by complementary nucleotides with 2'-O-alkyl modifications. It is possible.

Because the complementary sequence segments of the primer extension product (produced as a result of the enzymatic activity of the polymerase in a template-dependent manner) are unmodified, the primers are used when the polymerase binds complementaryly to such sequence segments. Can be recognized and stretched.

27-31 schematically show examples of arrangements and possible combinations including primer set 1 (P1.1 and P2.1) and primer set 2 (P3.1 and P4.1). One of the primers in the primer set 1 can be the same as one of the primers in the set 2 (eg, P4.1 in FIGS. 27-28 corresponds to P2.1). In particular, when a diluent of the reaction mixture is used after the first partial amplification and the components of the second amplification system are added after the first partial amplification, the primers of the second primer set have the same length as the primer set 1. It can be different (Fig. 30). Preferably, the structure of primer 3 is used that contains a sequence complementary to the controller oligonucleotide only in its 3'segment. The composition of the 5'segment of the third primer does not specifically include the complementary sequence segment to the controller oligonucleotide (FIG. 31).

FIGS. 32 to 35 schematically show the interaction of the components in the first partial amplification.

FIG. 32 describes the components of the structure shown in FIGS. 33-35.

FIG. 33 schematically shows the chain substitution mechanism.

FIG. 34 schematically shows the interaction of structures during strand substitution.

FIG. 35 schematically illustrates the structural interaction between the first nucleic acid amplification by amplification in one of the first oligonucleotide primer and the second oligonucleotide primer, as well as the action of the controller oligonucleotide and the resulting strand substitution. Shown in.

FIG. 36 shows the results of Example 1. FIG. 37 shows the results of Example 2. FIGS. 38-40 show the results of Example 3. 41 to 47 show the results of Example 4, and FIGS. 48 to 49 show the results of Example 5.

Figures 50-51 schematically show the preparation of starting nucleic acid chain 1.1.

FIGS. 52-54 schematically show specific embodiments of the amplification method.

To initiate the first amplification, a first nucleic acid polymer comprising a first target sequence M [and a sequence M'(reverse) complementary to M'] is provided, where M is in the 5'-3'direction. , Sequence segments MPL, MS and MPR are included most recently in succession.

In one embodiment, the first nucleic acid polymer comprises a starting nucleic acid chain. In another embodiment, the first nucleic acid polymer comprises a first amplified nucleic acid chain that is amplified. In another embodiment, the first nucleic acid polymer comprises a nucleic acid chain that is amplified by a second amplification.

In the embodiment, the target sequence M [and the (reverse) complementary sequence M'] in the 5'-3'direction comprises the sequence segments MPL, MS and MPR in the immediate vicinity. In one embodiment, the first nucleic acid polymer comprises such a target sequence M. In a further embodiment, the starting nucleic acid chain comprises such a target sequence M. In a further embodiment, the nucleic acid strand amplified by the first amplification comprises such a target sequence M. In a further embodiment, the amplified fragment 1.1 of the first amplification comprises such a target sequence M. In a further embodiment, the amplified nucleic acid strand of the second amplification comprises such a target sequence M. In a further embodiment, the amplified fragment 2.1 of the second amplification comprises such a target sequence M.

The first left oligonucleotide primer PL1 shown herein is (essentially) identical to the MPL of target sequence M. Such PL1 is a specific embodiment of the second oligonucleotide primer (P2.1).

The first right oligonucleotide primer PR1 shown here is a specific embodiment of the first oligonucleotide primer (P1.1). PR1 immediately contains the sequence segment PCR and PMR in the 5'-3'direction and PMR (in this embodiment, corresponds to the first region of said target sequence M contained in the amplified nucleic acid). Is an essentially complementary [hybridizing] sequence [which can bind essentially sequence-specifically] and sequence segment PCR (in the embodiment, the segment is a second region of a first primer. (Corresponding to) does not bind to the target sequence M [or, for example, the sequence of the first starting nucleic acid closest to the MPR with respect to sequence M in 3'], and PR1 (especially at the PCR site) Includes a modified nucleotide building block so that does not function as a template for the activity of the first template-dependent nucleic acid polymerase.

The controller oligonucleotide CR shown herein comprises the sequence segments CSR, CPR and CCR in the most recent contiguous direction in the 5'-3'direction. The sequence segment CSR corresponds to the third region of the controller oligonucleotide. The sequence segment CPR corresponds to the second region of the controller oligonucleotide. The sequence segment CCR corresponds to the first segment of the controller oligonucleotide.

In one embodiment, the sequence segment CSR is identical to the MS segment of the target sequence located at 5'of the MPR [and is first read at the start of the polymerase of PR1, i.e., the CSR is a polymerization product of primer PR1. (Can bind to the extension product of the first primer)].

In one embodiment, the sequence segment CPR is complementary to the PMR of the first primer (and is identical to the MPR of the target sequence M).

In one embodiment, the sequence segment CCR (first segment of controller oligonucleotide) is complementary to PCR (second region) of the first primer.

In one embodiment, the controller oligonucleotide (CR) comprises a modified nucleotide building block in its third segment (CSR), so that the CSR can serve as a template for template-dependent nucleic acid polymerase activity. Can not.

First Primer Extension Product (PR1'): This first primer extension product is the target sequence M in the region adjacent to the MPR at 5'in addition to the sequence region PCR and PMR (of the first oligonucleotide primer). Contains a synthetic region PSR that is essentially complementary to the 5'-3'direction.

Second Primer Extension Product (PL1'): In addition to the sequence region MPL, this primer extension product contains a synthetic region PSL that is essentially identical to the target sequence M in the region adjacent to the MPL at 3'.

PR1'and PL1'together form an amplified fragment 1.1 and serve as a template (starting nucleic acid 2.1) for the second amplification.

Second Left Oligonucleotide Primer PL2: Such PL2 is a particular embodiment of the fourth oligonucleotide primer (P4.1). In one embodiment, the PL2 is identical to the first secondary primer binding region MPL2. This MPL2 in this type of embodiment is included in the P4.1 Ext of the second amplified fragment 2.1. In another embodiment, MPL2 can be included in the second primer extension product. In a further embodiment, MPL2 can be included in the target sequence M.

Second Right Oligonucleotide Primer PR2: PR2 is a particular embodiment of a third oligonucleotide primer (P3.1) that is complementary to region MPR2 in one embodiment. The MPR2 can be included in an embodiment of a second primer extension product. In other embodiments, MPR2 can be included in the fourth primer extension product. In a further embodiment, MPR2 can be included in the target sequence M.

The arrangement of PL2 and PR2 can be different. In one embodiment, MPL2 and MPR2 will consist of the target sequence M. In one embodiment, the 3'end [at least 20 positions] of MPL2 is located at the 5'end 5'of MPR2. In a further embodiment, in particular, MPL2 is identical to MPL and / or MPR2 is identical to MPR.

In a further embodiment, the third (PR2) and fourth (PL2) oligonucleotide primers may include additional sequence segments (PCL2 or PCR2).

In the second amplification, the third (PR2) and fourth (PL2) oligonucleotide primers can bind to the corresponding complementary sites of the first amplification fragment, whereby these primers are second in the second amplification. It is extended by the polymerase of 2. As a result of the second amplification, the primer extension products (P3.1-Ext and P4.1-Ext) of the third and fourth primers are increased. The result is the second amplified fragment 2.1.

FIG. 55 schematically illustrates a particular embodiment of topography of the components of the first amplification system.

The starting nucleic acid 1.1 contains the following segments (in the 5'-3'direction): M1Y, M1.5, M1.4, M1.3, M1.2, M1.1, M1. X.

The first oligonucleotide primer comprises the following segment (in the direction of 5'-3'): the first segment (p1.1.1) and the second region (p1.1.2).

The controller oligonucleotide (C1.1) contains the following segments (in the 5'-3'direction): third region (C11.3), second region (C1.1.2), first. Region (C.1.1.1).

The controller oligonucleotide (C1.2) contains the following segments (in the 5'-3'direction): third segment (C1.2.3), second region (C1.2.2), first. Region (C.1.2.1).

The second primer P2.1 contains the next segment (in the 5'-3'direction): P2.1.1.

The second primer P2.2 contains the next segment (in the 5'-3'direction): P2.2.1.

The second primer P2.3 contains the next segment (in the 5'-3'direction): P2.3.1.

The first primer extension product (P1.1-Ext) contains the following segments (in the 5'-3'direction): P1.1E6, P1.1E5, P1.1E4, P1.1E3, P1.1E2, P1.1E1.

The second primer extension product (P2.1-Ext) contains the following segments (in the 5'-3'direction): P2.1E5, P2.1E4, P2.1E3, P2.1E2, P2.1E1.

The primer extension products P1.1-Ext and P2.1-Ext preferably obtained during the first amplification can form complementary duplexes and together represent the second amplification fragment 1.1. Can function as the starting nucleic acid 2.1 for the second amplification.

P1.1.1 can mainly bind complementarily to M1.1 and can be extended by the polymerase. P2.1.1 can complement P1.1E1 and can be extended by the polymerase. P1.1-Ext and P2.1-Ext represent the nucleic acid to be amplified, which functions as the starting nucleic acid 2.1 of the second amplification. P2.1, P2.2 and P2.3 represent variants of the second primer, resulting in the same amplified fragment.

Figures 56-57 schematically show specific embodiments of topography of the first and second amplification systems:

The third oligonucleotide primer contains the following segment (in the 5'-3'direction): P3.1.2, P3.1.1, where P3.1.2 is the starting nucleic acid 1.1 or Not complementary to P2.1-Ext. P3.1.1 is essentially complementary to P2.1E1 of P2.1-Ext.

The fourth oligonucleotide primer contains the following segment (in the 5'-3'direction): P4.1.2, P4.1.1, where P4.1.2 is the starting nucleic acid 1.1. Not complementary to complementary strands or P1.1-Ext. P4.1.1 is essentially complementary to P1.1E1 of P1.1-Ext.

Preferably, the primer extension products P3.1-Ext and P4.1-Ext obtained during the second amplification can form complementary duplexes, both representing the second amplification fragment 2.1. ..

P3.1-Ext includes the following segments (in the 5'-3'direction): P3.1E7, P3.1E6, P3.1E5, P3.1E4, P3.1E3, P3.1E2, P3.1E1. Segment P3.1. E5 to P3.1E3 are identical to the sequences of P1.1E4 to P1.1E2.

P4.1-Ext includes the following segments (in the 5'-3'direction): P4.1E7, P4.1E6, P43.1E5, P4.1E4, P4.1E3, P43.1E2, P4.1E1. Segment P4.1. E5 to P4.1E3 are identical to the sequences of P2.1E4 to P2.1E2.

FIG. 57 schematically illustrates specific embodiments of the third primer (P3.1, P3.2, P3.3) and the fourth primer (P4.1, P4.2, P4.3). The 3'segments of each primer can be shifted relative to each other.

Figures 58-59 schematically show specific embodiments of localized regions of oligonucleotide probes:
D1- is located at the 5'end of P4.1Ext. Therefore, a primer having a probe function can be used, for example, a Scorpion primer and a Lux primer. Primers are constructed in the 3'segment as in P4.1.

D4- is located at the 5'end of P3.1-Ext. Therefore, a primer having a probe function can be used, for example, a Scorpion primer and a Lux primer. Primers are constructed in the 3'segment as in P3.1.

Regions D2 and D3 are located in the central segment of P3.1-Ext and P4.1-Ext, respectively. Thus, hybridization probes (eg, molecular beacons, Taqman probes (5'-3'nuclease cleavable probes, or 2-oligonucleotide probes with FRET pairs) can be located here.

Since the D2 region does not overlap with the controller oligonucleotide, it is particularly suitable for possible probe localization (at P3.1-Ext or P4.1-Ext). Therefore, the probe can be used especially in the area of the uniform assay. The region contains the 3'segment of the third primer extension product. The probe for region D3 can also be provided for both P3.1-Ext and P4.1-Ext. However, if the concentration of C1.2 is relatively high (controller, eg 0.5 mol / l-5 μmol / l, eg homogeneous assay), such probes may interact with or be replaced by the controller. Must be considered so that it can be done. If the concentration of the controller is relatively low (eg, in the segment below 1 nmol / l), this interaction with the controller is negligible (eg, when diluting the first amplification before the second amplification).

FIG. 60 schematically illustrates a particular embodiment of the localization of probe segments capable of predominantly complementary binding to third (and perhaps first) primer extension products (P3.1-Ext and P1.1-Ext). Shown in. We show that there are several possibilities for possible probe localization.

FIG. 61 schematically illustrates a particular embodiment of the localization of probe segments capable of predominantly complementary binding to fourth (and perhaps second) primer extension products (P4.1-Ext and P2.1-Ext). Shown in. We show that there are several possibilities for possible probe localization.

Figures 62-63 schematically show specific embodiments of the probe:

Probes localized to D1: The probes S3.1, S3.2, S3.3 contain a fluorescent reporter (R) / fluorescent quencher (Q) pair, which is the corresponding segment of the primer extension product. When hybridized to, it results in an increase in signal (spatial separation of reporter / quencher). The probes S3.1 and S3.3 can be extended as primers to obtain primer extension products based on the primers having a probe function (S3.1-Ext and S3.3.Ext in FIG. 63).

FIGS. 64-67 schematically show specific embodiments of oligonucleotide probes and how widespread they are: S3.4 is a 5'-3'nuclease fibrable probe ("Taqman probe"). Represent. The probe is cleaved by Taq polymerase after hybridization to P3.1Ext and P4.1 elongation occurs simultaneously.

Probe S3.6 represents a probe system in which two oligonucleotides can or need to bind to P3.1Ext. Thus, one oligonucleotide is probe S3.6 with a fluorescent reporter (R) and the other probe is controller oligonucleotide C1.2, which contains a donor fluorescent dye molecule in the 5'region. If both oligonucleotides bind to P3.1-Ext at the same time, there is sufficient spatial proximity (less than 25 NT) for FRET to occur, which results in a signal.

Probe S3.7 schematically represents a probe with self-complementary parts (“molecular beacons”). In the unbound state, the signal from the fluorescent reporter (R) is quenched preferentially by the fluorescent quencher (Q) under selected reaction conditions. Complementary binding results in structural deconvolution, resulting in spatial separation of R from Q, which usually results in an increase in signal.

FIGS. 68-71 show the starting nucleic acid 1.1, the amplified fragment 1.1 (P1.1-Ext and P2.1-Ext), and the second amplified fragment 2.1 (P3.1-Ext and P4.1). Specific embodiments of localization of target sequences 1 to 3 in −Ext) are schematically shown.

Claims (22)

A method of amplifying nucleic acid (Fig. 56):
• Contains the first target sequence M1 [and sequence M1'(reverse) complementary to M1], where M is the sequence segments M1.5, M1.4, M1.3, M1.2 in the 5'-3'direction. In the first amplification step, a sample containing the first nucleic acid polymer containing the most recent contiguously and M1.1 was prepared with the following components:
First template-dependent nucleic acid polymerases, especially DNA polymerases, and substrates for template-dependent nucleic acid polymerases (particularly ribonucleoside triphosphates or deoxyribonucleoside triphosphates) and suitable cofactors, especially magnesium ions,
The first (right) oligonucleotide primer P1.1 containing the sequence segments P1.1.2 and P1.1.1 in the 5'-3'direction in the immediate vicinity, wherein P1.1.1 is , Has a [hybridizing] sequence complementary to M1.1 [which can bind essentially sequence-specific], and sequence segment P1.1.2 is M1 [or 3'direction with respect to sequence M1. The sequence immediately following .1] cannot be attached, and P1.1 (especially in segment P1.1.2) contains a modified nucleotide building block, whereby P1.1.2 is the first template-dependent nucleic acid. It cannot serve as a template for activating polymerase; and is (essentially) identical to M1.5 [and an inverse complementary sequence of M1.5 or an extension of P1.1 on the opposite strand of M1. , P1.1-Ext (called P1.1E1) can bind sequence-specifically] Second (left) oligonucleotide primer P2.1;
A controller oligonucleotide C1.2 containing the sequence segments C1.2.3, C1.2.2 and C1.2.1 in the 5'-3'direction in the immediate vicinity, wherein C1.2.3 is. Identical to segment M1.2 of M1 located in the 5'direction of M1.1 [and first read at the start of polymerase of P1.1, C1.2.3 is the polymer product of the primer P1.1-Ext Can bind to], C1.2.2 is complementary to P1.1.1 (and is identical to M1.1), and C1.2.1 is complementary to P1.1.2.
And, C1.2 contains a nucleotide building block modified with C1.2.1 Thus, C1.2.1 cannot function as a template for activating a template-dependent nucleic acid polymerase.
Contact with
And, in the second amplification step, the sample is subjected to the following components:
a) Containing sequence segment 3.1.1 at the 3'end [or essentially consisting of 3.1.1] and (reverse) complementary to M1.1 of M1 [complementary to P2.1-Ext] Can bind], 3rd (right) oligonucleotide primer P3.1,
b) Identical and / or essentially identical to M1.5 of M1 [can complementarily bind to P1.1-Ext], containing sequence segment 4.1.1 at the 3'end [or essentially] Consists of 4.1.1] Fourth (left) oligonucleotide primer P4.1,
c) A second template-dependent nucleic acid polymerase, especially a DNA polymerase, and optionally a template-dependent nucleic acid polymerase substrate (particularly ribonucleoside triphosphate or deoxyribonucleoside triphosphate) and suitable cofactors.
Contact,
The method. The first primer extension product, P1.1-Ext, contains the sequence segments P1.1E4, P1.1E3, P1.1E2 and P1.1E1 5'in addition to the sequence regions P1.1.2 and P1.1.1. The first primer extension product P1.1-Ext containing the synthetic region contained in the -3'direction in the 5'-3'direction was obtained, and P1.1E4, P1.1E3, P1.1E2 and P1.1E1 , Which are essentially complementary to the sequence segments M1.2, M1.3, M1.4 and M1.5 of the target sequence M1, respectively, and the second primer extension product P2.1-Ext is the sequence region P2. In addition to 1.1, the second primer extension product P2.1-containing a synthetic region P2.1-Ext that is essentially identical to the target sequence M1 in the region adjacent to M1.5 in the 3'direction. Ext is obtained,
And,
P1.1-Ext can form a double strand with M1 or P2.1-Ext, P1.1-Ext can form a double strand with C1.2, and P1.1 with P2.1-Ext So that the formation of the P1.1-Ext double strand with C1.2 is prioritized over the formation of the -Ext double strand.
− The reaction conditions of the first amplification step are selected and / or − P1.1.2 and, if applicable, lengths and / or M1.4, M1.3, M1.2 and M1.1. Melting temperature is selected,
The method for amplifying the nucleic acid according to claim 1. In the absence of controller oligonucleotide C1.2, the first primer extension product P1.1-Ext does not separate at all or essentially does not separate from the second primer extension product P2.1-Ext.
-The reaction conditions of the first amplification step are selected and / or-P1.1.2 and, where applicable, the lengths of M1.4, M1.3, M1.2, and M1.1 and / Or the melting temperature is selected,
The method for amplifying the nucleic acid according to claim 2. The method according to any one of claims 1 to 3, wherein the modified nucleotide building block comprises a 2'-O-alkylribonucleoside building block, particularly a 2'-O-methylribonucleoside building block. .. The method according to any one of claims 1 to 4, wherein the first amplification step is performed essentially isothermally, particularly at a temperature in the range of 20 ° C to 50 ° C. The first amplification step is carried out until P1.1-Ext exists in a copy number in the range of 10 to 1e12, particularly 100 to 1e10, particularly 1000 to 1e8, according to any one of claims 1 to 5. The method described. P3.1 and P4.1 contain a second region P3.1.2 and P4.1.2, respectively, adjacent to the 5'end of regions P3.1.1 and P4.1.1, respectively. The method according to any one of claims 1 to 6, wherein the region 2 is not complementary to P1.1-Ext and P2.1-Ext, respectively. During the second amplification step, the reaction temperature is periodically increased and decreased, especially between temperatures in the low temperature range of 20 ° C. to 75 ° C. and temperatures in the range of 85 ° C. to 105 ° C. The method according to any one of claims 1 to 7. The method according to any one of claims 1 to 8, wherein the first polymerase and the second polymerase are the same. -P1.1 and P3.1 are essentially the same, and / or-P2.1 and P4.1 are essentially the same.
The method according to any one of claims 1 to 9, wherein the method is characterized by. The method according to any one of claims 1 to 10, wherein the first amplification and the second amplification are carried out in the same reaction batch. A second polymerase, a third oligonucleotide primer (P3.1) and / or a fourth oligonucleotide primer (P4.1) can be activated and / or a controller oligonucleotide (C1. The method according to claim 7, wherein 1) can be deactivated. The method according to any one of claims 1 to 6, wherein the first amplification is carried out in a first reaction batch and the second amplification is carried out in a second reaction batch. .. The method of claim 9, wherein the aliquot of the first reaction batch or all the first reaction batches are added to the second reaction batch. Any of claims 1-14, the controller oligonucleotide (C1.1) comprises a fourth region capable of sequence-specific binding to a third oligonucleotide primer (P3.2). The method described in item 1. The first oligonucleotide primer (P1.1) is one or more that terminates the first polymerase in the second region, especially immediately after the first region of the first primer oligonucleotide, in the second region. The method according to any one of claims 1 to 15, which comprises the modification of. a. The array segment is
i. It is identical to the sequence of M1 located in the sequence segments M1.2, M1.3, and M1.4; or ii. Complementary to the sequence of M1 located in sequence segments M1.3 and M1.4,
Contains the sequence segment
b. Fluorescent dye,
iii. Form a donor-quencher pair or FRET pair with a second probe oligonucleotide linked to a first probe oligonucleotide; or iv. Forming a donor-quencher pair or FRET pair with a fluorescent dye linked to a second probe oligonucleotide that can bind in close proximity to the binding site of the first probe oligonucleotide.
A first probe oligonucleotide that binds to the fluorescent dye,
The method according to any one of claims 1 to 16, wherein the sample is further contacted. A method of amplifying nucleic acids:
a) A sample containing a first nucleic acid polymer containing the first target sequence M1 in which M1 in the 5'-3'direction is sequence segments M1.5, M1.4, M1.3, M1.2. And M1.1 in the most recent contiguously, the sample is subjected to the following components in the first amplification step:
b) A first template-dependent nucleic acid polymerase, particularly a DNA polymerase, and a substrate for a template-dependent nucleic acid polymerase,
c) The first (right) oligonucleotide primer P1.1 containing the sequence segments P1.1.2 and P1.1.1 in the 5'-3'direction in the immediate vicinity, P1.1.1. Has a [hybridizing] sequence that is complementary to M1.1, and sequence segment P1.1.2 cannot bind to M1, and P1.1 (especially in segment P1.1.2) is modified. The first (right) oligonucleotide primer P1.1; and d, which contain a nucleotide building block, whereby P1.1.2 cannot function as a template for activating the first template-dependent nucleic acid polymerase. ) Second (left) oligonucleotide primer P2.1, which is (essentially) identical to M1.5;
e) A controller oligonucleotide C1.2 containing the sequence segments C1.2.3, C1.2.2 and C1.2.1 in the 5'-3'direction in the immediate vicinity, C1.2.3. Is identical to the segment M1.2 of M1 located in the 5'direction of M1.1, and C1.2.2 is complementary to P1.1.1 (and identical to M1.1). And C1.2.1 is complementary to P1.1.2,
And C1.2 comprises a nucleotide building block modified with C1.2.1 so that C1.2.1 cannot function as a template for activating a template-dependent nucleic acid polymerase, said controller. Oligonucleotide C1.2,
Contact with
The sample is further a. The array segment is
i. It is identical to the sequence of M1 located in the sequence segments M1.2, M1.3, and M1.4; or ii. Complementary to the M1 sequence located in sequence segments M1.3 and M1.4,
Contains the sequence segment
b. Fluorescent dye,
i. Form a donor quencher pair or FRET pair with a second probe oligonucleotide linked to a first probe oligonucleotide; or ii. A first that binds to the fluorescent dye forming a donor-quencher pair or FRET pair with a fluorescent dye linked to a second probe oligonucleotide that can bind close enough to the binding site of the first probe oligonucleotide. Contact with probe oligonucleotides,
The method. Claim that the sequence segment of the first probe oligonucleotide does not hybridize with P1.1, P2.1, P3.1 and P4.1, or with C1.2 under the reaction conditions of the second amplification step. 17 or 18. A kit for carrying out the method according to any one of claims 1 to 19.
a) The first (right) oligonucleotide primer P1.1 containing the sequence segments P1.1.2 and P1.1.1 in the 5'-3'direction in the immediate vicinity, wherein P1.1. 1 binds to the primer binding site M1.1 of the genome sequence M1 of the eukaryotic or pathogenic bacteria, especially mammals, especially human target sequences, and M1 in the 5'-3'direction is the most recent contiguous. Containing sequence segments M1.5, M1.4, M1.3, M1.2 and M1.1, and sequence segment P1.1.2 is the sequence immediately following M1.1 with respect to sequence M1 in M1 [or 3'. ], And P1.1 (particularly segment P1.1.2) is modified nucleotide building so that P1.1.2 cannot function as a template for activating the first template-dependent nucleic acid polymerase. The first (right) oligonucleotide primer P1.1; and b) containing the block are (essentially) identical to M1.5 [and the inverse complementary sequence of M1.5 on the opposite strand of M1]. Alternatively, it can bind to P1.1-Ext (called P1.1E1), an extension product of P1.1, in a sequence-specific manner.] Second (left) oligonucleotide primer P2.1;
c) A controller oligonucleotide C1.2 containing the sequence segments C1.2.3, C1.2.2, and C1.2.1 in the 5'-3'direction in the most recent sequence, C1.2. 3 is located at 5'of M1.1 [and is first read at the start of the polymerase of P1.1, so C1.2.3 can bind to the polymerization product P1.1-Ext of the primer] M1 Is identical to segment M1.2, C1.2.2 is complementary to P1.1.1 (and identical to M1.1), and C1.2.1 is complementary to P1.1.2. Is,
C1.2 comprises a nucleotide building block modified with C1.2.1 so that C1.2.1 cannot function as a template for activating the first template-dependent nucleic acid polymerase.
And d) Containing sequence segment 3.1.1 at the 3'end [or essentially consisting of 3.1.1] and (reverse) complementary to M1.1 of M1 [to P2.1-Ext] Can complementarily bind], 3rd (right) oligonucleotide primer P3.1,
e) Containing [or essentially] sequence segment 4.1.1 at the 3'end, which is identical and / or essentially identical to M1.5 of M1 [can complementarily bind to P1.1-Ext]. Consists of 4.1.1] Fourth (left) oligonucleotide primer P4.1,
And optionally, a second template-dependent nucleic acid polymerase, particularly DNA polymerase, and optionally a template-dependent nucleic acid polymerase substrate (particularly ribonucleoside triphosphate or deoxyribonucleoside triphosphate) and suitable cofactors, particularly thermophilic. Template-dependent polymerases, especially thermophilic template-dependent polymerases with 5'-3'exonuclease activity;
Including
And / or a. The array segment is
i. The sequence is identical to the sequence of M1 located in the sequence segments M1.2, M1.3, and M1.4; or ii. Containing said sequence segment complementary to the sequence of M1 located at sequence segments M1.3 and M1.4.
And b. Fluorescent dye,
i. Form a donor quencher pair or FRET pair with a second probe oligonucleotide linked to a first probe oligonucleotide; or ii. Forming a donor quencher pair or FRET pair with a fluorescent dye linked to a second probe oligonucleotide that can bind in close proximity to the binding site of the first probe oligonucleotide.
The kit comprising a first probe oligonucleotide that binds to the fluorescent dye. A first template-dependent nucleic acid polymerase, especially a DNA polymerase, and optionally a substrate for the DNA polymerase (especially ribonucleoside triphosphate or deoxyribonucleoside triphosphate) and suitable cofactors, especially a medium-temperature template-dependent polymerase, especially 5 '-3' The kit according to claim 20, further comprising a medium temperature template-dependent polymerase having no exonuclease activity. The kit of claim 20 or 21, wherein the modified nucleotide building block comprises a 2'-O-alkylribonucleoside building block, particularly a 2'-O-methylribonucleoside building block.

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