JPH05192197A - Nucleic acid detection method and kit therefor - Google Patents

Abstract

PURPOSE:To provide a process for the assay of a nucleotide containing a specific multiple primer and to provide a kit therefor. CONSTITUTION:The subject assay is composed of several steps. A number of primers are bonded to a number of target sequences obtained from a biological sample and extended in the 5'-3' direction by an enzyme by capturing a residue from a mixture of a nucleoside triphosphate containing at least one nucleoside triphosphate modified to enable the detection or separation. The extension with the enzyme can continue beyond a separately started target site under specific conditions to result in the chain substitution dissociation of the separate extension product. The captured nucleoside modified to enable the detection is detected by a nonradioactive method after separating the extension product.

Description

Detailed Description of the Invention

[0001]

This invention relates to DNA technology and is intended for use as a technology in molecular biology, and clinical diagnostics and testing. In particular, it relates to sensitive assays and kits for amplifying and detecting low copy number target nucleotide sequences in nucleic acids (DNA, RNA, or both) of biological samples.

[0002]

2. Description of the Related Art Although DNA or RNA probe technology has some products on the diagnostic market,
Even today, it is still a means mainly for experimental research. Several companies have launched traditional types of DNA probes and kits in this market, and in recent years polymerase
Chain Reaction (PCR) probes have been approved for limited use in relevant laboratories for the detection and confirmation of HIV.

Classical protocols for detecting nucleic acids often involve immobilizing the target on a solid substrate, hybridizing a radiolabeled DNA probe to the sequence of the target, washing, and stabilizing. Detection of various hybrids (Sambrook, j, Fritsch, EF, and M
aniatis T. Molecular Cloning: a Laboratory Manual, 2n
ed., Cold Spring Harbor Laboratory Press, 1989).
A common assay method involves the detection of target nucleic acids on filter membranes (Meinkoth, J. and Wahl, G., Anal. Bioche.
m.138, pp267-284, 1984). Two types of this method are:
Southern blot hybridization or Northern blot hybridization. In the Southern type, the target D in which the DNA probe was immobilized on the filter
Bound to NA. In the Northern form, the target on the filter is RNA. In both methods, probe binding and detection methods are the same. Other types also exist. D
NA probe technology is used for nucleic acid detection in situ, eg, permea binding to microscope slides.
It is used to detect nucleic acids on bilized cells).

The probe used in the above assay can be labeled through incorporation or transfer of a component originally carried by a modified deoxynucleoside 5'triphosphate. Probes are often nick-translated (Kelly, RB et al.,
J. Biol. Chem. 245, pp39-45, 1970), or purified DN
Random priming of A fragment (Feinberg, AP. And V
ogelstein, B., Anal. Biochem. 132, pp6-13, 1983), or synthetic oligonucleotides 3'or 5
´ End labeling (Sambrook, J, Fritsch, EF, and M
aniatis, T.Molecular Cloning: A Laboratory Manual, 2n
ed., Cold Spring Harbor Laboratory Press, 1989). Labeled probes can also be produced by various types of chemical modifications (Symons, RH, Nucleic Acid Probes., CRC Press, Inc. 1).
989). Many recent assays include the polymerase chain reaction (PCR) (Mullis, US Pat. No. 4,683,202), the Q-beta replicase system (Lomeli, H. et al., Clin. Chem. 35). , pp1826-18
31, 1989), and a DNA-RNA-DNA probe cycling scheme (Duck, P. et al., BioTechniques 9,
pp142-147, 1990) etc. are included. These are each
The method for detecting a specific target nucleic acid generated at a low copy number is conceptually different. Amplification of a particular target sequence via a PCR-based method involves hybridization of several pairs of probes to the target, primer-enzyme synthesis of DNA copies, thermal denaturation of the strand, and multiple cyclic repeats of the process. Detection using PCR generally involves hybridizing the product with a labeled third probe. The Q-beta replicase system requires the presence of the target sequence to cause amplification of the RNA probe sequence itself. This method is PCR
Detection is done via an intercalating agent without the need for a thermal cycle such as. The DNA-RNA-DNA system involves using a single-stranded probe that is a chimeric nucleic acid having an RNA sequence with DNA located at each end. When this hybridizes with the DNA target, the enzyme R
Nase H can cut out the RNA portion of the probe. Since the DNA portion located on the side of the probe cannot remain in a hybridized state, it dissociates from the target. This can result in more of the above cycles. The production of short digested fragments of the probe is detected by gel electrophoresis.

A system for the detection of nucleic acid probes is
Often use autoradiography,
Alternatively, non-radioactive ones are possible (Symons, RH, Nucl
eicAcid Probes., CRC Press, Inc. 1989). Non-radioactive detection systems used in place of radioactive systems include fluorescent or enzymatic schemes involving the conversion of colorless compounds into a visible color, or the generation of light by bioluminescence or chemiluminescence. Improvements in chemiluminescence have now led to a level of sensitivity that is fairly close to that of radioactive methods. One such report is the chemiluminescent substance PPD (4
-Methoxy-4- (3-phosphate phenyl) spiro [1,2, -dioxetane-3,2'-adamantane]) is used (Bronstein, I. et al. Anal. Bioche
m.180, pp95-98, 1989).

A closer prior art to our invention is Vary e.
U.S. Pat. No. 4,851,331 by Tal. 1989. In this US patent, specific oligonucleotide primers are hybridized with a target sequence. DNA
Is enzymatically extended from the 3'end of the primer, resulting in the incorporation of labeled nucleotides. In that example, the product is then immobilized on a solid gel substrate and detected radioactively.

[0007]

The main object of the present invention is to:
It is to provide a sensitive and fast method for detecting the presence of a target nucleic acid by enzymatic synthesis which results in the amplification of a sequence complementary to the target.

While other nucleic acid probe methods are considerably more sensitive than immunological diagnostic methods, they have proven to be less suitable for the diagnostic market due to their relative complexity and slow procedure. The PCR method is highly sensitive but requires expensive temperature controlled heating / cooling units. The method also includes a final reprobing step that takes a long time. Similarly, the DNA-RNA-DNA probe system requires a gel electrophoresis step prior to final detection.

Another object of the invention is to provide a simple system that does not require temperature cycling and electrophoresis steps and does not include hybridizing the DNA probe with the product in the final reprobing step. ..

[0010]

The present invention is a method for amplifying and detecting a particular target sequence in nucleic acid of a biological sample, the method comprising:

(A) A plurality of oligonucleotide primers, which are a plurality of sets of sequences, and a nucleic acid derived from a biological sample containing both a target nucleotide sequence and a non-target nucleotide sequence, and the primer is used in a plurality of parts of the target nucleic acid. Contacting under conditions that form a hybrid by binding specifically and singly,

(B) by using one or more types of primer-dependent nucleic acid polymerases, the hybridized primer strands of the hybrid are advanced from the 3'end of each primer in the 5'-3 'direction. In which the unmodified and modified nucleoside triphosphates are incorporated into the strand that is complementary to the target strand from solution,

(C) by linking the extension product to a solid support coated with a ligand capable of binding the separably modified components incorporated into the product,
Separating the extension product of the primer from a non-target nucleotide sequence,

(D) The target originally present in the biological sample by quantifying the detectably modified nucleotide moieties incorporated into the extended chain during the synthesis of said chain by polymerase activity. A step of detecting the amount of sequence. Another object of the present invention is a kit for an amplification assay of a target sequence of a nucleic acid of a biological sample, comprising: (a) multiple sites of a target nucleotide sequence forming a template for a primer-dependent nucleic acid polymerase. A number of complementary primers,

(B) a mixture of nucleoside triphosphates necessary for primer synthesis, comprising at least one detectably modified or separably modified nucleoside triphosphate,

(C) A large number of primers can be synthesized at the 3'end and 5'to the complementary strand to the target nucleic acid
A kit comprising one or more sets of selected primer-dependent nucleic acid polymerases capable of extending in the −3 ′ direction.

Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be apparent from or may be understood by practice of the invention. Let's do it. The objects and advantages of the invention may be realized by means of the instruments and combinations particularly pointed out in the appended claims.

The preferred embodiments of the invention are illustrated by the use of the accompanying drawings, which are incorporated in and constitute a part of this specification, along with the means and examples for solving the problems set forth below.

There are four variants of the basic protocol which differ mainly in the method of immobilizing the product on the solid support and the method of detection. Therefore, other aspects of the protocol are different in each version to accommodate these differences. Version 1 is illustrated by Figures 1-3. This method involves incorporating modified dNTPs into the DNA that is synthesized for the target nucleic acid. The newly synthesized DNA 'is immobilized on a solid support. These are detected either by fluorescence or by first attaching a detection system via the modified residue and then adding a chemiluminescent substrate. In the isolation step, nucleic acid (total DNA or total RNA or both) is isolated from the patient and contains target sequence 1 (DNA or RNA). Next, the same or different primer 2 is added to the sample and hybridized with the target nucleic acid existing at many specific positions in the hybridization step. Examples of buffers for hybridization are shown in Table 4 below. The appropriate hybridization buffer to use depends on which enzyme is chosen for the extension step of the assay. Prior to the extension step, the appropriate amounts of ingredients are added to bring the final reaction conditions as illustrated in Table 5. Additives include buffer, strand-displacing polymerase, normal labeled deoxynucleoside 5'-triphosphate (dNT).
Ps) 3 and modified dNTPs4,5. An example of a suitable nucleotide mixture for the version 1-4 assay is shown in Table 1.

DNTPs are deoxyadenosine 5'-
Triphosphate (dATP), deoxycytidine 5'-triphosphate (dCTP), deoxyguanosine 5'-triphosphate (dGTP), deoxythymidine 5'-triphosphate (dT
TP). The modified dNTPs are any of the labeled dNTPs or deoxyuridine-5.
A derivative of ′ -triphosphate (dUTP) may also be used.

The primer preferably used here is
Should have a short nucleic acid sequence (eg 10-100 nucleotides) relative to the target nucleic acid sequence in the sample,
These may be other lengths. A specific primer means many different primers that can hybridize to different regions of a target nucleic acid strand, or many identical primers that hybridize to different regions of a target nucleic acid strand.

[0022]

[Table 1]

The combinations and concentrations of normal dNTPs3 and modified dNTPs 4, 5 shown in Table 1 are examples only. Other variations are possible. In the extension process,
As the polymerase extends the synthesis in the 5'-3 'direction from the 3'end of the primer, the analog is incorporated into the growing DNA complementary to the target, and the downstream double-stranded nucleic acid 6 is chain- Dissociated by displacement dissociation activity. Solid substrate 7 coated with a ligand 8 such as streptavidin
May be present from the beginning of the assay but should be added at this point. Then, dNTP4 modified to allow separation like biotinylated dNTP4.
Are bonded to the solid substrate 7 in the separation step. afterwards,
The solid substrate 7 is washed to remove unbound substances, unincorporated residues, and contaminants. Depending on the analog used in the reaction mixture, detection of the product is done in one of the following ways. Detectably modified dN
When fluorescently labeled dNTP is selected as TP5, the sample is exposed to one wavelength of the incident light 9 from the excitation lamp 10 and the fluorescence detection step of detecting the emitted light 11 at the second wavelength is performed directly. Alternatively, this is done by linking components of the detection system to the DNA containing the analog at this point. It is also possible to change the detection system in this basic version. If the detectably modified dNTP5 is a dNTP labeled with a hapten (eg, digoxigenin), an enzyme-labeled anti-hapten ligand 12 (eg, an anti-digoxigenin antibody conjugated with alkaline phosphatase) is added and bound. In the process, the ligand binds to the hapten. Following the wash, PPD (4-methoxy-4- (3
A chemiluminescent substrate such as -phosphate phenyl) spiro [1,2, -dioxetane-3,2'-adamantane] is added and detected by exposure to the film or by counting photons in the chemiluminescent process Is done.

Version 2 of the basic protocol is described below. Here, the conditions are essentially the same as for FIGS. 1-3, except that the separably modified dNTP is modified with a hapten such as digoxigenin, the solid substrate is coated with an anti-hapten antibody, and detection is performed. Differences in that the system contains a streptavidin-alkaline phosphatase complex that can bind to DNA via a biotinylated residue, or a fluorescent residue is used instead of the biotinylated residue (see Table 1). .. Otherwise the assay conditions are the same as for FIGS.

In another version of the assay, assay version 3, FIG. 4 shows the initiation (priming) with a labeled oligonucleotide. A biotin-labeled primer can immobilize newly synthesized DNA on a streptavidin-coated solid support. Primers such as (A) and (B) can be biotinylated internally via photobiotin or at the 3'or 5'ends. Two nucleotide mixtures compatible with biotinylated primers are shown in Table 1. The conditions are shown in Fig. 1.
Similar to basic assay ~ 3. Other means can be used to chemically modify the primer or other means for immobilizing the DNA to the solid support 7.

Version 4 of the basic protocol is
It is shown in FIGS. This version is used as an anti-ligand 8 for immobilizing the product on a solid substrate 7.
Double-stranded DNA antibody (anti-dsDNA antibody) 20 is used. Many features are the same as the other versions, but the initiation (priming) is required to occur from both strands of the double-stranded template 21. In the other three versions,
The use of double-stranded template is optional, but in this version it is highly desirable to use double-stranded template, as shown below. In version 4, another feature that differs from the other versions is that it was newly synthesized to allow displacement-dissociated amplification products to be captured with anti-dsDNA antibodies after strand-dissociation dissociation synthesis. The DNA has to be denatured into single-stranded DNA (ssDNA), which has to be rehybridized with newly synthesized complementary DNA. If the target is not a rare DNA, omit the denaturation and rehybridization steps,
Some ds extension products can be captured directly on the solid support 7 via the anti-dsDNA antibody 20. However, products whose strands have been dissociated will be excluded from the amplification process as described above. Table 1 shows this version 4
Shows a mixture of nucleotides compatible with. Also, the assay conditions are the same as for FIGS. Features associated with all versions of the fast DNA assay
Trait

7-10 relate to details of primer and target formation. The numbers of primers shown below for the various methods are only conceptual. Therefore,
The number of primers (and targets), as well as the specific combinations of repeated or different primers (repeated or different targets) shown below, can be varied from the conditions indicated. However, the greater the number of target sites that act as loci for primer hybridization, the greater the amount of synthesis and signal that can be achieved by these methods.

One feature applicable to versions 1 to 4 of the basic assay is, as shown in FIG. 7, different specific sites (a '), (b'), (c ') of the target sequence 1. And different sequences (A) that hybridize to (d '),
Use of specific primers of (B), (C), and (D) is included. Also, all conditions are similar to the basic protocol conditions.

FIG. 8 shows another feature applicable to versions 1-4. Here, the use of a primer of the same sequence (A), which has the same sequence (a) and hybridizes to a specific site of target 1 which is complementary to the primer is illustrated. This may provide the advantage of economically exploiting the presence of any repetitive sequences that may be present in Target 1. Thus, if a repeat sequence is present, a single primer sequence can be used to start at more than one site. All other conditions of the basic protocol can be applied here.

Another feature universally applicable to the four basic protocols is shown in FIG. 9, which is a combination of the features illustrated in FIGS. 7 and 8. This includes primers specific for both the same sequence (primer (A)) and different sequences (primers (B), (C) and (D)). Otherwise, this assay is identical to any of the basic protocols. Since we want to start from as many sites as possible in the target, this feature makes the basic method economical by hybridizing the same primers at repeat sites and hybridizing different primers where no repeats are present. Sex can be increased.

FIG. 10 shows the most commonly used version for versions 1-4. In this aspect,
The target is double-stranded (ds) nucleic acid (dsDNA or dsRN).
A) 21. This ds nucleic acid is denatured (strands are separated) by alkali, heat, etc. (when alkali is used, the pH is readjusted to 7.0 after that, the nucleic acid is precipitated, dried, and suspended again in a buffer solution. To). In the hybridization step, the primer (A),
(B), (C), (d), (e), (f), and (g)
And hybridize. The resulting binding to both strands causes priming from both strands to achieve maximization of the signal. The figure shows different multiplex primers (A), (C), (d), (e), (f), and (g).
And the same primer (B) both hybridize to the target sequence. The other assay conditions are the same as those described for FIGS.

Some examples of the requirements for the polymerase used are given in Table 2. Polymerases other than those listed in the table can also be used as long as they have the required properties.

[0033]

[Table 2] Figures 11-13 relate to the properties of the polymerase used.

The results of using the preferred polymerase are shown in FIG.
Shown in. The polymerase should be devoid of 5'-3 'or 3'-5' exonuclease degrading activity, and may be naturally occurring enzymes, or modified to lack these activities. In FIG. 12, for comparison, the results of using a polymerase having exonuclease activity are shown. A polymerase having exonuclease activity is
Since 5'-3 'exonuclease digestion product 25 and 3'-5' exonuclease digestion product 26 are removed from the hybrid product,
4 is not preferable.

An ideal polymerase should have strand-dissociation dissociation properties. As illustrated in FIG. 13, some outcomes of this process are displacement dissociated primer 30, displacement dissociated extension product 31, and partially displacement dissociated primer 32. Strand-dissociation dissociation results in more DNA synthesis than if the normal 5'-3 'synthetic movement of the polymerase is stopped by striking the nucleic acid of the downstream duplex. That is, the polymerase is able to move aside both the primer located downstream of the migrating enzyme and the newly synthesized DNA, and without physical dissociation of the upstream polymerase from the template. Must be able to continue to synthesize. According to this route, the amplification is D
Achieved through increased synthesis of NA, which also results in the uptake of higher amounts of modified dNTPs.

We have, for example, synthesized T4 DNA polymerase with a cofactor protein, T5 DNA polymerase, T5 DNA polymerase, to synthesize products by strand displacement dissociation, if necessary.
Type I T7 DNA polymerase, E. Klenow fragment of E. coli DNA polymerase, calf thymus DNA polymerase beta, or other strand-dissociating dissociative polymerases and cofactor proteins can be used.

Also, the enzyme selected should have high processivity and high polymerization rate. K
lenow fragment, Sequenase ™, and Tag
The properties of the three types of enzymes of DNA polymerase are shown in Table 3.

[0038]

[Table 3]

In addition to the need to have high processivity and polymerization rate, the polymerase compensates for any other property that reduces its efficiency and thus the selection of such enzymes for the assay. It must be possible to incorporate various analogs of deoxy-nucleoside-5'-triphosphate into the growing chain with sufficient efficiency to do so.

[0040]

【Example】

 <Synthesis of primer>

Primers OCWK1, OCWK2, and OCWK3 are synthesized on the Applied Biosystems Synthesizer. The sequence of the oligonucleotide OCWK1 is 5'-CCTGAGTGAGAACTTGCCTG.
It is C-3 '. OCWK2 is 5'-CGGTTTTA
GGCAAAGGGGGAGC-3 ', and OCWK3
Sequence is 5'-GGCTGAAGTCCAGAGTG
It is TCC-3 '. Primer OCWK1 was used to clone the cloned human HLA gene (ATCC clone 45.
1DRbeta008) is designed to anneal near the 3'-end of the antisense strand. Primer O
CWK2 and OCWK3 are prepared so as to hybridize to a site separated by 242 nucleotides on the sense strand of the same gene, or a corresponding site of mRNA synthesized from this gene. <Preparation of nucleic acid>

A. Pisum sativum va
r. Total cellular DNA and plasmid DNA from Alaska (including chromosomes and chloroplast DNA) (plasmid p carrying clone 45.1DRbeta008)
cDV1 over pL2) (American Type Culture Collection, Rockville, transcription synthesis of RNA in isolation, and in vitro of MD available from), all standard methods (Sambrook, J., Fritsh, EF , And Man
iatis, T.Molecular Cloning: a Laboratory Manual, 2nd
ed., T. Cold Spring Harbor Laboratory Press, 1989).

B. Mix target plasmid DNA, RNA, or both with 10 mg total cellular DNA and
After denaturing by alkaline treatment in 80 mM NaOH, 180 μM EDTA for 5 minutes, it is neutralized with 1 / 7.3 volume of 3 M sodium acetate (pH 4.8). Then add 2.3 volumes of cold ethanol to the nucleic acid and
Cool for 0 minutes and allow to settle. After centrifugation at 5 ° C for 10 minutes, the pellet is washed with cold 70% ethanol, centrifuged at 5 ° C for 5 minutes, lyophilized and resuspended in the appropriate annealing buffer shown in Table 4 immediately before use. <General conditions for hybridization>

Primers (eg, 5.1 pmol each) were added to 10 μl for 15 minutes at 37 ° C. as shown in Table 4.
Hybridize with denatured target nucleic acid (in 10 mg denatured chromosomal DNA) in IX annealing buffer.

[0045]

[Table 4] <Conditions for primer synthesis reaction> The enzymatic reaction is carried out under the final reaction conditions shown in Table 5. Enzymes are used alone or in pairs (see footnote in Table 5 for primer properties).

Taq DNA Polymerase and M-MLV
For the reaction containing RT, first, incubation is carried out at 37 ° C. for 5 minutes to allow the M-MLV RT enzyme to react, and then the temperature is shifted to 72 ° C. to react with Taq DNA polymerase.

[0047]

[Table 5] <Covalent coating of plate with anti-digoxigenin antibody>

Anti-digoxigenin antibody was applied to Neurat.
h and Stick, 1981 (J. Virol. Met.
Hods 3, pp155-165, 1981) and covalently linked to Microlite ™ (Dynatech Laboratories, Inc.), an opaque 96-well microtiter plate. Wells are incubated with methanesulfonic acid overnight, washed with distilled water and dried. The microplate is incubated with a 1: 1 mixture of glacial acetic acid and fuming nitric acid at 40 ° C. for 4 hours. The treated surface is rinsed with H ₂ O until the pH of the wash solution is above 6. Wells with 500 mM Na
Treat with 0.5% Na ₂ S ₂ O _{4 in} OH at 50 ° C. for 1 hour. After extensive washing, the plates are treated with 1% glutaraldehyde in 50 mM phosphate buffer (pH 8.5) for 2 hours at room temperature and then incubated for 16 hours at 4 ° C. After further washing, anti-digoxigenin antibody (100 μg / ml) in 50 mM phosphate buffer was added at 4 ° C.
Allow to bind to wells overnight. After that, the well is 1M
Treat with glycine-100 mM borate buffer (pH 8.5) for 4 hours at room temperature. Finally 10m plate
M Tris-HCl (pH 7.2), 0.9% NaC
Wash with 1 and store in the same buffer with 0.02% NaN ₃ . <Analysis of film by densitometer>

The film (Kodak XAR-5) exposed to the filter containing the chemiluminescent dots of the sample used in Example 2 below is analyzed by the following procedure. Any relative intensity values are determined by comparing the densitometer data for each dot with the data derived from the dots representing the serial dilutions of the standard chemiluminescent sample. The "on-scale" intensity value of the longest exposed film correlates with data derived from the same dot on another short-exposed film. This allows the data over the entire range from the lowest intensity dots to the highest intensity dots to be linearly correlated, compensating for the non-linearities resulting from overexposure. Example 1 DN in a reactor coated with anti-digoxigenin antibody
Detection of enzymatic synthesis initiated from A and / or RNA targets

The following are the results of a typical assay using a non-clinical set of probes and targets. In this assay, RNA and DNA targets are isolated and denatured as described above in the presence of chromosomal DNA. Primer OCW
K1, OCWK2, and OCWK3 are hybridized with the target DNA and / or RNA in 10 μl of an appropriate annealing buffer. These hybridizations and sequential reactions are performed either in separate tubes or in opaque 96-well microtiter plates coated with antibody. After annealing the probe and target nucleic acid, 10 μl of a suitable reaction mixture containing biotinylated and digoxigenin-labeled dNTPs
1 is added to bring the ingredients to the final concentrations listed in Table 5.
Paired enzymes are added and primed from RNA and DNA targets, respectively, according to the primer properties and temperatures shown in Table 5. When the reaction is carried out in antibody-coated microtiter plates, binding of the digoxigenin-containing DNA occurs during the primer synthesis incubation. When carrying out the reaction in normal test tubes, the nucleic acid is then precipitated with ethanol and
Samples of labeled DNA were resuspended in 0 mM Tris (pH 7.5), 1 mM EDTA and labeled with 25
The wells are allowed to bind to the anti-digoxigenin antibody coated at 15 ° C. for 15 minutes. After binding, the wells were placed in blocking buffer (0.2% casein, 80 mM NaHPO ₄ (p
Wash twice with H7.5), 7 mM NaCl). Streptavidin-alkaline phosphatase complex (B
(Available from RL),
Add 0.17 units alkaline phosphatase / 1 ml blocking buffer and allow adsorption of binding for 30 minutes at 25 ° C. The microtiter plate wells were then washed once with blocking solution, 80 mM NaHPO ₄
(PH 7.5), 7 mM NaCl, 0.25% Twe
Wash 3 times with en-20 and then assay buffer (0.
1M Tris-HCl (pH 9.5), 1 mM MgC
l ₂ ) wash 3 times. Finally, the detection substrate is 3 μM
Add as 75 μl of assay buffer containing PPD. The positive reaction is chemiluminescence, which is detected with a luminometer (Microlite ™ ML1000 Microplate Luminometer, Dynatech Laboratories).

Table 6 shows the results of Klenow and M.M. lutes
Figure 3 shows typical results of experiments for detection of DNA and / or RNA in a single reaction mixture using u DNA polymerase.

[0052]

[Table 6] Example 2 Demonstration of Strand-Displacement Dissociation Synthesis Initiated by Multiplex Oligonucleotides Hybridized to Target Nucleic Acid

Each of the primers OCWK2 and OCWK3, or both, are hybridized with 25 fmole of the target nucleic acid in the presence of chromosomal DNA as described above. After hybridization, 10 μl of the appropriate reaction mixture containing only one of the polymerases and biotin-14-dATP is added to give the final concentrations shown in Table 5. The reaction is allowed to proceed at 37 ° C and, in the case of Taq DNA polymerase, the temperature is then raised to 72 ° C for 1 minute. The reaction is stopped by taking aliquots of the reaction at regular intervals and diluting 1/100 in 1M sodium acetate (pH 7.0). The diluted aliquot is then heat denatured at 100 ° C. for 3 minutes and immediately placed in an ice bath. These are dot blotted onto a nylon membrane presoaked with 1M sodium acetate. Membranes are air dried and processed using the PPD-based PhotoGene ™ nucleic acid detection system protocol sold by BRL / Life Technologies.
Briefly, this involves incubating the filters in a blocking solution containing heat-inactivated BSA and running the detection system (streptavidin-alkaline phosphatase complex) in a buffer containing Tween-20. Bind, wash, add detection reagent containing PPD and enhancer. The detection of chemiluminescent dots is performed by exposing the film directly to Kodak XAR-5 film for a short time or for a long time, and the measurement is performed by scanning the film with a densitometer. A typical experiment with Taq DNA polymerase (Table 7) shows strand displacement dissociation. Displacement dissociation of the strands in the reaction involving the primers OCWK2 and OCWK3 results in individually initiated nearly additive synthetic reactions. If no displacement dissociation occurs,
Due to the proximity of the primers, the double-stranded primer reaction results in a fairly short stoichiometric synthesis.

[0054]

[Table 7]

In its broader aspects, the invention is not limited to the specific details and examples described herein. Additional advantages and modifications can be readily made by those skilled in the art. Therefore, various modifications may be made without departing from the spirit and scope of the general inventive concept as defined in the claims.

[Brief description of drawings]

FIG. 1 shows version 1 of the basic rapid DNA probe assay of the present invention.

FIG. 2 shows version 1 of the basic rapid DNA probe assay of the present invention.

FIG. 3 shows version 1 of the basic rapid DNA probe assay of the present invention.

FIG. 4 shows version 3 of the basic DNA probe assay of the present invention.

FIG. 5 shows version 4 of the basic DNA probe assay of the present invention.

FIG. 6 shows version 4 of the basic DNA probe assay of the present invention.

FIG. 7 shows hybridization with multiple primers of different sequences used in versions 1-4 of the invention.

FIG. 8 shows hybridization with multiple primers of the same sequence used in versions 1-4 of the present invention.

FIG. 9 shows hybridization using multiple primers of the same or different sequences used in versions 1 to 4 of the present invention.

FIG. 10 shows the processing of double-stranded DNA or RNA targets used in versions 1-4 of the present invention.

FIG. 11 is a diagram showing DNA synthesis by a polymerase lacking exonuclease activity.

FIG. 12: 5 ′ to 3 ′ by polymerases having 5′-3 ′ and 3′-5 ′ exonuclease activity simultaneously.
Showing DNA synthesis into DNA.

FIG. 13 is a diagram showing DNA synthesis by a polymerase having strand-dissociation dissociation activity.

[Explanation of symbols]

1 ... Target sequence, 2 ... Primer, 3 ... dNTPs, 4 ...
Modified dNTPs, 5 ... Modified dNTPs, 6
... Downstream double-stranded nucleic acid, 7 ... Solid substrate, 8 ... Ligand, 9
... incident light, 10 ... excitation lamp, 11 ... emitted light, 12 ... anti-hapten ligand, 20 ... anti-double-stranded DNA antibody, 21 ...
Double-stranded nucleic acid, 25 ... 5'-3 'exonuclease digestion product, 26 ... 3'-5' exonuclease digestion product, 30 ... Dissociated primer, 31 ... Dissociated extension product, 32 ... Part Extension products that are dissociated.

Claims (26)

[Claims] 1. A method for amplifying and detecting a specific target sequence in a nucleic acid of a biological sample, comprising: (a) a plurality of oligonucleotide primers which are a plurality of sets of sequences, and a target nucleotide sequence and a non-target nucleotide sequence. Contacting a nucleic acid from a biological sample that contains both target nucleotide sequences under conditions such that the primer singly and specifically binds to multiple parts of the target nucleic acid to form a hybrid; ) Extending one or more types of primer-dependent nucleic acid polymerases so that the hybridized primer strands of the hybrid proceed from the 3'end of each primer in the 5'-3 'direction. Which allows unmodified and modified nucleoside triphosphates to be introduced from solution into the strand complementary to the target strand. A non-target nucleotide sequence by attaching the extension product to a solid support coated with a ligand capable of binding the separably modified component incorporated into the product. Separating the extension product of the primer from the organism, and (d) quantifying the detectably modified nucleotide moiety incorporated into the extended strand during the synthesis of the strand by polymerase activity. Detecting the amount of target sequence initially present in the biological sample. 2. A strand capable of extending the synthesized complementary nucleic acid beyond the 5 ′ end of the primer-synthesized strand at a distance where the primer-dependent nucleic acid polymerase is located.
-The method according to claim 1, which is a displacement dissociation enzyme, whereby a distant nucleic acid is dissociated from the double-stranded state and a sequence complementary to the original target nucleic acid is amplified in excess of that amount. 3. The method according to claim 1, wherein the primer-dependent nucleic acid polymerase lacks exonuclease degrading activity. 4. The method according to claim 1, wherein the step of denaturing the nucleic acid of the biological sample is performed prior to the contacting step (a). 5. The method according to claim 4, wherein the nucleic acid is double-stranded. 6. The method of claim 5, wherein the target sequence is on the complementary strand of the nucleic acid. 7. An extension product is synthesized using a strand displacement dissociative nucleic acid polymerase and then the nucleic acid is denatured.
The method according to claim 6, wherein the displacement product dissociated and dissociated is re-hybridized to a complementary strand, and is bound to a solid substrate via a ligand that is an anti-double-stranded nucleic acid antibody. 8. The method of claim 1, wherein the target nucleic acid sequence is RNA and the primer-dependent nucleic acid polymerase is reverse transcriptase. 9. The method of claim 1, wherein the target nucleic acid sequence is DNA and the primer-dependent nucleic acid polymerase is DNA polymerase. 10. The target nucleic acid sequence is DNA or RNA.
And the primer-dependent nucleic acid polymerase is both a reverse transcriptase and a DNA polymerase. 11. The method according to claim 1, wherein the plurality of primers have the same sequence and are complementary to a plurality of sites of the target nucleic acid. 12. The method according to claim 1, wherein the plurality of primers have non-identical sequences and are complementary to a plurality of sites of the target nucleic acid. 13. The method according to claim 1, wherein the plurality of primers have identical and non-identical sequences and are complementary to a plurality of sites of the target nucleic acid. 14. The method of claim 1, wherein the modified nucleoside triphosphate is separably and detectably modified. 15. The method of claim 1, wherein the primer is a binding agent-modified primer and the mixture of nucleoside triphosphates contains detectably modified nucleoside triphosphates. 16. The method of claim 1, wherein said primer is a primer modified with a detectable substance and said mixture of nucleoside triphosphates comprises separably modified nucleoside triphosphates. 17. The method of claim 1, wherein the separably modified nucleoside triphosphate is biotin and the ligand used to bind the extension product is avidin or streptavidin. 18. The method of claim 1, wherein the ligand is an antibody that binds an extension product having a hapten as a separably modified nucleotide. 19. Detection is incorporated during the synthesis of extension products from a mixture containing detectably modified triphosphates, or by using a primer modified with a detectable substance. The method of claim 1, wherein the method is performed via a fluorescent component. 20. Detection is accomplished via a chemiluminescent format using a detectably modified nucleoside triphosphate incorporated into an extension product or a detectably modified primer, said modification comprising chemiluminescence. 2. The method of claim 1, which is a reactive group or component capable of chemically interacting as part of a luminescence detection system. 21. Detection is accomplished via a chemiluminescent format using a detectably modified nucleoside triphosphate incorporated into an extension product or a detectably modified primer, wherein the extension results in an extension. The method of claim 1, wherein the product is capable of binding, directly or indirectly, to a component that forms a complex with an enzyme capable of catalyzing a reaction that produces a chemiluminescent product. 22. The detectably modified group of a nucleic acid product is a hapten that binds to an antibody, the antibody being capable of forming a complex with an enzyme, or with an antibody further bound to an enzyme. 22. The method of claim 21, which is 23. The extension product is modified so that it can be detected with biotin, and the biotinylated residue further forms a conjugate or a complex with an enzyme, or a complex having a detection component. 22. The method of claim 21, wherein a complex can be formed with an avidin or streptavidin component capable of interacting. 24. A kit for an amplification assay of a target sequence of a nucleic acid in a biological sample, comprising: (a) complementary to multiple sites of a target nucleotide sequence forming a template for a primer-dependent nucleic acid polymerase. A number of primers, (b) a mixture of nucleoside triphosphates necessary for primer synthesis, comprising at least one detectably modified or separably modified nucleoside triphosphate, and (c) a number of primers Comprising one or more sets of selected primer-dependent nucleic acid polymerases capable of initiating synthesis at the 3'end and extending the complementary strand to the target nucleic acid in the 5'-3 'direction. Kit to do. 25. The primer-dependent nucleic acid polymerase can dissociate a remotely initiated strand from a duplex by continuing the synthesis of a closely initiated strand, and associated therewith complementary to a target nucleic acid. That can amplify various nucleic acids-
The kit according to claim 24, which is a displacement-dissociative polymerase. 26. The method of claim 2, wherein a cofactor protein is used as required for enhancing or activating the strand-dissociation dissociation properties of the primer-dependent nucleic acid polymerase.