JP3145169B2 - Nucleic acid detection method and kit - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

This invention relates to DNA technology and is intended for use as a technique in molecular biology and in 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 a biological sample.

[0002]

2. Description of the Related Art DNA or RNA probe technology is notwithstanding several products on the diagnostic market,
Even today, it is still mainly a tool for experimental research. Several companies have launched traditional types of DNA probes and kits in this market, and in recent years
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 radioactively labeled DNA probe to the target sequence, washing, and stabilizing. Detecting hybrids (Sambrook, j, Fritsch, EF, and M
aniatis T. Molecular Cloning: a Laboratory Manual, 2n
ed., Cold Spring Harbor Laboratory Press, 1989).
A typical assay involves detection of a target nucleic acid on a filter membrane (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, a DNA probe is immobilized on a target D
Bound to NA. In the Northern type, the target on the filter is RNA. In both methods, the probe binding and detection methods are the same. Other types also exist. D
NA probe technology is used to detect nucleic acids in situ, such as permeable cells (permea
Used to detect nucleic acids on bilized cells).

[0004] Probes used in the above assays can be labeled through incorporation or translocation of components originally carried by the modified deoxynucleoside 5 'triphosphate. Probes are often generated by nick translation (Kelly, RB et al.,
J. Biol. Chem. 245, pp39-45, 1970), or purified DN
Random priming of fragment A (Feinberg, AP. And V
ogelstein, B., Anal. Biochem. 132, pp6-13, 1983) or 3 'or 5 of synthetic oligonucleotides.
´Terminal 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 generated by various types of chemical modification (Symons, RH, Nucleic Acid Probes., CRC Press, Inc. 1).
989). Many recent assays include the polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202 to Mullis), 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,
pp. 142-147, 1990). These are each
It is conceptually different as a method for detecting a specific target nucleic acid generated at a low copy number. Amplification of a particular target sequence via a PCR-based method involves hybridization of several pairs of probes to the target, enzymatic synthesis of DNA copy primers, heat denaturation of the strand, and multiple cycling of the process. Detection using PCR generally involves hybridizing the product to a labeled third probe. The Q-beta replicase system requires the presence of a target sequence to effect amplification of the RNA probe sequence itself. This method uses PCR
The detection is performed via an intercalating agent without the need for a thermal cycle as described above. DNA-RNA-DNA systems include the use of single-stranded probes that are chimeric nucleic acids having RNA sequences with DNA located at each end. When this hybridizes to the DNA target, the enzyme R
Nase H can excise the RNA portion of the probe. The DNA portion located on the side of the probe cannot remain in a hybridized state and is dissociated from the target. As a result, more cycles can be generated. The formation of a short digested fragment of the probe is detected by gel electrophoresis.

[0005] A system for detecting a nucleic acid probe comprises:
Often uses autoradiography,
Alternatively, non-radioactive ones are also 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 that involve the conversion of a colorless compound to a visible color or the generation of light by bioluminescence or chemiluminescence. Improvements in chemiluminescence methods have now led to levels of sensitivity that are quite close to radioactive methods. One such report is the chemiluminescent substance PPD (4
-Methoxy-4- (3-phosphatephenyl) spiro [1,2, -dioxetane-3,2'-adamantane]) (Bronstein, I. et al. Anal. Bioche).
m.180, pp95-98, 1989).

[0006] The closer prior art of our invention is Vary e
No. 4,851,331 to T. al.1989. In this patent, certain oligonucleotide primers are hybridized to a target sequence. DNA
Is enzymatically extended from the 3 'end of the primer, resulting in incorporation of the labeled nucleotide. In that embodiment, the product is then immobilized on a solid gel substrate and detected radioactively.

[0007]

SUMMARY OF THE INVENTION The main object of the present invention is to:
It is an object of the present invention to provide a sensitive and fast method for detecting the presence of a target nucleic acid by enzymatic synthesis resulting in the amplification of a sequence complementary to the target.

Other nucleic acid probe methods, while considerably more sensitive than immunological diagnostic methods, have proven to be less suitable for the diagnostic market because of their rather complex and slow procedures. The PCR method is sensitive, but requires an expensive temperature controlled heating / cooling unit. The method also includes a final reprobing step that takes a long time. Similarly, DNA-RNA-DNA probe systems require a gel electrophoresis step before final detection.

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

[0010]

SUMMARY OF THE INVENTION The present invention is a method for amplifying and detecting a specific target sequence in a nucleic acid of a biological sample, comprising:

(A) A plurality of sets of sequences of oligonucleotide primers and a nucleic acid derived from a biological sample containing both a target nucleotide sequence and a non-target nucleotide sequence are combined in a plurality of portions of the target nucleic acid. Contacting under conditions such that they bind alone and specifically to form a hybrid;

(B) using one or more types of primer-dependent nucleic acid polymerases to cause the hybridized primer strands of the hybrid to proceed in a 5'-3 'direction from the 3' end of each primer. Elongating the unmodified and modified nucleoside triphosphates from solution into said strand complementary to the target strand,

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

(D) quantifying the detectably modified portion of the nucleotides incorporated into the extended strand during the synthesis of said strand by polymerase activity, thereby allowing the target originally present in the biological sample to be quantified. Detecting the amount of the sequence. Another object of the present invention is a kit for an assay for the amplification of a target sequence of a nucleic acid in a biological sample, comprising: (a) providing a template for a primer-dependent nucleic acid polymerase; A large number of complementary primers,

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

(C) Synthesis can be initiated at the 3 'end of a large number of primers, and a 5'
It is an object of the present invention to provide a kit comprising one or more sets of selected primer-dependent nucleic acid polymerases that can be extended 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 obvious from the description, or may be learned by practice of the invention. Would. The objects and advantages of the invention may be realized by means and combinations particularly pointed out in the appended claims.

Preferred embodiments of the present invention will be illustrated by the accompanying drawings, which are incorporated in and constitute a part of this specification, together with the means and examples for solving the problems described below.

There are four variants of the basic protocol, differing mainly in the method of immobilizing the product on the solid support and the method of detection. Thus, other aspects of the protocol are different in each version to accommodate these differences. Version 1 is illustrated by FIGS. The method includes incorporating the modified dNTPs into DNA synthesized for a target nucleic acid. The newly synthesized DNA 'is immobilized on a solid support. These are detected either by fluorescence or by first binding the 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, which contains target sequence 1 (DNA or RNA). Next, the same or different primers 2 are added to the sample and hybridized to target nucleic acids present at many specific positions in the hybridization step. Examples of the buffer for hybridization are shown in Table 4 below. The appropriate hybridization buffer to use depends on which enzyme is selected for the extension step of the assay. Prior to the extension step, the appropriate amounts of components are added to 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. Examples of suitable nucleotide mixtures for Versions 1-4 assays are shown in Table 1.

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

The primers preferably used here are:
It should have a nucleic acid sequence that is shorter (eg, 10-100 nucleotides) compared to the target nucleic acid sequence in the sample,
These may be other lengths. By specific primer is meant many different primers that can hybridize to different regions of the target nucleic acid strand, or many identical primers that hybridize to different regions of the target nucleic acid strand.

[0022]

[Table 1]

The combinations and concentrations of normal dNTPs3 and modified dNTPs4,5 shown in Table 1 are only examples. Other variations are possible. In the elongation process,
As the polymerase extends synthesis in the 5'-3 'direction from the 3' end of the primer, the analog is incorporated into growing DNA complementary to the target, and the downstream double stranded nucleic acid 6 becomes strand- It is dissociated by displacement dissociation activity. Solid substrate 7 coated with ligand 8 such as streptavidin
May be present at the beginning of the assay but should be added at this point. Then, dNTP4 modified to allow separation like biotinylated dNTPs
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, the detection of the product is performed in one of the following ways. DN modified to be detectable
When a 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 emitted light 11 is detected directly at the second wavelength in the fluorescence detection step. Alternatively, this may be done by coupling the components of the detection system to the DNA containing the analog at this point. In this basic version, it is also possible to change the detection system. 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 to alkaline phosphatase) is added and bound. In the process, the ligand binds to the hapten. Following washing, PPD (4-methoxy-4- (3
A chemiluminescent substrate such as -phosphatephenyl) spiro [1,2, -dioxetane-3,2'-adamantane] is added and detected by exposing the film or by counting photons in the chemiluminescent process. Is performed.

Version 2 of the basic protocol is described below. Here, the conditions are essentially the same as for FIGS. 1-3, except that the modified dNTPs are modified with a hapten such as digoxigenin, the solid substrate is coated with an anti-hapten antibody and detected. The difference is that the system includes a streptavidin-alkaline phosphatase complex that can bind to DNA via a biotinylated residue, or uses a fluorescent residue instead of a 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 initiation (priming) with labeled oligonucleotides. Biotin-labeled primers can immobilize newly synthesized DNA on a streptavidin-coated solid support. Primers such as (A) and (B) can be biotinylated internally or via the 3 'or 5' end via photobiotin. Table 1 shows a mixture of two nucleotides that are compatible with the biotinylated primer. The conditions are shown in FIG.
Similar to the basic assays of Nos. 1-3. Other methods for chemically modifying the primer by other methods and for immobilizing the DNA on the solid support 7 can be used.

Version 4 of the basic protocol is:
This is shown in FIGS. This version provides an anti-antibody as a ligand 8 for immobilizing the product on a solid substrate
A double-stranded DNA antibody (anti-dsDNA antibody) 20 is used. Many features are the same as in other versions, but require that priming be performed from both strands of the double-stranded template 21. In the other three versions,
The use of a double-stranded template is optional, but in this version the use of a double-stranded template is highly desirable, as described below. Another feature that differs from the other versions in version 4 is that a newly synthesized, displaced dissociated amplification product can be captured using an anti-dsDNA antibody after strand-displacement dissociation synthesis. That is, the DNA must be denatured into single-stranded DNA (ssDNA), which must 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 to the solid support 7 via the anti-dsDNA antibody 20. However, the products whose strands have been dissociated will be excluded from the amplification process as described above. Table 1 shows this version 4.
Shows a nucleotide mixture that conforms to The assay conditions are the same as those for FIGS. Features relevant to all versions of high-speed DNA assays
Sign

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

One feature applicable to versions 1-4 of the basic assay is that, as shown in FIG. 7, different specific sites (a '), (b'), (c '), And (d ') different sequences hybridizing to (A),
This includes using specific primers of (B), (C), and (D). All conditions are the same as those of the basic protocol.

FIG. 8 shows other features applicable to versions 1-4. Here is illustrated the use of a primer of the same sequence (A) that has the same sequence (a) and hybridizes to a specific site of target 1 that is complementary to the primer. This may have the advantage of economically utilizing the presence of any repetitive sequences that may be present in target 1. Thus, if a repetitive 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 that can be universally applied to the four basic protocols is shown in FIG. 9, which is a combination of the features illustrated in FIGS. This includes primers specific for both the same sequence (primer (A)) and different sequences (primers (B), (C) and (D)). Otherwise, the assay is identical to any of the basic protocols. Since we want to start from as many sites on the target as possible, this feature is an economic of the basic method by hybridizing the same primers at repeated sites and hybridizing different primers where no repeats are present. Performance can be increased.

FIG. 10 shows the most commonly used version for versions 1-4. In this aspect,
The target is a double-stranded (ds) nucleic acid (dsDNA or dsRN
A) It is 21. This ds nucleic acid is denatured (strands are separated) by alkali, heat, etc. (if alkali is used, the pH is then readjusted to 7.0, the nucleic acid is precipitated, dried and suspended again in buffer. Do). In the hybridization step, the primer (A)
(B), (C), (d), (e), (f), and (g)
And hybridized. The resulting binding to both strands causes priming from both strands and achieves signal maximization. The figure shows different multiplex primers (A), (C), (d), (e), (f), and (g).
And the same primer (B) hybridizes to the target sequence. 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 can also be used as long as they have the required properties.

[0033]

[Table 2] Figures 11 to 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 lack 5'-3 'or 3'-5' exonuclease degrading activity, and may be a naturally occurring enzyme or one which has been modified to lack these activities. FIG. 12 shows the results of using a polymerase having exonuclease activity for comparison. A polymerase having exonuclease activity is
Since the digestion product 25 by 5′-3 ′ exonuclease and the digestion product 26 by 3′-5 ′ exonuclease are removed from the hybrid product, version 1
4 is not preferred.

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

[0036] We may use, for example, T4 DNA polymerase with cofactor protein, T5 DNA polymerase,
Type I T7 DNA polymerase, E. coli. Klenow fragments of E. coli DNA polymerase, bovine thymus DNA polymerase beta, or other strand-displacement dissociative polymerases and cofactor proteins can be used.

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

[0038]

[Table 3]

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

[0040]

[Example] <Synthesis of primer>

The primers OCWK1, OCWK2, and OCWK3 are synthesized on an Applied Biosystems synthesizer. The sequence of oligonucleotide OCWK1 is 5'-CCTGAGTGAGACTTGCCTG
C-3 '. OCWK2 is 5'-CGGTTTA
GGCAAAGGGGGAGC-3 ′ and OCWK3
The sequence of 5′-GGCTGAAGTCCAGAGTG
TCC-3 '. Primer OCWK1 was cloned from the 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 chromosomal and chloroplast DNA) (plasmid p carrying clone 45.1 DRbeta008).
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 of total cellular DNA and mix
After denaturation by alkaline treatment in 80 mM NaOH, 180 μM EDTA for 5 minutes, the mixture is neutralized with 1 / 7.3 volume of 3M sodium acetate (pH 4.8). Thereafter, the nucleic acid was added by adding 2.3 volumes of cold ethanol.
Cool for 0 minutes and allow to settle. After centrifugation at 5 ° C. for 10 minutes, the pellet is washed with 70% cold 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>

As shown in Table 4, 10 μl of the primers (for example, 5.1 pmol each) was added at 37 ° C. for 15 minutes.
With denatured target nucleic acid (in 10 mg of denatured chromosomal DNA) in IX annealing buffer.

[0045]

[Table 4] <Conditions for Primer Synthesis Reaction> The enzyme reaction is performed under the final reaction conditions described in Table 5. Enzymes are used alone or in pairs (see footnotes in Table 5 for primer properties).

Taq DNA polymerase and M-MLV
For the reaction including RT, first, the mixture is incubated at 37 ° C. for 5 minutes, and after the M-MLV RT enzyme is reacted, the temperature is shifted to 72 ° C. in order to react Taq DNA polymerase.

[0047]

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

An anti-digoxigenin antibody was purchased from Neurat
h and Stick, 1981 (J. Virol. Met).
hods 3, pp155-165, 1981), is covalently linked to Microlite ™ (Dynatech Laboratories), an opaque 96-well microtiter plate. The wells are incubated overnight with methanesulfonic acid, 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, until the pH of the wash solution is 6 or more rinsed with H ₂ O. 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 at 4 ° C. for 16 hours. After further washing, anti-digoxigenin antibody (100 μg / ml) in 50 mM phosphate buffer was added at 4 ° C.
To bind wells overnight. Then the wells are
Treat with glycine-100 mM borate buffer (pH 8.5) at room temperature for 4 hours. Finally the plate is 10m
M Tris-HCl (pH 7.2), 0.9% NaC
and store in the same buffer with 0.02% NaN ₃ . <Film densitometer analysis>

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 data derived from the dots representing a series of dilutions of a 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 data over the entire range from the lowest intensity dots to the highest intensity dots to be linearly correlated, compensating for non-linearities resulting from overexposure. Example 1 DN in a reactor coated with anti-digoxigenin antibody
Detection of enzyme synthesis initiated from A and / or RNA targets

The following shows 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 target DNA and / or RNA in 10 μl of appropriate annealing buffer. These hybridizations and sequential reactions are performed either in separate tubes or in opaque 96-well microtiter plates coated with antibodies. After annealing the probe and target nucleic acids, a suitable reaction mixture containing biotinylated and digoxigenin-labeled dNTPs, 10 μl,
Add 1 to bring the components to the final concentrations listed in Table 5.
A pair of enzymes is added and primed from the RNA and DNA targets, respectively, according to the primer properties and temperature shown in Table 5. If the reaction is performed in a microtiter plate coated with the antibody, binding of the DNA containing digoxigenin occurs during the incubation of the primer synthesis. When performing the reaction in a normal test tube, the nucleic acid is then precipitated with ethanol and
Resuspend in 0 mM Tris (pH 7.5), 1 mM EDTA, and label DNA sample
Allow to bind anti-digoxigenin antibody coated on the wells for 15 minutes at C. After binding, the wells were blocked with blocking buffer (0.2% casein, 80 mM NaHPO ₄ (p
H7.5), 2 × with 7 mM NaCl). Streptavidin-alkaline phosphatase complex (B
(Available from RL)
Add 0.17 units of alkaline phosphatase / 1 ml of blocking buffer and allow binding to occur at 25 ° C. for 30 minutes. 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 three times with en-20, then assay buffer (0.
1 M Tris-HCl (pH 9.5), 1 mM MgC
Wash 3 times with l ₂ ). Finally, 3 μM of the detection substrate was used.
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 Klenow and M.K. lutes
Figure 4 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 Multiple Oligonucleotides Hybridized to a Target Nucleic Acid

Each of the primers OCWK2 and OCWK3, or both of them, is hybridized with a 25 f mole 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 a final concentration as 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. Aliquots of the reaction are taken at regular intervals and diluted 1/100 in 1M sodium acetate (pH 7.0) to stop the reaction. The diluted aliquot is then heat denatured at 100 ° C. for 3 minutes and immediately ice bathed. These are dot blotted onto a nylon membrane presoaked with 1 M sodium acetate. The membrane is air dried and processed using the PPD-based PhotoGene ™ nucleic acid detection system protocol available from 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 conjugate) in a buffer containing Tween-20. Binding, washing, and adding detection reagents and enhancers containing PPD. The detection of chemiluminescent dots is performed by exposing the film to Kodak XAR-5 film for a short time or a long time, and the measurement is performed by scanning the film using a densitometer. A typical experiment using Taq DNA polymerase (Table 7) shows strand displacement dissociation. Displacement dissociation of the strands in a reaction involving the primers OCWK2 and OCWK3 results in substantially additional synthetic reactions initiated individually. If no dissociation occurs,
Since the primers are nearby, the duplex primer reaction results in a rather short stoichiometric synthesis.

[0054]

[Table 7]

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

[Brief description of the drawings]

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

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

FIG. 3 shows version 1 of the basic fast 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 using multiple primers of different sequences used in versions 1 to 4 of the present invention.

FIG. 8 is a diagram showing hybridization using multiple primers having the same sequence used in versions 1 to 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 to 4 of the present invention.

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

FIG. 12: 5 ′ to 3 ′ by polymerase having simultaneous 5′-3 ′ and 3′-5 ′ exonuclease activity
FIG.

FIG. 13 is a diagram showing DNA synthesis by a polymerase having strand-displacement 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 ... emission 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 Elongation products that have been dissociated.

──────────────────────────────────────────────────の Continuation of the front page (56) References JP-A-59-62600 (JP, A) JP-A-4-504203 (JP, A) Biol. Chem. , 258, 11174 (1983) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/68 C12N 15/09 G01N 21/76 BIOSIS (DIALOG) MEDLINE (STN)

Claims (26)

(57) [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 sets of sequenced oligonucleotide primers; Contacting a nucleic acid from a biological sample containing both target nucleotide sequences under conditions such that the primers individually and specifically bind to multiple parts of the target nucleic acid to form a hybrid; (b) E) extending the hybridized primer strands of said hybrids in a 5'-3 'direction from the 3' end of each primer by using one or more types of primer-dependent nucleic acid polymerases; To thereby convert unmodified and modified nucleoside triphosphates from solution into said strand complementary to the target strand. (C) binding the extension product to a solid support coated with a ligand capable of binding to a separably modified component incorporated into the product, thereby providing a non-target nucleotide sequence. (D) quantifying the detectably modified nucleotide moiety incorporated into the extended strand during synthesis of the strand by polymerase activity, Detecting the amount of the target sequence originally present in the biological sample. 2. A strand wherein the primer-dependent nucleic acid polymerase is capable of extending the synthesized complementary nucleic acid beyond the 5 'end of the remotely primer synthesized strand.
-The method according to claim 1, wherein the dissociation enzyme dissociates a distant nucleic acid from a double-stranded state and amplifies a sequence complementary to the original target nucleic acid beyond its 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 said nucleic acid is double-stranded. 6. The method according to claim 5, wherein the target sequence is on a complementary strand of the nucleic acid. 7. An elongation product is synthesized using a strand displacement dissociable nucleic acid polymerase, and then the nucleic acid is denatured.
7. The method according to claim 6, wherein the dissociated dissociated extension product is re-hybridized to a complementary strand, and bound to a solid substrate via a ligand that is an anti-double-stranded nucleic acid antibody. 8. The method of claim 1, wherein said target nucleic acid sequence is RNA and said primer-dependent nucleic acid polymerase is reverse transcriptase. 9. The method of claim 1, wherein said target nucleic acid sequence is DNA and said primer-dependent nucleic acid polymerase is a DNA polymerase. 10. The method according to claim 10, wherein the target nucleic acid sequence is DNA or RNA.
The method according to claim 1, wherein 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 of claim 1, wherein said plurality of primers have non-identical sequences and are complementary to a plurality of sites of said target nucleic acid. 13. The method of claim 1, wherein said plurality of primers have identical and non-identical sequences and are complementary to a plurality of sites of said target nucleic acid. 14. The method of claim 1, wherein said modified nucleoside triphosphate is separably and detectably modified. 15. The method of claim 1, wherein said primer is a primer modified with a binding agent, and said mixture of nucleoside triphosphates comprises a detectably modified nucleoside triphosphate. 16. The method according to claim 1, wherein said primer is a primer modified with a detectable substance, and said mixture of nucleoside triphosphates contains a nucleoside triphosphate modified so as to be separable. 17. The method of claim 1, wherein said separably modified nucleoside triphosphate is biotin and said ligand used to bind the extension product is avidin or streptavidin. 18. The method of claim 1, wherein said 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 an extension product from a mixture containing a detectably modified triphosphate or by using a primer modified with a detectable substance. 2. The method of claim 1, which is performed via a fluorescent component. 20. Detection is performed via a chemiluminescent format using a detectably modified nucleoside triphosphate incorporated into the extension product, or a detectably modified primer, wherein the modification comprises The method of claim 1, wherein the reactive group or component is a chemically interactable part as part of a luminescence detection system. 21. Detection is performed via a chemiluminescent format using a detectably modified nucleoside triphosphate incorporated into the extension product, or a detectably modified primer, wherein said modification causes the extension to occur. The method of claim 1, wherein the product can be directly or indirectly associated with 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 the nucleic acid product is a hapten that binds to an antibody, said antibody being conjugated to an enzyme, or capable of forming a complex with an antibody further conjugated to an enzyme. 22. The method of claim 21, wherein 23. The extension product is modified so as to be detectable with biotin, and the biotinylated residue is bound to or conjugated with an enzyme or forms a complex with a complex having a detection component. 22. The method of claim 21, wherein a complex can be formed with the interactable avidin or streptavidin component. 24. A kit for an assay for amplification of a target sequence of a nucleic acid in a biological sample, comprising: (a) complementary to a number of sites of the target nucleotide sequence forming a template for a primer-dependent nucleic acid polymerase. A number of primers; (b) a mixture of nucleoside triphosphates required for primer synthesis, comprising at least one detectably modified or separably modified nucleoside triphosphate; (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 strand complementary to the target nucleic acid in the 5'-3' direction. Kit to do. 25. The primer-dependent nucleic acid polymerase is capable of dissociating a remotely initiated strand from a duplex by continuing synthesis of a nearby initiated strand, and concomitantly complementary to a target nucleic acid. Strand that can amplify various nucleic acids
The kit according to claim 24, which is a dissociative dissociative polymerase. 26. The method according to claim 2, wherein a cofactor protein is used as necessary for enhancing or activating the strand-displacement dissociation property of the primer-dependent nucleic acid polymerase.