JP3301876B2 - Method for detecting specific polynucleotide and kit for detection used in the method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polynucleotide containing a specific nucleotide sequence present in a sample effective for diagnosing a genetic disease or an infectious disease (hereinafter referred to as a target polynucleotide).
And a kit for detecting a polynucleotide used in the detection method.

[0002]

2. Description of the Related Art An analysis method based on the complementarity of nucleic acid base sequences can directly analyze genetic characteristics. Therefore, it is a very effective means for identifying genetic diseases, canceration, microorganisms, and the like. In addition, in some cases, a time-consuming and labor-intensive operation such as culture can be omitted in order to detect the gene itself.

However, detection when the amount of a target gene is small in a sample is generally not easy, and it is necessary to amplify the target gene itself or the detection signal. One of the methods for amplifying a target gene is PCR (Pol
The ymerase chain reaction) method is known. The PCR method is the most common technique for in vitro nucleic acid amplification. However, in the PCR method, a special temperature controller is required for performing the method; there is a problem in the quantification because the amplification reaction proceeds logarithmically; Further, there are known problems such as susceptibility to contamination, in which a nucleic acid erroneously mixed functions as a template.

That is, in a reaction such as PCR that amplifies DNA up to several million times, erroneous results tend to be provided because a small amount of contaminating DNA is similarly amplified. This is a serious problem, especially when dealing with a large number of specimens simultaneously. As a countermeasure against such contamination, a laboratory is distinguished or a uracil group is incorporated in a PCR reaction, and the sample is treated with uracil glycosylase before a new PCR reaction is performed. Although some measures have been taken to decompose only the amplification product of the reaction system, such measures are not always sufficient.

As a method for amplifying a detection signal, a method for amplifying a signal RNA using Qβ replicase (Bio / Technology, 6, 1197-1202 (1988), PMLizardi)
et al.) have been reported. However, in this method, it is necessary to insert the sequence to be amplified into the sequence recognized by the replicase, and there are problems such as restrictions on the insertion position and sequence due to restrictions on the three-dimensional structure. Also, the method of amplifying signal RNA with Qβ replicase has the same contamination problem as the PCR method, and cannot be said to be a fundamental solution.

The above-mentioned method is a method for amplifying a base sequence to be detected, and a signal amplification method for detecting a degradation product as described below has also been devised. For example,
A signal amplification method for hybridizing an oligonucleotide probe DNA to a target nucleic acid, treating with a restriction enzyme, and detecting the cleaved probe fragment is known (EP-04).
55517 / A1). Although this method has a lower detection sensitivity than the PCR method, it has advantages such as excellent quantification, and does not require a special device for implementation. However, in this method, it is necessary to coexist a second oligonucleotide having a specific sequence other than the probe DNA with a specific sequence for causing the reaction to occur repeatedly in the system. In addition, there is a drawback in that the site to be detected requires a restriction enzyme cleavage site, so that the detectable site is limited.

[0007] In addition, a cycling assay method using λ exonuclease, which specifically cleaves double-stranded DNA, has been developed (BioTechniques, Vol. 13, No. 6, 8).
82-892 (1992), CG Copley et al.). This method uses a λ exonuclease to act on a double-stranded DNA formed by hybridization of an oligonucleotide probe with a complementary sequence, and when the probe DNA is degraded to such an extent that the double-stranded DNA cannot be maintained, a new probe is produced. The cycling reaction occurs by replacing the DNA and subsequently degrading the new probe. This method can determine the presence or absence of a specific DNA sequence by detecting a degradation product of the probe. In addition, the method using a restriction enzyme is simple in that the reaction principle is simple and the sequence of the detection site is not restricted (EP-0455517 /
It is more advantageous than A1).

[0008] However, λ exonuclease requires a probe DNA whose 5 ′ end is phosphorylated as a substrate. When a probe DNA is chemically synthesized using a DNA synthesizer, the 5 ′ end is not phosphorylated, and thus it is necessary to phosphorylate the end again after synthesis. And 5 '
Since it is difficult to confirm whether the end is completely phosphorylated, a problem remains in the reproducibility of probe preparation. Furthermore, the cycling assay method using λ exonuclease requires that the probe DNA repeatedly hybridize to the template DNA, but since the hybridization reaction is unlikely to occur at a constant temperature, this becomes the rate-limiting in the entire reaction system, and There is also a problem that the number of reaction turnovers is small (about 500 times / hour in the literature).

As a cycling assay using exonuclease, a method described in JP-A-5-130870 is also known. The method described in this publication is a method in which a 5 ′ → 3 ′ exonuclease is allowed to act together with a complementary strand synthesis reaction starting from a primer to degrade the primer in the reverse direction. That is, a new primer hybridizes in place of the primer degraded by 5 ′ → 3 ′ exonuclease, the synthesis of the complementary strand by the DNA polymerase and the reaction of removing the previously synthesized strand progress, and the cycling reaction is established. I do. In the method, the PCR
Although complicated temperature control such as the reaction is not required, the problem remains that it is difficult to increase the number of turnovers (the number of repetitions of hybridization of the probe DNA) because the primer hybridization step needs to be repeated.

[0010] The present inventors have proposed to use a specific reaction accelerator for exonuclease III in order to solve these problems.
A nucleotide sequence detection method was developed for use with
327499). This method is an excellent method that can easily synthesize a probe, does not require special temperature control at the time of a reaction, and can detect a base sequence with almost no influence of contamination. However, in this technique, although the reaction accelerator has the effect of increasing the number of turnovers (the number of repetitions of hybridization of the probe DNA as described in the cycling assay method using λ exonuclease), the effect of the probe DNA is recognized. There is no change in the need to repeatedly hybridize, and the problem remains that high sensitivity cannot always be obtained.

In addition, in the gene amplification method described so far, the oligonucleotide added as a primer becomes an amplification product and does not function as a primer. Oligonucleotides must be provided as primers. Particularly in a system in which amplification products are generated logarithmically, such as the PCR method, it was necessary to prepare a very large amount of oligonucleotide. Regardless of whether the oligonucleotide primer is chemically synthesized or a raw material is required for a biological material, it is preferable to use a small amount of the oligonucleotide primer from the viewpoint of the cost of the reagent component.

[0012]

SUMMARY OF THE INVENTION An object of the present invention is to provide a technique for detecting a base sequence which is not easily affected by contamination in a simple reaction system, regardless of complicated temperature control requiring a special apparatus. I do. Further, the present invention has no restriction on the base sequence to be detected in order to achieve these objects,
Another object is to provide a detection technology that is excellent in versatility. Another object of the present invention is to provide a base sequence detection technique capable of obtaining high sensitivity and quantification depending on the selection of a detection system.

[0013]

Means for Solving the Problems The present inventors have made intensive studies for the purpose of solving the above-mentioned problems. As a result, only the double-stranded DNA was cut without acting on the single-stranded DNA, and the nuclease, which is not limited to the nucleotide sequence, was converted to DN.
The present invention was completed by developing a signal amplification method using A polymerase.

That is, the present invention has the following matters as its gist. (1) A polynucleotide whose nucleotide sequence to be detected is known is hybridized with an oligonucleotide primer having a sequence complementary to a part of the polynucleotide and having nuclease resistance, and then at least one kind thereof is added thereto. A deoxynucleoside triphosphate, a DNA polymerase and a nuclease are added to form a complementary strand for the base species adjacent to the 3 ′ end of the primer and complementary to the polynucleotide, followed by decomposition, and The method for detecting a polynucleotide, wherein the pyrolysis or deoxynucleoside monophosphate generated is detected by repeating the decomposition at least once or more.

(2) The oligonucleotide primer, wherein the oligonucleotide primer is phosphorothioated at the 3 'end of the oligonucleotide primer.
(1) The method for detecting the polynucleotide according to (1).

(3) The DNA polymerase as described above, wherein the DNA polymerase is a DNA polymerase selected from the group consisting of DNA polymerase I, Klenow fragment of DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase and Phi29 DNA polymerase.
The method for detecting the polynucleotide according to (1) or (2).

(4) The nuclease is exonuclease III
The method for detecting a polynucleotide according to any one of the above (1) to (3), wherein

(5) β of deoxynucleoside triphosphate
(1) to (1) to (1) to (3), wherein a molecule or atom other than the phosphoric acid molecule at the γ-position and the γ-position is labeled with a radioisotope, and detects deoxynucleoside monophosphate generated by a reaction with a nuclease. 4) The method for detecting a polynucleotide according to any one of the above.

(6) The method according to any one of the above (1) to (4), wherein the deoxynucleoside monophosphate produced by the reaction with the nuclease is separated by chromatography and the deoxynucleoside monophosphate is optically measured. A method for detecting the polynucleotide according to the above.

(7) In the method for detecting a polynucleotide according to any one of the above (1) to (4), the pyrophosphate produced by complementary strand synthesis by DNA polymerase may be adenosine-5'-phosphosulfate and adenosine trilin. A method for detecting a polynucleotide, comprising reacting with acid sulfurylase to generate adenosine triphosphate and detecting the adenosine triphosphate.

(8) The method for detecting a polynucleotide according to (7), wherein adenosine triphosphate is measured by a luciferin-luciferase reaction.

(9) The following components (1) to (4): 1. an oligonucleotide primer having a sequence complementary to a part of a polynucleotide whose base sequence to be detected is known and having nuclease resistance; 2. DNA polymerase, At least one kind of deoxynucleoside triphosphate and a nuclease having an activity of decomposing a double-stranded DNA in a 4.3 ′ → 5 ′ direction. A kit for detecting a polynucleotide used in the method for detecting a polynucleotide according to any one of the above (1) to (8), comprising:

(10) The polynucleotide detection kit according to the above (9), wherein the detection kit contains a reagent for detecting deoxynucleoside monophosphate.

(11) The polynucleotide detection kit according to (9), wherein the detection kit contains a pyrophosphate detection reagent.

Hereinafter, the present invention will be described in detail. A. The detection target of the present invention is a polynucleotide whose base sequence is known. The detection target of the present invention is an animal, a plant,
It extends to polynucleotides from bacteria, yeast, filamentous fungi, mycoplasma, rickettsia, viruses and anything else. As a polynucleotide, not only genomic nucleic acid but also cDNA derived from RNA virus or mRNA can be detected. When a sample is actually analyzed, there may be a problem with the sequence of DNA contained in the sample other than the detection target or the presence of a sequence that can serve as a primer for DNA synthesis. It is also possible that DNA polymerase, nuclease activity inhibitor, and deoxynucleoside triphosphate are contaminated. Further, when pyrophosphate is to be detected, pyrophosphate coexisting in the sample also becomes a factor that hinders the analysis.

Therefore, in the present invention, an amplification reaction is performed.
It is preferable to remove the contaminants as much as possible
No. For example, using a capture probe bound to a solid phase
DNA, followed by washing to remove the impurities
Then, when the present invention is applied, there is no background
A highly sensitive detection system becomes possible. At this time, the capture probe
By binding to the solid phase at the 5 'end (Eur. J. Immunol.,Two
Three, 1895-1901 (1993), H. Kohsaka et al .; Nucleic Acid
s Research,twenty one, 3469-3472 (1993), H. Kohsaka et al.
 ), The capture probe itself as a primer
The method can also be applied. The pyrroline
Enzymatic removal of acid by pyrophosphatase
Removal is also possible.

In a preferred method of the present invention, in particular, in which the extension and decomposition of one base is repeated as one cycle,
It can be applied to the detection of point mutation as described later. As a method for detecting a point mutation, Japanese Patent Application Laid-Open No. 59-208
The method described in Japanese Patent Publication No. 465 is already known. This method is based on a reaction principle that a nucleotide derivative is incorporated only when a point mutation exists (or does not exist) by using a nucleotide derivative as a substrate in a synthesis reaction of a complementary sequence following a primer. is there. That is, when a nucleotide derivative is incorporated as a substrate, the reaction product acquires resistance to exonuclease. Therefore, a point mutation can be detected by specifying the presence or absence of nucleotide degradation by the nuclease.

Certainly, some of the elements used in the method are common to the present invention. However, while the method is characterized in that the presence or absence of nuclease resistance acquisition is used as an indicator of the presence of a point mutation, the present invention provides an indicator of the presence or absence of a point mutation whether or not the reaction occurs repeatedly. Is essentially different in that it is used as The detection of a point mutation by the method of the present invention is more favorable and advantageous in sensitivity than the detection of the mutation by the method.

B. The present invention is characterized in that an oligonucleotide primer having a sequence complementary to a part of the polynucleotide and having nuclease resistance is hybridized to the known polynucleotide.
In the present invention, “complementary” refers to a state in which a base sequence can form a double strand by hydrogen bonding with another nucleic acid according to the Watson-Crick rule of base pairs. Specifically, thymine (T) has a corresponding complementarity to adenine (A) and cytosine (C) to guanine (G). The primer used in the present invention does not need to be completely complementary to the base sequence of the target polynucleotide, and at least the oligonucleotide can hybridize to the target polynucleotide as a whole. Is enough. Specifically, it is essential that the base at the 3 ′ end of the oligonucleotide corresponds to the base of the target polynucleotide,
It is necessary that at least 70% of the total bases of the oligonucleotide have said complementarity in relation to the target polynucleotide. If the complementarity is less than 70%, the hybridization itself becomes difficult, which is not preferable. On the other hand, the condition that the base at the 3 ′ end corresponds to the base of the target polynucleotide is an essential condition for the present invention to carry out the base addition reaction at the 3 ′ end. If the base does not correspond at the 3 'end and the primer is single-stranded, the polymerase or nuclease cannot be recognized.

The oligonucleotide primer used in the present invention is a “part” of a known target polynucleotide.
And is complementary to This is because the method of the present invention is a method in which DN
This is characterized in that a specific deoxynucleotide added in advance by the A polymerase and present in the reaction system is detected. That is, if the nucleotide serving as a template for the deoxynucleotide added to the 3 'end of the primer is not known, it is difficult to cause a specific deoxynucleotide to exist in the system in advance,
The oligonucleotide primer used in the present invention needs to be complementary to a "part" of a known target polynucleotide.

The oligonucleotide primer used in the present invention needs to have resistance to nuclease. This is because, in the present invention, it is necessary to allow a nuclease to be present in the system, and if the oligonucleotide primer is degraded by the nuclease, it becomes difficult to carry out the method of the present invention. The method for imparting nuclease resistance to the oligonucleotide primer is not particularly limited, and any method commonly used in this field can be used.

Specifically, for example, when an oligonucleotide serving as a primer is synthesized by a DNA synthesizer, phosphorothioate (Phosp
Introducing a horothioate) linkage can impart nuclease resistance to the oligonucleotide primer. As such a method, for example, solid phase phosphoramidite (P
hosphoramidite)
By performing an oxidation treatment with an appropriate S-forming reagent (phosphorothioate-forming reagent) instead of the oxidation step with iodine water or the like, a phosphorothioate bond can be introduced into the oligonucleotide instead of a normal phosphodiester bond. As the S-forming reagent, 3H-1,2-benzodithiole-3-one
1,1-dioxide (Beaucage's Reagent), TETD / Acetonitril
e (TETD: tetraethylthiuram disulfide) and the like are known. According to this method, a phosphorothioate bond can be introduced at any site of the oligonucleotide.

Alternatively, D
When synthesizing NA, a phosphorothioate bond can be introduced into an oligonucleotide by using, as a substrate, a substance in which oxygen bonded to the phosphorus atom at the α-position of deoxyribonucleoside triphosphate is replaced with sulfur.
Such substituted compounds include α-S-deoxythymidine triphosphate, α-S-deoxycytosine triphosphate, α-S-deoxyadenine triphosphate and α-S-
Deoxyguanine triphosphate and the like (these substituted compounds are collectively abbreviated as SdXTP) can be exemplified. When performing a synthesis reaction by DNA polymerase,
By using SdXTP as a substrate instead of deoxynucleoside triphosphate (hereinafter abbreviated as dXTP),
The synthesized oligonucleotide is phosphorothioated and acquires nuclease resistance. When a phosphorothioate bond is introduced by a DNA polymerase,
This reaction can be allowed to proceed in a state where it is actually hybridized to the polynucleotide to be detected. That is, the nuclease-resistant oligonucleotide primer in the present invention does not necessarily need to be prepared in advance, and a method of acquiring nuclease resistance during the reaction can be adopted.

Once nuclease resistance is established, subsequent reactions can proceed according to the reaction principle described below.
In this case, first, SdXTP is added, and then dX
The addition of TP requires an extension reaction of at least two bases, but the method of the present invention can be carried out because nuclease acts on the second dXTP. In any case, by making the phosphodiester bond near the 3 ′ end of the primer a phosphorothioate bond,
This is an oligonucleotide primer that is resistant to nucleases that attack from the 3 'side. However, if the entire oligonucleotide is phosphorothioate-bonded, the degree of nuclease resistance further increases, but at the same time, the efficiency of hybridization decreases. Therefore, the number of phosphorothioate bonds is preferably one to several from the terminal.
The phosphorothioate bond may be only one terminal,
If several, preferably three, phosphorothioate bonds are successively formed, more complete nuclease resistance can be expected.

However, as described above, the efficiency of incorporation into DNA by a DNA polymerase depends on whether SdXTP is dXT.
Since it is much lower than P, dXTP for decomposition is added after the incorporation of SdXTP first, or SdXT
In some cases, it is necessary to devise a method of setting a higher P concentration.

Means for imparting nuclease resistance include methylphosphonate (M
ethylphosphonate) bond, phosphoramidate (Phospho
roamidate) bond, Polyamide nucleic acid
cid (PNA)) binding and the like. By these bonds, the phosphate binding site of the nucleotide, the ribose site, and the base site of the nucleic acid are modified or the structure is modified to acquire nuclease resistance.

In addition, an RNA primer can be used as an oligonucleotide primer. R
NA is almost resistant to nucleases acting on DNA. Therefore, if an RNA primer is used, it is possible to prepare an oligonucleotide primer having nuclease resistance that acts on DNA without special modification. However, some nucleases and DNA polymerases acting on DNA are RNases.
Some also have H activity, in which case R
Since the NA primer itself is decomposed, it may be difficult to obtain high sensitivity depending on the combination of enzymes.

The oligonucleotide primer of the present invention has a base number of at least 6 or more, preferably 10 to 5 bases.
0, particularly preferably about 15 to 30. When it is less than 6, not only does it not easily hybridize under normal conditions, but also the probability of stochastically leading to a nonspecific reaction increases. On the other hand, oligonucleotide primers that have been made nuclease-resistant by phosphorothioation, for example, have a reduced affinity (Tm) for the template, so that it is necessary to provide a certain length to compensate for this decrease.
On the other hand, if it exceeds 30, the yield is likely to decrease during chemical synthesis, which is economically disadvantageous and is not preferred. Furthermore, when a primer longer than necessary is used, two
The main chain is easily formed, and a non-specific reaction easily occurs. Such an unnecessarily long primer is not preferable because it may cause non-specific amplification or the like.

The DNA polymerase which can be used in the present invention will be described later. Some DNA polymerases, such as DNA polymerase I, which have a weak 5 ′ → 3 ′ exonuclease activity on double-stranded DNA also exist. When this type of enzyme is used as a DNA polymerase, the hybridized probe is degraded from the 5 'side, so the 5' side should also be made nuclease resistant. In addition, even with the same DNA polymerase, Klenow fragment and Ph
Since i29 DNA polymerase does not have this activity, it is not necessary to particularly modify the 5 ′ side, and this is preferable.

The oligonucleotide primer is used in such an amount as to be sufficient for the nucleotide sequence to be detected. In the conventional method of amplifying a nucleotide sequence, the oligonucleotide added as a primer becomes an amplification product and does not function as a primer, so it was necessary to use a large excess of the amount of the nucleotide sequence as a template. In the present invention, a once-hybridized oligonucleotide primer repeatedly functions as a primer, so that a quantitative reaction is possible if at least equimolar to the base sequence serving as a template. In practice, it is preferable to utilize a sufficient amount of oligonucleotide primer to tilt the equilibrium towards the hybridizing state. It is difficult to predict the amount of the target sequence to be detected in advance, but at least an equimolar amount, preferably a 5-fold or more excess amount, of the desired detection range is used. This is a preferable condition for securing high sensitivity.

The conditions for hybridization are not particularly limited, but it is necessary to pay attention to the fact that after hybridization, all reactions need to proceed in the presence of DNA polymerase and nuclease. In particular, nucleases do not have a high degree of heat resistance unlike Taq polymerase used in the PCR method, so it is necessary to adopt conditions that can maintain the enzyme activity of the nuclease. In addition, selection of a buffer other than temperature, setting of pH, and the like should be performed under conditions that allow not only hybridization but also sufficient nuclease activity to be expressed. As an example of specific conditions, when Tris-HCl buffer or the like is used as a buffer, the pH is about 7 to
The reaction is carried out at 20 to 55 ° C. Particularly preferred conditions include a pH of 7.5 to 8.5 and a temperature of 30 to 45 ° C.

For example, as shown in the examples, 0.1 pmol
Primers, 50 mM Tris / HCl buffer (pH 7.
5) After heat treatment (100 ° C. for 5 minutes) in a solution consisting of only 10 mM MgCl ₂ , annealing is performed at 65 ° C. for 10 minutes. In this case, the DNA polymerase reaction and nuclease reaction can immediately proceed at 37 ° C.
In addition, when pyrophosphoric acid finally accumulated in the reaction system is enzymatically measured, suitable reaction conditions must be provided for this enzymatic reaction. That is, the pH, salt concentration, temperature and the like are set so as to be suitable for the enzyme reaction.

Further, a stabilizer for DNA polymerase or nuclease can be added to the reaction solution. Such stabilizers include, for example, bovine serum albumin (BS
A), dithiothreitol (DTT), β-mercaptoethanol and the like. The amount of the stabilizer added is about 10 to 500 μg / ml for BSA, about 1 mM for DTT, and about 10 mM for β-mercaptoethanol. When the polynucleotide to be detected is in a double-stranded state, it must be previously denatured to a single-stranded state. Examples of such a denaturation method include generally known denaturation methods such as heat denaturation, acid denaturation, and alkali denaturation. In view of the simplicity and certainty of the method, heat denaturation (5 ° C at 90 ° C to 100 ° C).
(Heating for more than one minute).

In addition, DNA other than the target primer
The exonuclease treatment can be carried out before the addition of the DNA polymerase in order to suppress the hybridization of DNA or to remove the non-specifically hybridized DNA to suppress the non-specific reaction.

C. The invention then comprises adding, after the hybridization, at least one deoxynucleoside triphosphate, a DNA polymerase and a nuclease to the system to add a base adjacent to the 3 'end of the primer and complementary to the polynucleotide. The method is characterized in that the species is synthesized with a complementary strand and then decomposed, and the complementary strand synthesis and degradation are repeated at least once. The reaction principle of the present invention will be described below. The reaction principle of the present invention is shown in FIG.

[0046]

Embedded image

First, based on the oligonucleotide primer hybridized to the single-stranded nucleic acid to be detected, the synthesis of the complementary strand is started by the action of DNA polymerase. At this time, one molecule of pyrophosphoric acid (hereinafter sometimes abbreviated as PPi) is generated with the addition of one molecule of dXTP. On the other hand, when a nuclease that degrades from the 3 ′ end of the double-stranded DNA is acted on in this state, a reaction for decomposing the extended portion proceeds. However, since the oligonucleotide primer is nuclease resistant, the primer portion remains undegraded and becomes the starting point of the extension reaction by the DNA polymerase again. As a result of such repeated reactions, pyrophosphate or deoxynucleoside monophosphate generated by the decomposition of nuclease accumulates in the reaction system.

As described above, DNA was used during the reaction.
By the action of the polymerase, the oligonucleotide primer can be subjected to the above step as nuclease resistance. By detecting this pyrophosphate or deoxynucleoside monophosphate generated by degradation by nuclease, the presence or absence of the base sequence to be detected can be detected or quantified.

Further, it is possible to construct a system for detecting a point mutation by the detection method of the present invention. That is, when a site where a point mutation occurs is known in advance, a sequence complementary to a region adjacent to the 5 ′ side of the site is used as an oligonucleotide primer. If only one type of substrate is added as dXTP as a substrate for the elongating chain so that the elongation occurs only when the sequence is normal, the reaction is stopped if a point mutation occurs because the elongation cannot be performed. In this way, the presence or absence of a point mutation can be easily confirmed (formula 2 below).

[0050]

Embedded image

Further, if a mutation occurs in the region to which the oligonucleotide primer should hybridize, the efficiency of the hybridization decreases as a matter of course, so that the whole reaction becomes difficult to proceed. Can be. The DNA polymerase used in the above catalyzes a reaction of extending a complementary strand in a 5 ′ → 3 ′ direction starting from a double-stranded portion formed by hybridizing a complementary strand to a single-stranded nucleic acid serving as a template. DNA polymerase is used. In fact, currently known DNA polymerases all have a DNA synthetase activity in the 5 ′ → 3 ′ direction. In addition, a primer is always required for DNA synthesis to occur, and if no primer is present or hybridization is impossible, DNA synthesis cannot occur. This is one of the reasons that the present invention has high specificity depending on the primer and the template strand. Specific examples of the DNA polymerase used in the present invention include DNA polymerase I, Klenow fragment of DNA polymerase I, T
4 DNA polymerase, T7 DNA polymerase, Ph
Examples thereof include i29 DNA polymerase and the like and mutants of these DNA polymerases.

As a DNA polymerase, T4DN was used.
By using a compound having strong 3 ′ → 5 ′ exonuclease activity such as A polymerase or T7 DNA polymerase, it is possible to save the amount of nuclease added separately, or to use one type of enzyme acting on nucleic acids. DNA having a 3 ′ → 5 ′ exonuclease activity
The reaction system of the present invention can be constituted by adding only a polymerase. However, since these DNA polymerases also decompose nuclease-resistant phosphorothioate bonds to some extent, high sensitivity may not be expected in some cases. Therefore, in this case, it is desirable to modify the phosphodiester bond near the terminal of the primer over a plurality of bonds or to make it nuclease resistant by a modification other than phosphorothioation.

The nuclease used in the above is 2
It is a nuclease that has the activity of degrading main-strand DNA in the 3 ′ → 5 ′ direction. Such nucleases include
Exonuclease III is known. Exonuclease III is currently commercially available from Escherichia coli, but is not limited to this, and other microorganisms or those obtained by genetic recombination can also be used. Exonuclease II
When I is used, DNA polymerase I exemplified as DNA polymerase, its Klenow fragment,
Since the enzyme activity is exhibited under substantially the same reaction conditions as T4 DNA polymerase or the like, the reaction can proceed simultaneously in the same reaction solution in parallel with the reaction by the DNA polymerase. Exonuclease III converts double-stranded DNA to 3 '→
It is specifically degraded in the 5 'direction, does not act on single-stranded DNA, so that the sequence or primer to be detected is not attacked, and the primer can be easily made nuclease resistant by SdXTP. Very suitable enzymes for the circulation of the reaction cycle of the invention.

Deoxynucleoside triphosphate (dXTP) is used as a substrate for a DNA extension reaction starting from an oligonucleotide primer by a DNA polymerase. In the present invention, analysis can be performed if at least one base is previously extended for an apparent portion of the sequence, so that a desired reaction can be performed by using only one kind of nucleotide corresponding to at least one base. Can be done. The substrate concentration is added so as to be sufficient for the number of moles of the base sequence to be detected. In general, it is difficult to accurately predict the amount of a target to be detected. Therefore, if nucleotides of at least 0.1 μM, preferably 1 μM or more are added, in most cases, a situation where the substrate is insufficient can be avoided.

D. The present invention is characterized in that the generated pyrophosphate or deoxynucleoside monophosphate is detected. Detection of Deoxynucleoside Monophosphate Produced In the method of the present invention, the deoxynucleoside monophosphate (hereinafter, referred to as dXMP) generated by a nucleotide chain degradation reaction by nuclease is detected, that is, added by a DNA polymerase elongation reaction. By detecting dXMP corresponding to dXTP,
The desired polynucleotide can be detected.

By substituting and labeling the α-position phosphorus atom of dXTP used as a substrate with a radioactive isotope ( ³² P or ³³ P); the α-phosphoric acid, deoxyribose or base hydrogen atom can be replaced with ³ H By displacement labeling with;
Alternatively, by measuring the radioactivity of dXMP, which is a decomposition product, by labeling the carbon atom of deoxyribose with ¹⁴ C, separating dXTP and dXMP chromatographically with an ion exchange resin or the like, and tracking the radioactivity. Is possible. It should be noted that at present the phosphorus atom at the α-position is labeled with ³² P.
As XTP, dATP, dCTP, dGTP and d
TTP is commercially available. Also, the phosphorus atom at the α-position is
³³ P The labeled dXTP, dATP and dCTP
Are commercially available. Examples of the bases labeled with ³ H include dATP, dCTP, dGTP and dTTP.
Are commercially available.

DXMP with radioisotope label
The radioactivity can be easily and qualitatively detected by autoradiography if it is separated by thin layer chromatography using an ion exchange resin. Further, as shown in the examples, quantitative analysis is possible by removing the radioactive spot and counting it with a scintillation counter. Further, even if a substrate labeled with an isotope is not necessarily used, dX can be performed by the following operation.
MP can be separated and measured. That is, the reaction solution is separated by thin-layer chromatography coated with a commercially available fluorescent dye, and when this is irradiated with ultraviolet light, the nucleotide absorbs the ultraviolet light, so that dXMP can be visually recognized as a non-fluorescent spot. In addition, dXMP can be quantitatively detected by separation by liquid chromatography and measurement of ultraviolet absorption.

Quantification of Pyrophosphate Generated by Chain Elongation Reaction with DNA Polymerase In the method of the present invention, a desired polynucleotide can be detected by measuring the production of PPi accompanying the addition of dXMP by the DNA polymerase. This D
According to the method of tracking PPi produced by the addition of dXMP by NA polymerase, enzymatic measurement can be performed, and the operation of separating the reaction solution by chromatography or the like can be omitted. Become.
Specifically, a reaction represented by the following formula 3 is known. The reaction of the formula 3 (J. Immunological Methods, 156, 55-60 (19
92), T. Tabary et al.) Not only enables highly sensitive measurement but also allows easy analysis because it can be analyzed in a homogeneous system.

[0059]

Embedded image

As another method for measuring pyrophosphoric acid,
The method cited in "Anal. Biochem., 94 , 117-120 (1979)" is known.

E. Specific Examples Hereinafter, a specific oligonucleotide primer sequence and a reaction system according to the present invention using the sequence will be exemplified (Formula 4 below).

[0062]

Embedded image

Human cytomegalovirus (hereinafter referred to as CMV)
Taking CMV genomic DNA as an example,
The primer is a sequence specific to the CMV genome, which is derived from the D fragment of the EcoRI cleaved fragment therein.
The specific arrangement is as shown in Equation 4. Although any part of the array shown in Equation 4 may be used, in the following description, Equation 4
A complementary strand that hybridizes to a portion sandwiched by → ← is used as a primer (SEQ ID NO: 1). The phosphodiester bond between the 3 'ATs is referred to as a phosphorothioate bond. By measuring PPi or the degradation product dAMP, it is possible to know whether the CMV sequence is present in the sample.

In particular, the preferred method of the present invention, in which the extension-decomposition of one base is repeated as one cycle, can be applied to the detection of point mutation as described above. The following is an example of detection of a point mutation as a human oncogene Ki-ras.
12 shows a detection system of / 12. In Ki-ras / 12, a mutant represented by the following formula 5 is known (the following formula 5)
The dots shown in the above are normal arrangement portions where no abnormality has occurred.)

[0065]

Embedded image

As the primer, the sequence (SEQ ID NO: 2) shown in Formula 5 is used. A phosphorothioate bond is formed between the 3'-side TGs to make them nuclease-resistant, and the substrate for DNA polymerase synthesis is d
Add only GTP. In this example, if the detection target is normal, the reaction of incorporating dGTP proceeds, and PPi and dGTP
GMP is accumulated. However, when the detection target is a mutant, the reaction does not proceed because there is no dTTP necessary for the synthesis of the complementary strand. Conversely, if the reaction proceeds when only dTTP is added to the substrate, it is understood that the sequence targeted for detection contains a point mutation. In the present invention, it is possible to detect a point mutation in this manner (formula 6 below).
reference).

[0067]

Embedded image

In the formulas 5 and 6, the primer is shown in the upper row and the template sequence is shown in the lower row. The display is different from the above formulas 1, 2 and 4 (the template sequence is in the upper row and the primer is in the lower row). I have.

F. Kit for Detecting Polynucleotide of the Present Invention The various components required for the present invention as described above can be supplied in the form of reagents previously combined. The configuration of the reagent for detecting a base sequence according to the present invention is shown below again. The reagents shown below can be combined with any necessary components such as materials necessary for the detection of the label, a buffer constituting the reaction solution, or a negative or positive control to form a kit.

The configuration of the kit for detecting the polynucleotide of the present invention is as follows. 1. 1. an oligonucleotide primer having a sequence complementary to a part of a polynucleotide whose base sequence to be detected is known and having nuclease resistance; 2. DNA polymerase, At least one kind of deoxynucleoside triphosphate; a nuclease having an activity of decomposing a double-stranded DNA in a direction of 4.3 ′ → 5 ′;

A kit containing any of the above 1 to 4 can contain a detection reagent in accordance with the above-mentioned index for detecting a polynucleotide. That is, when the detection of dXMP is intended, a reagent for detecting deoxynucleoside monophosphate can be contained. Specific examples of such a detection kit include, for example, the following detection kits.

That is, phosphorothioated oligonucleotides, DNA polymerase I Klenow fragment, deoxynucleoside triphosphate (labeled with ³² P), exonuclease III, Tris / H
Includes Cl buffer, MgCl ₂ , BSA and DTT; EDTA as a reaction stop solution; ion exchange cellulose for thin layer chromatography (PEI = polyethyleneimine cellulose) and LiC as reagents for detecting dXMP
l (developing solvent) or as a reagent for detecting pyrophosphate,
Examples include a kit for detection containing TP sulfylase, adenosine-5'-phosphosulfate, luciferin and luciferase.

Hereinafter, the mechanism of action and the practical effects of the present invention will be summarized and described. The oligonucleotide primer of the present invention specifically hybridizes to a base sequence to be detected and serves as a starting point of an extension reaction by DNA polymerase. In addition, by preliminarily making nuclease-resistant or by adding a deoxynucleotide derivative (SdXTP or the like) by the action of DNA polymerase to make it nuclease-resistant, deoxynucleoside triphosphate (dXT
It is possible to repeatedly cause two reactions, namely, the addition of P) to the decomposition by the action of a nuclease. Since this reaction repeatedly occurs linearly, in the present invention, nuclease degradation products accumulate in the reaction system, and a quantitative and highly sensitive detection system can be realized.

Even if the primer hybridizes to a non-specific sequence due to mismatch, if dXTP prepared in advance as a substrate is not compatible in the step of synthesizing the complementary strand of the region adjacent thereto, the subsequent reaction Has the advantage that erroneous results do not occur. Further, in the present invention, there is no need to repeat the denaturing operation of DNA by high-temperature treatment, so that thermal stability is not required for reagent components such as DNA polymerase, and complicated temperature control is not required. Therefore, there is also an advantage that the operation can be easily automated.

The DNA polymerase of the present invention synthesizes a strand complementary to a sequence adjacent to the region where the primer hybridizes. At the time of synthesis,
One mole of PPi is produced for the addition reaction of one mole of dXTP. Since PPi can be specifically measured by an enzymatic reaction, the present invention is a system that simultaneously generates signals by a synthetic reaction. In another embodiment, a product in which a part extended by this reaction is degraded by nuclease can be a detection target. DNA polymerase has a function of converting it to nuclease resistance by using a deoxynucleotide derivative as a substrate when a non-nuclease-resistant oligonucleotide primer is used.

The nuclease of the present invention has an action of specifically hydrolyzing a portion extended from an oligonucleotide primer hybridized to a sequence to be detected to the nuclease resistant site. This degradation does not occur when the target sequence is not present, and even if the target sequence is present, it is not hydrolyzed unless the hybridization of the oligonucleotide primer and the extension reaction by the DNA polymerase occur. Due to such characteristics of the nuclease, the degradation product accumulating in the reaction solution in the present invention increases specifically and linearly with respect to the detection target, and enables highly quantitative analysis.

Another advantage of the present invention is that it is hardly affected by contamination. That is, even if the sample after the reaction is erroneously mixed into the solution to be reacted, the accumulated degradation product itself does not function as a new template, so that the reaction does not proceed based on this. It is very advantageous in practice.

Further, a unique effect of the present invention is that a simple operation can be provided in a homogeneous system. PP generated by addition of dXTP by DNA polymerase
If the method of tracking i is adopted, a reaction system that does not require any operation such as hybridization with an additional probe or separation by electrophoresis can be provided. Since PPi can be specifically measured by a purely enzymatic reaction, it is possible to continue the reaction as it is in the reaction solution after the synthesis and decomposition reaction, which is very practical. Such advantages of the present invention are more evident than detection of amplification products by the PCR method requires hybridization with a probe or separation by electrophoresis.

Another advantage of using the primer hybridized to the sequence to be detected in the first stage of the reaction as it is for the reaction is that only a relatively small amount of primer is required. In a system where primers are consumed by amplification products such as PCR, a large excess of primers must be prepared, but in the present invention, it is sufficient to prepare a slight excess of primers with respect to the number of moles of the sequence to be detected. . The same can be said for deoxynucleoside triphosphate as a substrate. That is, for example, in PCR, all necessary substrates for the region to be extended must be prepared. The present invention has the advantage that the reagent configuration can be simplified because at least one kind of base complementary to the sequence adjacent to the hybridized region has to be prepared.

An additional effect of the present invention is that it can be applied to the detection of point mutations. That is, when a site that causes a point mutation is known in advance, a region adjacent to this portion is selected as a primer, and the reaction system is set so that the reaction does not proceed when the point mutation occurs, thereby performing the point mutation. Can be confirmed. As described above, the method for detecting a polynucleotide according to the present invention has various advantages, and can be expected to be an effective method in gene analysis.

[0081]

Hereinafter, the present invention will be described in more detail with reference to examples. However, it should not be construed that the technical scope of the present invention is limited by the embodiments. [Example] Detection of HBV-e antigen gene of HBV • DNA Detection of HBV-e antigen gene of HBV • DNA was attempted. The nuclease is exonuclease derived from E. coli.
III (manufactured by Takara Shuzo) and an oligonucleotide having the base sequence shown in SEQ ID NO: 3 was chemically synthesized and used as the primer DNA.

(1) -1 Preparation of Primer DNA The oligonucleotide having the above sequence (SEQ ID NO: 3)
Cyanoethylamidite method (J. Am. Chem. Soc., 112, 125
3-1254 (1990)). 3H as S reagent
-1,2-benzodithiole-3-one 1,1-dioxide (Beaucage's Re
A nuclease-resistant primer DNA was obtained by using a phosphorothioate bond between the TCs on the 3′-terminal side using an agent). Cyclone® P as a DNA synthesizer
rus DNA / RNA synthesizer (Nippon Millipore Limited) was used. The synthesized phosphorothioated oligonucleotide was purified by HPLC according to a conventional method and used.

(1) -2 Detection of Nucleotide Sequence The primer DNA was mixed with M13 phage DNA partially containing HBV DNA serving as a target sequence, and the reaction according to the present invention was actually carried out. The specific composition of the reaction solution was as shown below. The numerical values shown below are all final concentrations. In order to confirm whether or not the reaction actually proceeded, a reaction system excluding each component was provided and the results were compared. In order to confirm the necessity of a nuclease-resistant primer, an experiment was conducted with a primer having the same sequence but not having nuclease resistance. When the primers used in this example were hybridized, the sequence adjacent to the 3 ′ side was G, and therefore d
CTP is required. And the nuclease degradation product of this dCTP becomes dCMP.

<Composition of reaction solution> First reaction (5 μl) 10 fmol target sequence (HBV-e on M13 phage DNA)
Single-stranded DNA having antigen gene region) 1 pmol Primer DNA 50 mM Tris / HCl (pH 7.5) 10 mM MgCl ₂ Second reaction (10 μl) 1 unit DNA polymerase I Klenow fragment 5 units Exonuclease III 10 μM dCTP ( ³² P label, 5 P × 10 ⁴ cpm) 50 μg / ml bovine serum albumin (hereinafter abbreviated as BSA) 10 mM dithiothreitol

In the first reaction, the reaction solution (5 μl) was heated at 100 ° C. for 5 minutes to make the DNA single-stranded, and then left at 65 ° C. for 10 minutes to hybridize the primer DNA. Next, a reagent solution containing components necessary for the second reaction is added, and the mixture is further heated to 37 ° C.
Was reacted. After reacting for 1 hour, 1 mM of 20 mM EDTA was added.
l In addition, the reaction was stopped. The total amount of the reaction solution after the reaction was stopped was thin-layer chromatography (thin layer: PEI-Cellulose
F (manufactured by Merck), developing solvent: developing with 0.4 M LiCl for 40 minutes at room temperature, and dCMP, which is a nuclease degradation product, was confirmed by autoradiography.

As is apparent from FIG. 2, the sequence to be detected, Klenow fragment, exonuclease III
Is a nuclease degradation product dCM
No P was detected (lanes 2-4), confirming that the reaction of the present invention was specifically progressing (lane 1). Lane 1 is a system for the detection target sequence + all reagent components, Lane 2 is a system for only the reagent components, Lane 3 is a system for removing the Klenow fragment in the reagent components, and Lane 4 is a system for the reagent components. Medium, excluding exonuclease III.

When a primer having no nuclease resistance was used as a primer, it was confirmed that dCMP did not accumulate as a nuclease degradation product even in a reaction system satisfying the other components (FIG. 3: lane 1 A nuclease-resistant primer was used. Lane 2 uses an unmodified primer, that is, a nuclease-sensitive primer.) This is considered to be because dCTP cannot be added because the 3′-side region of the primer (that is, the dCTP addition site) is degraded by the nuclease.

(2) Template specificity Only when the primer has complementarity with the sequence to be detected,
The following experiment was performed to confirm that the decomposition of the substrate by nuclease occurred. That is, a reaction was actually carried out by the same operation as in the above (1) under two conditions, that is, a case where a sequence having no complementarity with the primer was used as a detection target sequence and a case where no detection target sequence was present.

As a result, FIG. 4 (lane 1: the sequence to be detected is complementary to the primer sequence, lane 2: the sense sequence to be detected is the same as the primer sequence, ie, not complementary, lane 3: does not contain the sequence to be detected) As shown in the figure, if the sequence to be detected and the primer DNA are complementary, the degradation product dCMP accumulates (lane 1), but the sense sequence, ie, the same sequence as the primer but not complementary (lane 2), When the target sequence was not present (lane 3), it was confirmed that no degradation product accumulated. This experiment demonstrated that the reaction of the present invention proceeds when the sequence to be detected has a sequence complementary to the primer.

(3) Substrate (deoxynucleoside triphosphate) specificity In order to confirm that degradation by nuclease occurs only at the time of dCTP in which the substrate, deoxynucleoside triphosphate, is incorporated into the primer, Was replaced with dGTP and reacted. As a result, FIG. 5 (lane 1: dCTP was used as a substrate, lane 2:
As shown in lane 3: dCMP and dCTP developed as markers, lane 4: dGMP and dGTP developed as markers), the substrate was dCT.
It was confirmed that the reaction proceeded only at the time of P and dCMP as a decomposition product was observed (lane 1), and that the reaction did not proceed when dGTP was added (no decomposition product was generated, lane 2). And from this result, it became clear that the detection method according to the present invention can be used for detection of point mutation.

(4) Detection sensitivity of the present invention The amount of DNA to be detected is reduced to 0 to 1 by the same operation as in (1).
The sensitivity of the detection method according to the present invention was confirmed by changing to pmol (see Table 1). FIG. 6 (Lane 1: 1 pmol of DNA containing the sequence to be detected was added and reacted; Lane 2: 0.1 pmol of DNA containing the sequence to be detected was added and reacted; Lane 3: 0.01 pmol of DNA containing the sequence to be detected Lane 4: DNA containing the sequence to be detected was added and reacted at 1 fmol. Lane 5: DNA containing the sequence to be detected.
Lane 6: DNA containing the sequence to be detected was reacted at 0.01 fmol, and lane 7 was reacted.
Lane 8: The DNA containing the sequence to be detected was added without reacting with 1 amol of the DNA containing the sequence to be detected.
1 fmol (lane 4) of DN
It was confirmed that A could be detected. From this result, it was found that the sensitivity of the detection method according to the present invention was extremely high. Further, in order to confirm the quantification of the nucleotide sequence detection method according to the present invention, each band was cut out and the radioactivity was measured by a liquid scintillation counter. Table 1 shows the results. In Table 1, the values of (A) and (B) indicate the radioactivity (CPM) of each spot. It was confirmed that the quantification could be ensured in the range of 0.1 fmol to 0.01 pmol.

In this example, ³² P-labeled dCTP was used.
Is adjusted in advance with an unlabeled dCTP in a range where a difference in the size of the spot of autoradiography is likely to occur. Therefore, higher sensitivity can be achieved by increasing the ratio of the labeling substance to dCTP.

[0093]

Table 1 Examination of detection sensitivity of the detection method of the present invention ───────────────────────────────────対 象 Detection target (A) (B) AB / Total dCMP accumulation rate DNA amount dCTP dCMP (%) (pmol) (dCMP / Target DNA) ─────────────────── ───────────────── 1pmol 6566 17883 70.9 70.9 70.9 0.1 8805 25 204 72.5 72.5 725 0.01 18381 15212 43.7 43.7 4370 1fmol 32552 1808 3.67 3.67 3670 0.1 31550 681 0.42 0.42 4200 none 35997 547 --────────────────────────────────────

(5) Detection method of the present invention using T4 DNA polymerase T4D is used in place of the Klenow fragment in the above (1).
The method for detecting a nucleotide sequence according to the present invention was attempted using NA polymerase. Since T4 DNA polymerase is an enzyme having both 3 ′ → 5 ′ exonuclease activity as described above, it is possible to constitute a reaction system without adding another enzyme as an enzyme having nuclease activity.

Using 4 units of T4 DNA polymerase,
The reaction system had the composition shown below. Otherwise, the reaction was carried out under the same conditions as in the above (1). The results are shown in FIG. 7 (lane 1: Klenow fragment + exonuclease alone and no detection target sequence, lane 2: Klenow fragment + exonuclease + detection target sequence, lane 3: T4DN
A polymerase alone, as shown in Lane 4: T4 DNA polymerase + sequence to be detected). Although the spot was smaller than the result of the above (1) shown as a control, production of dCMP, which is a nuclease degradation product, was confirmed. As described above, it was found that the use of T4 DNA polymerase allows the reaction according to the present invention to proceed without adding another substance as an enzyme having nuclease activity.

<Composition of reaction solution> First reaction (5 μl) 10 fmol Target sequence 1 pmol Primer DNA 67 mM Tris / HCl (pH8.8) 6.7 mM MgCl ₂ 16.7 mM (NH ₄ ) ₂ SO ₄ 6.7 mM EDTA Second reaction ( 10 μl) 10 mM β-mercaptoethanol 10 μM dCTP ( ³² P label, 5 × 10 ⁴ cpm) 50 μg / ml BSA 4 units T4 DNA polymerase (Takara Shuzo)

[0097]

According to the present invention, a method for detecting a polynucleotide containing a specific base sequence present in a sample effective for diagnosing a genetic disease or an infectious disease, and a kit for detecting a polynucleotide used in the detection method Is provided.

[0098]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Origin Organism: Cytomegalovirus (CMV) Sequence CCCCGAAATG GGACCCAGTA CGGAT

SEQ ID NO: 2 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Origin Organism: Human oncogene Ki-ras / 12 sequences ATAAACTTGT GGTAGTTGGA GCTG 24

SEQ ID NO: 3 Sequence length: 25 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Origin Organism name: HBV-HBV-e of DNA Antigen gene sequence AATGCCCCTA TCTTATCAAC ACTTC 25

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the reaction principle of the present invention.

FIG. 2 shows the results of autoradiography obtained by the method for detecting an HBV-e antigen gene according to the present invention.

FIG. 3 shows the results of autoradiography in which the necessity of nuclease-resistant primers in a reaction system according to the present invention was investigated.

FIG. 4 is a diagram showing the results of autoradiography in which the relationship between the complementarity of the primer sequence and the nucleotide sequence of the polynucleotide to be detected and the nuclease degradation product was investigated.

FIG. 5 shows the results of autoradiography in which the relationship between a substrate and a nuclease degradation product was investigated.

FIG. 6 shows the results of autoradiography in which the sensitivity was investigated by changing the concentration of a target polynucleotide.

FIG. 7 is a diagram showing the results of autoradiography investigated for the case where T4 DNA polymerase was used.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-6-327499 (JP, A) JP-A-59-208465 (JP, A) WO 93/21340 (WO, A1) ANALYTICAL BIOCHE MISTRY, 1988, Vol. 174, p.423-436 ANALYTICAL BIOCHE MISTRY, 1993, Vol. 208, pp. 171-175 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/68 BIOSIS / WPI (DIALOG)

Claims (11)

(57) [Claims] 1. A polynucleotide whose base sequence is known to be detected is hybridized with an oligonucleotide primer having a sequence complementary to a part of the polynucleotide and having nuclease resistance. A deoxynucleoside triphosphate, a DNA polymerase and a nuclease are added to synthesize a complementary strand of a base species adjacent to the 3 'end of the primer and complementary to the polynucleotide, and subsequently decompose the complementary strand; The method for detecting a polynucleotide, wherein the synthesis and the decomposition are repeated at least once or more, and the generated pyrophosphate or deoxynucleoside monophosphate is detected. 2. The oligonucleotide primer according to claim 1, wherein the oligonucleotide primer is phosphorothioated at the 3 ′ end of the oligonucleotide primer.
A method for detecting the polynucleotide according to the above. 3. The DNA polymerase according to claim 1 or 2, wherein the DNA polymerase is a DNA polymerase selected from the group consisting of DNA polymerase I, Klenow fragment of DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase and Phi29 DNA polymerase. A method for detecting a polynucleotide. 4. The nuclease is exonuclease III.
The method for detecting a polynucleotide according to any one of claims 1 to 3, wherein 5. A method for detecting deoxynucleoside monophosphate in which a molecule or atom other than a phosphoric acid molecule at the β-position and γ-position of deoxynucleoside triphosphate is substituted and labeled with a radioisotope, and which is generated by a reaction with a nuclease. The method for detecting a polynucleotide according to any one of claims 1 to 4, characterized in that: 6. The method according to claim 1, wherein the deoxynucleoside monophosphate generated by the reaction with the nuclease is separated by chromatography, and the deoxynucleoside monophosphate is optically measured. A method for detecting a polynucleotide. 7. The method for detecting a polynucleotide according to any one of claims 1 to 4, wherein pyrophosphate produced by complementary strand synthesis by a DNA polymerase is converted to adenosine-5'-phosphosulfate and adenosine triphosphate sulfurylase. A production method of adenosine triphosphate, and detecting the adenosine triphosphate. 8. A method for converting adenosine triphosphate to luciferin-
The method for detecting a polynucleotide according to claim 7, wherein the measurement is performed by a luciferase reaction. 9. The following components (1) to (4): 1. an oligonucleotide primer having a sequence complementary to a part of a polynucleotide whose base sequence to be detected is known and having nuclease resistance; 2. DNA polymerase, At least one kind of deoxynucleoside triphosphate and a nuclease having an activity of decomposing a double-stranded DNA in a 4.3 ′ → 5 ′ direction. A polynucleotide detection kit used in the polynucleotide detection method according to any one of claims 1 to 8. 10. The polynucleotide detection kit according to claim 9, wherein the detection kit contains a reagent for detecting deoxynucleoside monophosphate. 11. The polynucleotide detection kit according to claim 9, wherein the detection kit contains a pyrophosphate detection reagent.