JPH07231799A - Method for detecting specific polynucleotide and kit for detection used for the same method - Google Patents

Abstract

PURPOSE:To provide a method for detecting a polynucleotide containing a specific basic sequence existing in a sample effective in diagnosis for genetic diseases or infectious diseases and a kit for detecting the polynucleotide used in the detection method. CONSTITUTION:This method for detecting a polynucleotide comprises hybridizing a polynucleotide in which the basic sequence to be detected is known with an oligonucleotide primer having a sequence complementary to a part of the polynucleotide and having nuclease resistance, adding at least one kind of deoxynucleoside triphosphoric acid, a DNA polymerase and a nuclease thereto, synthesizing a complementary strand of a base series adjacent to 3' end of a primer and complementary to the polynucleotide and successively decomposing the strand and repeating synthesis and decomposition of the complementary strand one or more times and detecting the produced pyrophosphate or deoxynucleoside monophosphoric acid. This kit for detection is used for the method for detecting the polynucleotide.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD 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 Analysis methods based on the complementarity of nucleic acid base sequences can directly analyze genetic characteristics. Therefore, it is a very effective means for genetic diseases, canceration, identification of microorganisms, and the like. In addition, since the gene itself is the detection target, it may be possible to omit a time-consuming and troublesome operation such as culturing.

However, it is generally not easy to detect when the amount of the target gene in the sample is small, and it is necessary to amplify the target gene itself or the detection signal. As one of the methods to amplify the target gene, PCR (Pol
ymerase Chain Reaction) method is known. The PCR method is the most general method for amplifying nucleic acids in vitro. However, in the PCR method, a special temperature control device is required for implementation; there is a problem in quantitativeness because the amplification reaction proceeds logarithmically; the sample and the reaction solution are contaminated from the outside. However, it is known that nucleic acids that are erroneously mixed in are likely to be affected by contamination that functions as a template.

That is, in a reaction such as PCR in which DNA is amplified up to several million times, a small amount of contaminated DNA is also amplified, so that an erroneous result tends to be provided. This is a serious problem especially when a large number of specimens are handled at the same time. As a countermeasure against such contamination, different laboratories may be used, or a uracil group may be incorporated in a PCR reaction, and the sample is treated with uracil glycosylase before a new PCR reaction, so that other samples erroneously mixed in the sample may be used. Although measures have been taken to decompose only the amplification product of the reaction system, these measures are not always sufficient.

As a method for amplifying the detection signal, a method for amplifying signal RNA with Qβ replicase (Bio / Technology, 6, 1197-1202 (1988), PM Lizardi
et al.) has 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 is a problem that the insertion position or sequence is restricted due to the restriction on the three-dimensional structure. Further, the method of amplifying the signal RNA by Qβ replicase has the same contamination problem as the PCR method, and it cannot be said to be a fundamental solution.

The above 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 is known in which an oligonucleotide probe DNA is hybridized with a target nucleic acid, followed by treatment with a restriction enzyme and detection of the cleaved probe fragment (EP-04.
55517 / A1). Although this method has a lower detection sensitivity than the PCR method, it has the advantages that it is excellent in quantification and that no special device is required for its implementation. However, in this method, in addition to the probe DNA, it is necessary to allow a second oligonucleotide having a specific sequence for allowing repeated reactions to coexist in the system. Further, it has a drawback that the detectable site is limited because the site to be detected requires a restriction enzyme cleavage site.

In addition, a cycling assay method using λ exonuclease that specifically cleaves double-stranded DNA has been developed (BioTechniques, Vol. 13, No. 6, 8).
82-892 (1992), CG Copley et al.). In this method, a double-stranded DNA formed by hybridizing an oligonucleotide probe with a complementary sequence is reacted with λ exonuclease, and when the probe DNA is decomposed to the extent that the double-stranded DNA cannot be maintained, a new probe is produced. Cycling reaction occurs due to the fact that this new probe is replaced by DNA and is subsequently decomposed. This method can determine the presence or absence of a specific DNA sequence by detecting the degradation product of the probe. In addition, since the reaction principle is simple and the sequence of the detection site is not limited, a method using a restriction enzyme (EP-0455517 /
It has an advantage over A1).

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

As a cycling assay method using exonuclease, the method described in JP-A-5-130870 is also known. The method described in this publication is a method in which a primer is decomposed from the reverse direction by allowing a 5 ′ → 3 ′ exonuclease to act together with a synthetic reaction of a complementary strand starting from a primer. That is, a new primer is hybridized in place of the primer decomposed by 5 '→ 3' exonuclease, the synthesis of complementary strand by DNA polymerase and the reaction of removing the previously synthesized strand proceed, and a cycling reaction is established. To do. In this method, PCR
Although a complicated temperature control such as a reaction is not required, it is still difficult to increase the turnover number (probe DNA hybridization repetition number) because it is necessary to repeat the primer hybridization step.

The present inventors have used a specific reaction promoter as an exonuclease III for the purpose of solving these problems.
We have developed a nucleotide sequence detection method for use with
327499 publication). This method is an excellent method because the probe can be easily synthesized, special temperature control is not required during the reaction, and the base sequence can be detected with almost no influence of contamination. However, in this technique, the reaction promoter has the effect of increasing the turnover number (the number of repetitions of hybridization of probe DNA similar to the description in the cycling assay method using λ exonuclease), but There was no change in that they had to be hybridized repeatedly, and there was a problem that high sensitivity could not always be obtained.

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

[0012]

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a technique for detecting a base sequence that is not easily affected by contamination in a simple reaction system, regardless of complicated temperature control that requires a special device. To do. Further, the present invention has no limitation on the base sequence to be detected in order to achieve these objects,
It is also an object to provide a detection technique having excellent versatility. Furthermore, it is another object to provide a technique for detecting a base sequence that can obtain high sensitivity and quantification depending on the selection of the detection system.

[0013]

Means for Solving the Problems The inventors of the present invention have made extensive studies for the purpose of solving the above problems. As a result, a nuclease that does not act on single-stranded DNA, cuts only double-stranded DNA, and is not limited to a base sequence is DN
The present invention was completed by developing a signal amplification method used together with A polymerase.

That is, the gist of the present invention is as follows. (1) A polynucleotide whose base 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 Deoxynucleoside triphosphates, DNA polymerase and nuclease are added to synthesize complementary strands of base species adjacent to the 3'end of the primer and complementary to the polynucleotide, and subsequently decomposed, and the complementary strand synthesis and The method for detecting a polynucleotide, wherein the pyrophosphate or deoxynucleoside monophosphate produced is detected by repeating the decomposition at least once or more.

(2) The oligonucleotide primer is phosphorothioated at the 3'end of the oligonucleotide primer.
(1) The method for detecting a polynucleotide as described.

(3) 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.
(1) or the method for detecting a polynucleotide according to (2).

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

(5) β of deoxynucleoside triphosphate
A molecule or atom other than the phosphate molecule at position and γ-position is substituted and labeled with a radioisotope, and is characterized by detecting deoxynucleoside monophosphate produced by a reaction with nuclease (1) to ( The method for detecting a polynucleotide according to any one of 4).

(6) The deoxynucleoside monophosphate produced by the reaction with a nuclease is separated by chromatography to optically measure the deoxynucleoside monophosphate. In any one of the above (1) to (4) A method for detecting a polynucleotide as described.

(7) In the method for detecting a polynucleotide according to any one of (1) to (4) above, pyrophosphate generated by complementary strand synthesis by a DNA polymerase is converted into adenosine-5'-phosphosulfate and adenosinetrilin. A method for detecting a polynucleotide, which comprises reacting with acid sulfurylase to generate adenosine triphosphate and detecting the adenosine triphosphate.

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

(9) Components 1 to 4 below: 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. DNA polymerase, 3. At least one kind of deoxynucleoside triphosphate, and a nuclease having an activity of degrading double-stranded DNA in the 4.3 ′ → 5 ′ direction. A kit for detecting a polynucleotide used in the method for detecting a polynucleotide according to any one of (1) to (8) above, which comprises:

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

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

The present invention will be described in detail below. 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, yeasts, filamentous fungi, mycoplasma, rickettsia, viruses and everything else. As the polynucleotide, not only genomic nucleic acid but also cDNA derived from RNA virus or mRNA can be used as a detection target. When actually analyzing a sample, the presence of a sequence of DNA other than the detection target contained in the sample and a sequence that can serve as a primer for DNA synthesis may pose a problem. In addition, there is a possibility that DNA polymerase, nuclease activity inhibitor, and deoxynucleoside triphosphate are also mixed. Furthermore, when pyrophosphoric acid is the detection target, pyrophosphoric acid coexisting in the sample also becomes a factor that interferes with the analysis.

Therefore, in the present invention, it is preferable to remove the contaminants as much as possible in order to carry out the amplification reaction. For example, when the target DNA is captured by using a capture probe or the like bound to a solid phase, and then the impurities are removed by washing, and then the present invention is applied, a highly sensitive detection system without background becomes possible. At this time, if the capture probe is bound to the solid phase at the 5'end (Eur.J.Immunol., 2
3 , 1895-1901 (1993), H. Kohsaka et al .; Nucleic Acid
s Research, 21 , 3469-3472 (1993), H. Kohsaka et al.
), The method of this experiment can be applied using the capture probe itself as a primer. The pyrophosphate can be removed enzymatically with pyrophosphatase.

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

Indeed, some of the elements used in the method are common with the present invention. However, in the first place, the method is characterized in that the presence or absence of nuclease resistance is used as an index for the presence of a point mutation, whereas the present invention determines whether the reaction is repeated or not by the presence of a point mutation. Are essentially different in that they are used as. The detection of the point mutation by the method of the present invention is advantageous in terms of sensitivity as compared with the detection of the mutation by the method, which is advantageous.

B. The present invention is characterized in that the known polynucleotide is hybridized with an oligonucleotide primer having a sequence complementary to a part of the polynucleotide and having nuclease resistance.
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 pairing. Specifically, it is a state in which thymine (T) corresponds to adenine (A) and cytosine (C) corresponds 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 as a whole can hybridize to the target polynucleotide. That's 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 all the 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 a base addition reaction at the 3'end. If the base does not correspond to the 3'end and the primer is single-stranded, polymerase and nuclease cannot be recognized.

The oligonucleotide primer used in the present invention is a "part" of a known target polynucleotide.
It is characterized by being complementary to. This is because the method of the present invention is
Due to the feature of detecting a specific deoxynucleotide added to the reaction system in advance and added by A polymerase. That is, if the nucleotide serving as the template for the deoxynucleotide added to the 3′-terminal side of the primer is not known, it is difficult to allow a specific deoxynucleotide to exist in the system in advance,
The oligonucleotide primer used in the present invention needs to be complementary to a "portion" of a known target polynucleotide.

Further, the oligonucleotide primer used in the present invention is required to have resistance to nuclease. This is because, in the present invention, it is necessary to allow a nuclease to exist in the system, and therefore, if the oligonucleotide primer is decomposed by such a 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 synthesizing an oligonucleotide serving as a primer in a DNA synthesizer, a phosphorothioate (Phosp
Introducing a horothioate) bond can impart nuclease resistance to the oligonucleotide primer. Examples of such a method include solid-phase phosphoramidite (P
hosphoramidite) method to synthesize DNA,
Instead of the oxidation step with iodine water or the like, an oxidation treatment with an appropriate S-forming reagent (phosphorothioate-forming reagent) can be carried out to introduce a phosphorothioate bond into the oligonucleotide instead of the usual phosphodiester bond. As the S 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, DNA polymerase
When synthesizing NA, a phosphorothioate bond can be introduced into an oligonucleotide by using, as a substrate, deoxyribonucleoside triphosphate in which the oxygen bonded to the phosphorus atom at the α-position is replaced with sulfur.
Examples of such a substitution compound include α-S-deoxythymidine triphosphate, α-S-deoxycytosine triphosphate, α-S-deoxyadenine triphosphate and α-S-.
Examples thereof include deoxyguanine triphosphate and the like (these substituted compounds are generically abbreviated as SdXTP). When performing a synthetic 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 introducing a phosphorothioate bond by DNA polymerase,
This reaction can be allowed to proceed in the state in which it hybridizes with the polynucleotide that is actually the detection target. That is, the nuclease-resistant oligonucleotide primer of the present invention does not necessarily have to be prepared in advance, and a method of acquiring nuclease resistance during the reaction can be adopted.

Once nuclease-resistant, the subsequent reaction can proceed according to the reaction principle described later.
In this case, SdXTP is added first, and then dX
Although TP is added and an extension reaction of at least 2 bases is required, the nuclease acts on the second dXTP, so that the method of the present invention can be carried out. In any case, by converting the phosphodiester bond near the 3'end of the primer into a phosphorothioate bond,
It serves as an oligonucleotide primer resistant to a nuclease attacking from the 3'side. However, if the entire oligonucleotide is phosphorothioate-bonded, the nuclease resistance will increase further, but at the same time, the efficiency of hybridization will decrease. Therefore, the number of phosphorothioate bonds is preferably 1 to several from the end.
The phosphorothioate bond may be only one terminal,
More complete nuclease resistance can be expected if several, preferably three, phosphorothioate bonds are consecutively formed.

However, as described above, the efficiency of incorporation into DNA by the DNA polymerase is such that SdXTP is dXT.
Since it is much lower than P, SdXTP is first taken in and then dXTP for decomposition is added, or SdXT
In some cases, it is necessary to devise to set the P concentration higher.

As a means for imparting nuclease resistance, methylphosphonate (M
ethylphosphonate) bond, phosphoramidate (Phospho
roamidate binding, Polyamide nucleic acid
The introduction of a cid (PNA)) bond or the like can be shown. These bonds modify the phosphate binding site of the nucleotide, the ribose site, and the base site of the nucleic acid, or modify the structure to acquire nuclease resistance.

In addition to this, it is also possible to use an RNA primer as the oligonucleotide primer. R
NA is almost resistant to nuclease acting on DNA. Therefore, by using an RNA primer, it is possible to prepare an oligonucleotide primer having nuclease resistance that acts on DNA without any special modification. However, among nucleases and DNA polymerases that act on DNA, RNase
Some of them also have H activity, and in such a case R
Since the NA primer itself is degraded, it may be difficult to obtain high sensitivity depending on the combination of enzymes.

The oligonucleotide primer of the present invention has at least 6 or more bases, preferably 10 to 5 bases.
The value is 0, particularly preferably about 15 to 30. If it is less than 6, not only is it difficult to hybridize under normal conditions, but there is an increased possibility that a stochastic non-specific reaction will occur. On the other hand, an oligonucleotide primer made nuclease resistant by phosphorothioation, for example, has a low affinity (Tm) with respect to the template, and therefore it is necessary to provide a certain length to compensate for this decrease.
On the other hand, if it exceeds 30, the yield tends to decrease when chemically synthesized, which is economically disadvantageous and is not preferable. In addition, when using a primer longer than necessary, it is possible to
A single strand is easily formed and a nonspecific reaction is likely to occur. Such an unnecessarily long primer is not preferable because it may cause nonspecific amplification or the like.

The DNA polymerase which can be used in the present invention will be described later. Among them, there is a DNA polymerase such as DNA polymerase I which has a weak 5 ′ → 3 ′ exonuclease activity for double-stranded DNA. When this kind of enzyme is used as a DNA polymerase, the hybridized probe is decomposed from the 5'side, so it is preferable to make the 5'side resistant to nucleases. Even with the same DNA polymerase, Klenow fragment and Ph
Since i29 DNA polymerase does not have this activity, it is not necessary to modify the 5'side, and this is preferable.

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

The conditions of hybridization are not particularly limited, but it is necessary to pay attention to the fact that after the hybridization, all the reactions need to proceed in the presence of DNA polymerase and nuclease. In particular, nucleases do not have a high degree of heat resistance as Taq polymerase used in the PCR method, so it is necessary to adopt conditions that can maintain the enzymatic activity of the nuclease. In addition, selection of a buffer solution, which is an element other than temperature, and setting of pH should select conditions under which not only hybridization but also nuclease activity can be sufficiently expressed. For example, when Tris-hydrochloric acid buffer is used as the buffer, the pH is about 7 to
9 and the reaction is carried out at 20 to 55 ° C. Particularly preferable conditions are pH 7.5 to 8.5 and temperature of 30 to 45 ° C.

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

Further, a stabilizing agent for DNA polymerase or nuclease can be added to the reaction solution. Examples of such stabilizers include bovine serum albumin (BS
A), dithiothreitol (DTT), β-mercaptoethanol and the like can be mentioned. The amount of the stabilizer added is 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 needs to be denatured into a single strand in advance. Examples of such denaturing methods include generally known denaturing methods such as heat denaturation, acid denaturation, and alkali denaturation. In view of the simplicity and certainty of the method, heat denaturation (at 90 ° C to 100 ° C,
It is preferable to adopt heating for more than a minute).

DNA other than the intended primer
In order to suppress the non-specific reaction by suppressing the hybridization of the DNA or the non-specifically hybridized DNA is removed, the exonuclease treatment can be performed before the addition of the DNA polymerase.

C. The present invention then comprises the addition of at least one deoxynucleoside triphosphate, a DNA polymerase and a nuclease to the system after said hybridizing to the base adjacent to the 3'end of said primer and complementary to said polynucleotide. The species is characterized by synthesizing complementary strands, subsequently degrading, and repeating the synthesis and degrading of the complementary strands at least once or more. The reaction principle of the present invention is shown below. The reaction principle of the present invention is shown in FIG. 1 and the following formula 1.

[0046]

[Chemical 1]

First, the synthesis of the complementary strand is started by the action of DNA polymerase based on the oligonucleotide primer hybridized to the single-stranded nucleic acid to be detected. At this time, one molecule of pyrophosphoric acid (hereinafter sometimes abbreviated as PPi) is generated along with the addition of one molecule of dXTP. On the other hand, when a nuclease that decomposes from the 3'end of the double-stranded DNA acts in this state, the reaction of decomposing the extended portion proceeds. However, since the oligonucleotide primer is nuclease resistant, the primer portion remains as it is without being decomposed, and becomes the starting point of the extension reaction by the DNA polymerase again. By repeating the reaction in this manner, pyrophosphate or deoxynucleoside monophosphate produced by the decomposition of nuclease accumulates in the reaction system.

As mentioned above, DNA is used during the reaction.
It is also possible to subject the oligonucleotide primer to nuclease resistance in the above step by the action of a polymerase. The presence or absence of the base sequence to be detected can be detected or quantified by detecting this pyrophosphate or deoxynucleoside monophosphate generated by decomposition with nuclease.

Furthermore, it is possible to construct a system for detecting point mutations 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 dXTP, which serves as a substrate for the chain to be extended, is added to only one type of substrate so that extension will occur only in the case of a normal sequence, the reaction will be stopped when point mutation occurs and the reaction cannot be performed. In this way, it becomes possible to easily confirm the presence or absence of a point mutation (Equation 2 below).

[0050]

[Chemical 2]

Further, if a mutation occurs in a region to be hybridized with an oligonucleotide primer, the efficiency of hybridization is naturally lowered, so that the whole reaction is difficult to proceed, whereby it is possible to know the presence or absence of a point mutation. You can The DNA polymerase used in the above catalyzes a reaction of extending a complementary strand in the 5 ′ → 3 ′ direction starting from a double-stranded portion formed by hybridizing the complementary strand to a single-stranded nucleic acid serving as a template. A DNA polymerase is used. In fact, all of the currently known DNA polymerases have DNA synthase activity in the 5 ′ → 3 ′ direction. In addition, a primer is always required for DNA synthesis to occur, and DNA synthesis cannot occur if there is no primer or if hybridization is not possible. This is one of the reasons why the present invention has high specificity depending on the primer and 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, or mutants of these DNA polymerases.

As a DNA polymerase, T4DN was used.
If one that also has a strong 3 '→ 5' exonuclease activity, such as A polymerase or T7 DNA polymerase, is used, it is possible to save the amount of nuclease added separately, or one type of strong enzyme acting on the nucleic acid. DNA with excellent 3 '→ 5' exonuclease activity
It is also possible to construct the reaction system of the present invention by adding only a polymerase. However, since these DNA polymerases also decompose phosphorothioate bonds that are nuclease resistant 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 end 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 single-stranded DNA in the 3 '→ 5'direction. Such nucleases include:
Exonuclease III is known. As the exonuclease III, those derived from Escherichia coli are commercially available at present, but the exonuclease III is not limited to this, and other microorganisms or those obtained by gene recombination can also be used. Exonuclease II
When I is used, DNA polymerase I exemplified as the DNA polymerase, Klenow fragment thereof,
Since the enzyme activity is exhibited under almost the same reaction conditions as T4 DNA polymerase and 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 into 3 '→
Due to the fact that it decomposes specifically in the 5'direction, it does not act on single-stranded DNA, the sequence or primer to be detected is not attacked, and that SdXTP makes the primer easily nuclease resistant. It is a very suitable enzyme for circulation in the reaction cycle of the invention.

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

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

By substituting and labeling the phosphorus atom at the α-position of dXTP used as a substrate with a radioisotope ( ³² P or ³³ P), the hydrogen atom at the phosphate, deoxyribose, or base at the α-position is ³ H. By substitution labeling with;
Alternatively, by labeling the carbon atom of deoxyribose with ¹⁴ C, dXTP and dXMP can be chromatographically separated by an ion exchange resin or the like and the radioactivity can be traced to measure the radioactivity of the degradation product dXMP. Is possible. At present, the phosphorus atom at the α-position is labeled with ³² P d
As XTP, dATP, dCTP, dGTP and d
TTP is commercially available. In addition, the phosphorus atom at the α position is currently
³³ P The labeled dXTP, dATP and dCTP
Is commercially available. Further, as having been ³ H labeled base moiety, dATP, dCTP, dGTP and dTTP
Is commercially available.

DXMP with radioisotope label
The radioactivity can be easily and qualitatively detected by autoradiography by separation by thin layer chromatography using an ion exchange resin. Further, as shown in the examples, if the spot having the radioactivity is scraped off and counted by a scintillation counter, a quantitative analysis is possible. Even if a substrate labeled with an isotope is not necessarily used, dX
It is possible to separate and measure MP. That is, when the reaction solution is separated by thin layer chromatography coated with a commercially available fluorescent dye and irradiated with ultraviolet rays, the nucleotides absorb the ultraviolet rays, so that dXMP can be visually recognized as a non-fluorescent spot. In addition, dXMP can be quantitatively detected by separating by liquid chromatography and measuring ultraviolet absorption.

Quantification of Pyrophosphate Generated by Chain Extension Reaction with DNA Polymerase In the method of the present invention, the desired polynucleotide can be detected by measuring the production of PPi accompanying the addition of dXMP by DNA polymerase. This D
According to the method of tracing PPi generated by the addition of dXMP by NA polymerase, it is possible to enzymatically measure it, and the work of separating the reaction solution by chromatography or the like can be omitted. Become.
Specifically, the reaction of the following formula 3 and the like are known. Reaction of the formula 3 (J. Immunological Methods, 156, 55-60 (19
92), T. Tabary et al.) Enables highly sensitive measurement, and the operation is simple because it allows analysis in a homogeneous system.

[0059]

[Chemical 3]

As another method for measuring pyrophosphate,
The method and the like cited in "Anal. Biochem., 94 , 117-120 (1979)" are known.

E. Specific Example The specific sequence of the oligonucleotide primer and the reaction system according to the present invention when the sequence is used are illustrated below (Formula 4 below).

[0062]

[Chemical 4]

Human cytomegalovirus (hereinafter CMV
For example, CMV genomic DNA
A sequence derived from the D fragment of the restriction enzyme EcoRI digested fragment and specific to the CMV genome is used as a primer.
The specific sequence is as shown in Formula 4. Although any part of the array shown in Expression 4 may be used, in the following description, Expression 4
The complementary strand that hybridizes to the part sandwiched between → ← is used as a primer (SEQ ID NO: 1). The phosphodiester bond between ATs on the 3'side is a phosphorothioate bond. By measuring PPi or the degradation product dAMP, it is possible to know whether or not the CMV sequence is present in the sample.

Particularly, the preferred method of the present invention in which the extension to the decomposition of one base is repeated in one cycle can be applied to the detection of point mutation as described above. As an example of detecting a point mutation, the human oncogene Ki-ras will be described below.
/ 12 detection system is shown. For Ki-ras / 12, mutants represented by the following formula 5 are known (formula 5 below).
In the figure, the dots indicate the normal sequence part where no abnormality has occurred).

[0065]

[Chemical 5]

As the primer, the sequence of the portion shown in Formula 5 (SEQ ID NO: 2) is used. The 3'side TG was phosphorothioate-bonded to make it nuclease-resistant, and d was used as a substrate for synthesis by DNA polymerase.
Only GTP is added. In this example, if the detection target is normal, the reaction that takes in dGTP proceeds and PPi and d
GMP is accumulated. However, when the detection target is a mutant, the reaction does not proceed because there is no dTTP required for the synthesis of the complementary strand. On the contrary, if the reaction proceeds when only dTTP is added to the substrate, it can be seen that the sequence to be detected contains a point mutation. In the present invention, it is possible to detect a point mutation in this way (Equation 6 below).
reference).

[0067]

[Chemical 6]

In Formulas 5 and 6, the primers are shown in the upper row and the template sequences are shown in the lower row, which is different from the expressions in Formulas 1, 2 and 4 (the template sequences are in the upper row and the primers are in the lower row). There is.

F. Polynucleotide Detection Kit of the Present Invention The various components necessary for the present invention as described above can be supplied in the form of pre-combined reagents. The constitution of the reagent for detecting a base sequence according to the present invention is shown below. The reagents shown below may be combined in the form of a kit with a combination of materials necessary for the detection of the label, a buffer that constitutes the reaction solution, or any component such as a negative or positive control.

The composition of the polynucleotide detection kit 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. DNA polymerase, 3. At least one type of deoxynucleoside triphosphate, a nuclease having the activity of degrading double-stranded DNA in the 4.3 ′ → 5 ′ direction.

The kit containing the above 1 to 4 may contain a detection reagent according to the above-mentioned index for detecting the 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 the following detection kits.

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

The mechanism of action and the actual effect of the present invention will be summarized below. 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 a DNA polymerase. In addition, by making it nuclease-resistant in advance, or by adding a deoxynucleotide derivative (SdXTP etc.) by the action of DNA polymerase to make it nuclease-resistant, deoxynucleoside triphosphate (dXT) by the action of the following DNA polymerase is obtained.
It is possible to repeatedly cause two reactions of addition of P) to decomposition by the action of nuclease. Since this reaction repeatedly and linearly occurs, in the present invention, a nuclease degradation product accumulates 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 a mismatch, if the dXTP prepared in advance as a substrate is not suitable in the step of synthesizing the complementary strand of the region adjacent thereto, the subsequent reaction Has the advantage that no erroneous results will occur. Further, in the present invention, it is not necessary to repeat the denaturation operation of DNA by high temperature treatment, so that the reagent components such as DNA polymerase are not required to have thermal stability, 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 the sequence adjacent to the region hybridized with the primer. At the time of synthesis,
For the addition reaction of 1 mol of dXTP, 1 mol of PPi is produced. Since PPi can be specifically measured by an enzymatic reaction, the present invention is a system that simultaneously generates a signal by a synthetic reaction. In another embodiment, a product obtained by decomposing a portion extended by this reaction with a nuclease can be used as a detection target. When a non-nuclease-resistant oligonucleotide primer is used as a DNA polymerase, it has the action of converting the deoxynucleotide derivative into a nuclease-resistant substrate by using it as a substrate.

The nuclease in the present invention has the action of specifically hydrolyzing the extended portion starting from the oligonucleotide primer hybridized with the sequence to be detected up 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 nuclease, the degradation products accumulated in the reaction solution in the present invention increase specifically and linearly with respect to the detection target, which enables highly quantitative analysis.

Further, one of the advantages of the present invention is that it is not easily affected by contamination. That is, even if the sample after the reaction is mistakenly mixed in 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 practical use.

Furthermore, as a unique effect of the present invention, it is possible to provide a simple system and a simple operation. PP produced by addition of dXTP by DNA polymerase
By adopting the method of tracking i, it is possible to provide a reaction system that does not require any operation such as hybridization with an additional probe or separation by electrophoresis. Since PPi can be specifically measured by a purely enzymatic reaction, the reaction can be continuously continued as it is in the reaction solution in which the synthesis and decomposition reactions have been performed, which is very practical. Such an advantage of the present invention is clearer than that hybridization with a probe or separation by electrophoresis is required to detect an amplification product by the PCR method.

Another advantage of continuously using the primer hybridized to the detection target sequence in the reaction in the first step of the reaction is that a relatively small amount of primer needs to be prepared. In a system in which 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 a sequence to be detected. . The same applies to deoxynucleoside triphosphate, which is a substrate. That is, for example, in PCR, it is necessary to prepare all necessary substrates for the region to be extended. The present invention has an advantage that the reagent composition can be simplified because at least one kind of base complementary to the sequence adjacent to the hybridized region needs 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 the site causing the point mutation is known in advance, the region adjacent to this part is selected as a primer and the reaction system is set so that the reaction does not proceed when the point mutation occurs. It is possible to confirm the presence or absence of. 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]

The present invention will be described in more detail based on the following examples. However, the technical scope of the present invention is not limited to the present embodiment. [Example] Detection of HBV-e antigen gene of HBV DNA An attempt was made to detect the HBV-e antigen gene of HBV DNA. Exonuclease derived from E. coli is used as the nuclease.
III (manufactured by Takara Shuzo Co., Ltd.) and an oligonucleotide having a base sequence shown in SEQ ID NO: 3 was chemically synthesized and used as a primer DNA.

(1) -1 Preparation of primer DNA The oligonucleotide of the above sequence (SEQ ID NO: 3) is β-
Cyanoethyl amidite 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 an agent) as a phosphorothioate bond between TCs on the 3′-terminal side. Cyclone (registered trademark) P as a DNA synthesizer
The lus DNA / RNA synthesizer (Japan Millipo Limited) was used. The synthesized phosphorothioated oligonucleotide was used after being purified by HPLC according to a conventional method.

(1) -2 Detection of Nucleotide Sequence The primer DNA was mixed with M13 phage DNA having a part of HBV DNA 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 also performed on a primer having the same sequence but not having nuclease resistance. When the primer used in this example was hybridized, the sequence adjacent to the 3'side was G, and therefore d was used for the synthesis of its complementary strand.
CTP is required. Then, 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 labeling, 5 × 10 ⁴ cpm) 50 μg / ml Bovine serum albumin (abbreviated as BSA below) 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, add a reagent solution containing the components necessary for the second reaction and further add 37 ° C.
It was made to react with. After reacting for 1 hour, add 1μ of 20mM EDTA.
l In addition, the reaction was stopped. Thin layer chromatography (thin layer: PEI-Cellulose)
F (manufactured by Merck), developing solvent: developed with 0.4 M LiCl for 40 minutes at room temperature), and dCMP as a nuclease degradation product was confirmed by autoradiography.

As is clear from FIG. 2, the sequence to be detected, the Klenow fragment, and the exonuclease III
DCM, which is a nuclease degradation product without any of
P was not detected (lanes 2-4), confirming that the reaction of the present invention proceeded specifically (lane 1). In addition, lane 1 is a system for detection target sequence + all reagent components, lane 2 is a system for reagent components only, lane 3 is a system for removing Klenow fragment in the reagent components, and lane 4 is a reagent component. Among them, it is a system 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 other components (Fig. 3: lane 1 shows A nuclease-resistant primer is used, and a lane 2 is an unmodified primer, that is, a nuclease-sensitive primer). It is considered that this is because dCTP cannot be added because the 3'side region of the primer (that is, the addition site of dCTP) is decomposed by the nuclease.

(2) Template Specificity Only when the primer has complementarity with the sequence to be detected,
The following experiment was conducted to confirm that the degradation of the substrate by nuclease occurred. That is, the reaction was actually performed by the same operation as the above (1) under the two conditions of using a sequence having no complementarity with the primer as the detection target sequence and not detecting the detection target sequence.

As a result, in FIG. 4 (lane 1: the detection target sequence is complementary to the primer sequence, lane 2: the detection target sequence is the same sense sequence as the primer sequence, that is, not complementary, lane 3: does not include the detection target sequence). As shown, if the target sequence and the primer DNA are complementary, dCMP, which is a degradation product, accumulates (lane 1), but the sense sequence, that is, the same sequence as the primer and not complementary (lane 2), and detection It was confirmed that the degradation product did not accumulate when the target sequence did not exist (lane 3). This experiment proved that the reaction of the present invention proceeds when the target sequence has a sequence complementary to the primer.

(3) Substrate (deoxynucleoside triphosphate) specificity In order to confirm that the degradation by nuclease occurs only when dCTP, which is the substrate deoxynucleoside triphosphate, is incorporated into the primer, the substrate in (1) above is used. Was replaced with dGTP and reacted. As a result, FIG. 5 (lane 1: using dCTP as a substrate, lane 2:
Using dGTP as a substrate, lane 3: dCMP and dCTP developed as markers, lane 4: dGMP and dGTP developed as markers)
It was confirmed that the reaction proceeded only when P and dCMP which is a decomposition product was confirmed (lane 1), and that when dGTP was added, the reaction did not proceed (decomposition product was not generated, lane 2). From this result, it became clear that the detection method according to the present invention can be used to detect a point mutation.

(4) Detection Sensitivity of the Present Invention The amount of DNA to be detected is 0 to 1 by the same operation as the above (1).
The sensitivity of the detection method according to the present invention was confirmed by changing to pmol (see Table 1). Figure 6 (Lane 1: 1 pmol of DNA containing the sequence to be detected was 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 detection target sequence, added and reacted, 1 fmol of DNA containing detection target sequence, lane 5: DNA containing detection target sequence
Was reacted by adding 0.1 fmol of lane 6: 0.01 fmol of DNA containing the sequence to be detected was added and reacted, lane 7:
1 amol of DNA containing the sequence to be detected was added and reacted, Lane 8: DNA containing the sequence to be detected was not added,
1 fmol (lane 4) of DN
It was confirmed that A could be detected. From this result, it was found that the detection method according to the present invention has extremely high sensitivity. Further, in order to confirm the quantitativeness of the method for detecting a nucleotide sequence according to the present invention, each band was cut out and the radioactivity was measured by a liquid scintillation counter. The results are shown in Table 1. In Table 1, the values of (A) and (B) show the radioactivity (CPM) of each spot. It was confirmed that the quantitative property could be secured in the range of 0.1 fmol to 0.01 pmol.

In this example, ³² P-labeled dCTP was used.
The amount used of is adjusted beforehand by dilution with unlabeled dCTP within a range in which a difference in spot size of autoradiography is likely to occur. Therefore, higher sensitivity can be realized by increasing the proportion of the labeled substance in dCTP.

[0093]

[Table 1] Table 1 Examination of detection sensitivity of the detection method of the present invention ──────────────────────────────────── ─ 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 25204 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 Instead of the Klenow fragment in the above (1), T4D
The method for detecting a nucleotide sequence according to the present invention was tried using NA polymerase. Since T4 DNA polymerase is an enzyme that also has a 3 ′ → 5 ′ exonuclease activity as described above, it is possible to configure a reaction system without adding another enzyme as an enzyme having a nuclease activity.

Using 4 units of T4 DNA polymerase,
The reaction system had the composition shown below. Other than that, the reaction was carried out under the same conditions as in (1) above. 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 only, lane 4: T4 DNA polymerase + target sequence for detection). Although the spot was smaller than the result of (1) shown as a control, the production of dCMP which is a nuclease degradation product was confirmed. Thus, it was revealed 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-labeled, 5 × 10 ⁴ cpm) 50 μg / ml BSA 4 units T4 DNA polymerase (manufactured by Takara Shuzo)

[0097]

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

[0098]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 25 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Origin Biological Name: Cytomegalovirus (CMV) Sequence CCCCGAAATG GGACCCAGTA CGGAT

SEQ ID NO: 2 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Origin organism name: 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 strand 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 drawings]

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

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

FIG. 3 is a diagram showing the result of autoradiography for investigating the necessity of a nuclease-resistant primer in the reaction system according to the present invention.

FIG. 4 is a diagram showing the result of autoradiography for investigating the relationship between the complementarity between the primer sequence and the nucleotide sequence of the polynucleotide to be detected and the nuclease degradation product.

FIG. 5 is a diagram showing the result of autoradiography for investigating the relationship between a substrate and a nuclease degradation product.

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

FIG. 7 shows the results of autoradiography investigated for the case of using T4 DNA polymerase.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12Q 1/66 6807-4B

Claims (11)

[Claims] 1. A polynucleotide having a known base sequence to be detected is hybridized with an oligonucleotide primer having a sequence complementary to a part of the polynucleotide and having nuclease resistance, and then at least this is hybridized. One kind of deoxynucleoside triphosphate, DNA polymerase and nuclease is 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 decomposed, and the complementary strand. The method for detecting a polynucleotide, which comprises repeating the synthesis and the decomposition at least once to detect the produced pyrophosphate or deoxynucleoside monophosphate. 2. The oligonucleotide primer is phosphorothioated at the 3 ′ end of the oligonucleotide primer.
A method for detecting a polynucleotide as described. 3. 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 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 molecule or atom other than the β- and γ-position phosphate molecules of deoxynucleoside triphosphate is substituted and labeled with a radioactive isotope, and deoxynucleoside monophosphate produced by the reaction with nuclease is detected. The method for detecting a polynucleotide according to any one of claims 1 to 4, wherein: 6. The deoxynucleoside monophosphate produced by the reaction with a nuclease is separated by chromatography, and the deoxynucleoside monophosphate is measured optically, according to any one of claims 1 to 5. 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 into adenosine-5'-phosphosulfate and adenosine triphosphate sulfurylase. A method for detecting a polynucleotide, which comprises reacting with adenosine triphosphate to produce adenosine triphosphate and detecting the adenosine triphosphate. 8. Adenosine triphosphate is added to luciferin-
The method for detecting a polynucleotide according to claim 7, which is performed by a luciferase reaction. 9. The following 1-4 components: 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. DNA polymerase, 3. At least one kind of deoxynucleoside triphosphate, and a nuclease having an activity of degrading double-stranded DNA in the 4.3 ′ → 5 ′ direction. A kit for detecting a polynucleotide used in the method for detecting a polynucleotide according to any one of claims 1 to 8, which comprises: 10. The kit for detecting a polynucleotide 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 reagent for detecting pyrophosphate.