JP2008278779A - Solution used for nucleic acid hybridization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reaction solution for decreasing hybridization sequence dependence and also improving the reaction rate in hybridizing a nucleic acid, and to provide a method for distinguishing a monobase polymorphism with high accuracy by applying the solution. <P>SOLUTION: Hybridization is promoted and also the sequence dependence on hybridization is reduced by hybridizing a specimen nucleic acid with a nucleic acid probe, in the presence of a melting temperature-adjusting agent of the nucleic acid and a cationic polymer. Also, by using a partial double-stranded nucleic acid molecule as the nucleic acid probe, the method for distinguishing the monobase polymorphism quickly, with further higher accuracy is achieved. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a hybridization solution used when hybridizing nucleic acids.

  In recent years, high throughput (HT) analysis of single nucleotide polymorphisms (SNPs) has been actively developed. This is because SNPs are expected as clues for knowing differences among individuals such as genetic factors, drug efficacy, and side effects of diseases. The development of technology for detecting SNPs quickly, simply and inexpensively is the key to dramatic progress in future tailor-made medicine and genome drug discovery. Also, in the treatment of infectious diseases, it is already practiced to quickly and accurately determine the genotypes of viruses and infectious pathogens and select treatment methods and drugs based on the results. All are required to detect single nucleotide polymorphisms and mutated sequences in genomic nucleic acids accurately, quickly and at low cost. Detection methods based on nucleic acid amplification such as PCR and LAMP methods, microarrays and DNA chips Has been actively developed and actively reported.

  One of the major properties of nucleic acid molecules is, for example, in the case of a double-stranded nucleic acid molecule, even if it is dissociated into a single-stranded nucleic acid molecule by a heating operation at about 70 to 100 ° C. The molecules form double-stranded nucleic acid molecules again by annealing. In this double-stranded nucleic acid, a helical stable structure is formed by hydrogen bonds formed between G base and C base and between A base and T base between complementary nucleic acid molecules.

The nucleic acid detection reaction using a single-stranded nucleic acid probe utilizes this property. For example, a synthetic oligonucleotide having 15 to 50 residues labeled with a fluorescent dye such as FITC, Cy3 or Cy5, a radioisotope such as ³² P, or an enzyme such as biotin-HRP, as a probe. Alternatively, hybridize with the target nucleic acid on the solid phase to form double-stranded nucleic acid, remove the excess probe, and then detect the remaining probe with a fluorescence scanner, fluorescence plate reader, absorbance meter, autoradiography, etc. Is the method. At this time, a nucleic acid of about 15 to 50 residues complementary to the target nucleic acid sequence is used as a probe nucleic acid. To determine a single nucleotide polymorphism or to detect and distinguish only a specific sequence, Conditions such as the length of the nucleic acid probe, GC content, salt concentration, and pH must be strictly set. In particular, in order to discriminate the sequence of a plurality of nucleic acids, it is necessary to design the nucleic acid probes so that the melting temperatures (Tm values) of all the probe nucleic acids are almost the same, and to strictly set the reaction conditions.

  A synthetic oligonucleotide of about 10 to 30 residues called a primer also in a method of amplifying a nucleic acid by a nucleic acid amplification method such as a PCR method or a LAMP method and discriminating a single nucleotide polymorphism based on the presence or absence of the amplified product And the target nucleic acid are hybridized, and a nucleic acid is amplified by performing an extension reaction with a polymerase from the 3 ′ end of the hybridized primer. In the case of the normal PCR method, a paired plummer set is used, and in the case of the LAMP method, a primer composed of the sequence of the 4 to 6 regions of the target nucleic acid or its complementary sequence is used. Even in this case, it is necessary to test the melting temperature of the used plummer and to set various reaction conditions strictly. In addition, when there are similar sequences in the target nucleic acid, or when single nucleotide polymorphisms are identified, design an appropriate primer sequence based on the melting temperature of the primer, and set the reaction conditions strictly. Must be done.

  Wallace et al. Reported a method for detecting a single nucleotide difference by accurately controlling stringency (Non-patent Document 1). This is because when the probe hybridizes with a sequence different from the target sequence to form a double-stranded nucleic acid (mismatch), the melting temperature is lower than that of the double-stranded nucleic acid (full match) between the probe and the target sequence. Is determined under a strict temperature control. In many cases, the difference in melting temperature between these single-base mismatched double-stranded nucleic acids and full-matched double-stranded nucleic acids is considered to be about 1 to 3 ° C.

  Japanese Patent No. 2783568 (International Patent Publication No. WO89 / 01049) (Patent Document 1) discloses a method for performing this stringency control more simply, but still requires strict temperature control. Yes.

  Japanese Patent Publication No. 2005-58107 (Patent Document 2) discloses a method for analyzing a melting curve of a hybrid of a nucleic acid probe and a target nucleic acid, and when the nucleic acid probe is set to a single nucleotide polymorphism part, The polymorphic type can be identified by determining the melting temperature. However, since this also requires strict temperature control, a highly accurate thermostat capable of temperature control is required.

  Japanese Published Patent No. 2003-528626 (International Patent Publication No. WO01 / 073118) (Patent Document 3) discloses a method for detecting and discriminating SNPs using a special nucleic acid probe called a beacon. In this method, the melting temperature curve is drawn by changing the reaction temperature between the mismatched nucleic acid molecule and the full-matched nucleic acid molecule to record the fluorescence intensity, and the SNPs are discriminated from the difference in melting temperature. In order to achieve this, a high-accuracy device capable of high-precision temperature control and fluorescence detection is required.

  International Patent Publication No. WO 2004/025257 (Patent Document 4) reports a method for estimating the melting temperature of a nucleic acid probe under a specific salt concentration.

  The melting temperature (Tm) is the temperature at which 50% of the hybridized double-stranded nucleic acid is dissociated into single-stranded nucleic acid, and depends on the salt conditions of the solution, the length of the nucleic acid, the content of the GC sequence, the composition, and the sequence. Dependent. In the nucleic acid sequence, when the GC content (rate) is high, the melting temperature tends to increase. When performing multiple hybridizations under the same conditions, it is better to keep the melting temperature of the nucleic acid probe constant, but it may be difficult to design the probe without losing the specificity of each nucleic acid probe. . In such cases, a melting temperature adjusting agent that destabilizes the duplex of the nucleic acid and serves to adjust the melting temperature is often used. A typical example is betaine (trimethylglycine). In Japanese Patent Publication No. 2002-345499 (Patent Document 5), proline, dimethyl sulfoxide, and trimethylamine N-oxide are used as melting temperature adjusting agents in addition to betaine. Japanese Patent Publication No. 2005-160387 (Patent Document 6) discloses dimethyl sulfoxide (DMSO), formamide or glycerol as a melting temperature adjusting agent in addition to betaine. The optimum concentration of the melting temperature adjusting agent is set under conditions that give appropriate stringency and reactivity in consideration of the reaction conditions. In general, betaine having a concentration of about 0.5 to 5M is used.

  Japanese Patent Application Publication No. 2005-87109 (Patent Document 7) discloses a nucleic acid buffer that can efficiently perform a nucleic acid hybridization reaction by shortening the reaction time. This buffer solution contains a phospholipid, and more preferably a buffer solution to which any of a surfactant, a nucleic acid component, betaine, formamide, and Denhardt's solution is added. However, nucleic acid hybridization requires 3 hours at a reaction temperature of 65 ° C., and there is no description about discrimination of single nucleotide polymorphisms. Moreover, the optimal temperature of hybridization is disclosed as 37-72 degreeC.

  Japanese published patent 2001-78769 (patent document 8) and Bioconjugate Chem., 8, 3-6 (1997) (non-patent document 2) include a cationic polymer polymer for promoting exchange between nucleotide sequences. It is disclosed. This is a polymer obtained by graft polymerization using a cationic polymer such as polylysine or polyarginine as a main chain and dextran or polyethylene glycol as a side chain. Examples thereof include α-PLL-g-Dex, ε-PLL-g-Dex, and PAA-g-Dex. It has been reported that in the presence of this polymer, nucleic acid strand exchange reaction and hybridization reaction are promoted.

Dextran side chain modified α-poly (L-lysine) (hereinafter abbreviated as α-PLL-g-Dex)

Dextran side chain modified ε-poly (L-lysine) (hereinafter abbreviated as ε-PLL-g-Dex)

Dextran side chain modified polyallylamine (hereinafter abbreviated as PAA-g-Dex)

Furthermore, in International Publication No. WO2003 / 018841 (Patent Document 9), a double-stranded nucleic acid containing a mismatch is formed when hybridization is performed between a double-stranded nucleic acid and a single-stranded nucleic acid in the presence of this polymer. A method for quickly and easily discriminating single nucleotide polymorphisms from the difference between the reaction rate and the reaction rate for forming a fully-matched double-stranded nucleic acid not containing a mismatch is disclosed.

A Proc. Natl. Acad. Sci. USA, 80, 278-282 (1983) Bioconjugate Chem., 8, 3-6 (1997) Japanese Patent No. 2783568 (International Patent Publication No. WO89 / 01049) Japanese Patent No. 2005-58107 Japanese Published Patent No. 2003-528626 (International Patent Publication No. WO01 / 073118) International Patent Publication No. WO2004 / 025257 Japanese published patent 2002-345499 Japanese Published Patent 2005-160387 Japanese Published Patent No. 2005-87109 Japanese published patent 2001-78769 International Patent Publication WO2003 / 018841

  An object of the present invention is to provide a reaction solution that reduces the sequence dependency of the nucleic acid to be hybridized and improves the reaction rate when the nucleic acid is hybridized. In addition, the present invention has an object to be solved by applying this to provide a single nucleotide polymorphism high-accuracy discrimination method.

  As a result of intensive studies to solve these problems, the present inventors have reduced the sequence dependency of a nucleic acid probe by coexisting with a melting temperature adjusting agent for nucleic acid and a cationic polymer, and have achieved hybridization reaction. It came to find the solution which improves speed. Furthermore, the present inventors have developed a method for accurately discriminating single base substitution (mismatch) in a buffer solution using this solution. That is, the present invention is as follows.

(1) A solution for use in hybridization of nucleic acid, the solution for nucleic acid hybridization comprising a nucleic acid melting temperature adjusting agent and a cationic polymer in the solution.

(2) The nucleic acid hybridization solution according to (1), wherein the melting temperature adjusting agent includes at least one of DMSO, proline, betaine, glycerol, formamide, trimethylamine N-oxide, and tetraalkylammonium salt.

(3) The nucleic acid high according to (1) or (2), wherein the cationic polymer is a copolymer composed of a polymer chain having a cationic group and a hydrophilic polymer chain. Hybridization solution.

(4) The nucleic acid hybridization solution according to (3), wherein the polymer chain having a cationic group is polylysine or polyallylamine.

(5) The nucleic acid hybridization solution according to (3) or (4), wherein the hydrophilic polymer chain is dextran or polyethylene glycol.

(6) The nucleic acid according to any one of (1) to (5), wherein the cationic polymer contains at least one of α-PLL-g-Dex, ε-PLL-g-Dex, and PAA-g-Dex. Hybridization solution.

(7) A hybridization method comprising hybridizing a sample nucleic acid and a nucleic acid probe in the nucleic acid hybridization solution according to any one of (1) to (6).

(8) A method for detecting a mismatch of a sample nucleic acid in the nucleic acid hybridization solution according to any one of (1) to (6), wherein (a) the sample nucleic acid and the nucleic acid probe are hybridized. Measuring the rate or rate at which a hybrid is formed, (b) measuring the rate or rate at which a reference nucleic acid and a nucleic acid probe are hybridized to form a fully matched hybrid, and (c) the rate or rate of (a) And (b) comparing the rate or rate, and (d) the sequence of the sample nucleic acid comprising determining that there is a mismatch in the sequence of the single-stranded nucleic acid molecule of the sample when the rate or rate of (a) is low How to detect.

(9) A method for detecting a full match of a sample nucleic acid in the nucleic acid hybridization solution according to any one of (1) to (6), wherein (a) the sample nucleic acid is hybridized with a nucleic acid probe. Measuring the rate or rate at which a hybrid is formed, (b) measuring the rate or rate at which a hybrid of a reference nucleic acid and a nucleic acid probe is formed to form a mismatched hybrid, and (c) the rate or rate of (a) And (b) comparing the speed or rate, and (d) detecting the sequence of the sample nucleic acid comprising determining that the sequence of the sample nucleic acid molecule is a full match when the rate or rate of (a) is high how to.

(10) The method according to any one of (7) to (9), wherein the nucleic acid probe is a partial double-stranded nucleic acid molecule.

  The nucleic acid solution of the present invention is a nucleic acid solution containing a nucleic acid melting temperature adjusting agent and a cationic polymer in the solution, and the sequence dependency is reduced at the time of hybridization between the sample nucleic acid and the nucleic acid probe, It is possible to accelerate the reaction rate, and it is effective for detecting the sequence of a sample nucleic acid using a nucleic acid probe method and discriminating a single nucleotide polymorphism with high accuracy.

Hereinafter, although this invention is demonstrated in detail, it is not limited to these.
In the present invention, the nucleic acid may include DNA, RNA, a nucleic acid derivative, a non-natural nucleotide of a modified base, or a non-natural nucleotide bond called PNA (Nature Biotechnology, 14.1700-1704 (1996)). The nucleic acid may be a single-stranded nucleic acid, a double-stranded nucleic acid, or a triple-stranded nucleic acid, unless otherwise specified. Double-stranded nucleic acids are complementary base pairs, such as adenine (A) and thymine (T) and guanine (G) and cytosine (C) in DNA, and A and uracil (U) and G in RNA. This means a higher order structure formed by pairing with C. In addition to DNA single-stranded / DNA single-stranded double-stranded DNA, RNA single-stranded / RNA single-stranded double-stranded RNA, DNA single-stranded / RNA single-stranded hybrid double-stranded nucleic acid may also be mentioned. In addition, the nucleic acid having a double-stranded portion may have a double-stranded structure formed by annealing complementary sequences within the self-molecules of the single-stranded nucleic acid.

  Nucleotide single nucleotide polymorphisms include nucleobase substitutions, deletions, and the like. A double-stranded nucleic acid being a full match means that the sequences of the target regions are complementary to each other, and a mismatch is a base substitution or deletion in one of the single-stranded nucleic acids that make up the duplex. It means a state that is not a full match due to or addition.

  The nucleic acid may be in the form of a solution or may be immobilized on a solid surface such as an ELISA plate or magnetic beads. For example, a partial double-stranded probe is immobilized on a 96-well or 384-well plate, and the target By performing hybridization by adding a nucleic acid, it is possible to construct a high-throughput evaluation system that measures changes in the fluorescence intensity of the partial double-stranded probe.

  As a method for measuring the hybridization process, a method of labeling a nucleic acid with a fluorescent dye such as FITC, TAMRA, DABCYL, Cy3, or Cy5 is often used. A method of measuring a change in fluorescence intensity by the FRET method by combining a plurality of fluorescent substances is also possible. Although the method of measuring the change of molecular weight by SPR method is also mentioned, it is not limited to these.

The solution of the present invention includes 0.01 to 10 × SSC buffer (sodium citrate buffer; 1 × SSC composition: 0.15M NaCl, 15 mM sodium citrate, pH 7.0), 2 to 10 × Denhardt's solution (Denhardt solution; 50 × Denhardt solution composition: 1% Bovine Serum Albumin, 1% Ficol, 1% polyvinil pyrrolidone), PBS buffer (10 mM sodium phosphate, 0.5 mM EDTA, 0.15 M NaCl, pH 7. 2) It is preferable to contain a commonly used nucleic acid buffer such as TE buffer (10 mM Tris-HCl, 1 mM EDTA (pH 8)). Moreover, you may add about 0.1-300 mM salt, for example, NaCl, as needed. Some divalent cations such as magnesium ions have a function of stabilizing the double strand of the nucleic acid. For example, about 1 to 5 mM MgCl ₂ may be added.

Further, a surfactant may be added to this solution. This is particularly effective for reducing non-specific adsorption of a fluorescently labeled or radioisotope-labeled nucleic acid probe to a solid phase such as a reaction vessel, a membrane or a magnetic bead. Surfactants are mainly classified into anionic, cationic, and nonionic, but nonionic and anionic are more preferable, and anionic is more preferable.
As the nonionic surfactant, polyoxyethylene sorbitan fatty acid ester, Triton X-100, polyethylene glycol and the like are often used. As an anionic surfactant, N-lauroyl sarcosine sodium, sodium N-dodecyl sarcosinate, alkylbenzene sulfonate and the like are preferably used. The optimum concentration of the surfactant is about 0.001 to 10% by weight, but is optimized depending on the buffer and hybridization reaction conditions.

  Normal hybridization conditions are performed at about 50 to 70 ° C. near the melting temperature of the nucleic acid probe. In the presence of formamide, hybridization is carried out by reducing the reaction temperature by about 0.5 to 0.6 ° C. per 1% formamide concentration. However, since the solution of the present invention contains a cationic polymer that promotes hybridization such as α-PLL-g-Dex, hybridization at a lower temperature is possible. In addition, if the hybridization reaction is performed near the melting temperature of the nucleic acid probe, the stability of the double-stranded nucleic acid is low, and thus there is a risk that the discrimination efficiency such as hybridization efficiency and mismatch will be reduced. Conversely, the reaction rate tends to decrease at low temperatures. That is, the reaction temperature of hybridization using the solution of the present invention is preferably 5 to 50 ° C, more preferably 10 to 36 ° C, and most preferably 15 to 25 ° C.

  The concentration of betaine in the solution of the present invention is preferably 0.1 to 10M, more preferably 3 to 7M, and most preferably 4.1 to 6M.

  In the case of α-PLL-g-Dex, the concentration of the cationic polymer in the solution of the present invention is preferably 0.01 to 10,000, more preferably 0.01 to 1000, and most preferably 0.1 to 1000. .

The present invention is specifically described by the following examples, but the present invention is not limited to the scope of the examples.
[Example 1] GC content and hybridization rate of double-stranded part of partial double-stranded nucleic acid probe Target nucleic acid and oligodeoxynucleotide (ODN) for nucleic acid probe are chemically synthesized and used after HPLC purification did. The oligodeoxynucleotide was dissolved in PBS buffer (10 mM sodium phosphate, 0.5 mM EDTA, 0.15 M NaCl, pH 7.2). Further, both the target nucleic acid and the partial double-stranded nucleic acid probe were prepared by mixing equal amounts of + and − strand oligonucleotides, heating at 90 ° C. for 1 minute, and then gradually cooling and annealing (FIG. 1). Partial double-stranded nucleic acid probes P-d15, P-d50, and P-d85 have a GC content of 15%, 50%, and 85%, respectively, at the double-stranded sites. Hybridization was performed by adding the target nucleic acid (final concentration: 60 nM) to the partial double-stranded nucleic acid probe (final concentration: 12 nM). Furthermore, the cationic polymer (α-PLL-g-Dex) was synthesized according to Japanese Patent Publication No. 2001-78769 and Bioconjugate Chem., 8, 3-6 (1997), and the synthesized cationic polymer (α-PLL). -G-Dex) and betaine (trimethylglycine) were added for hybridization. The 5 'and 3' ends of oligodeoxynucleotides for nucleic acid probes are labeled with a fluorescent dye such as TAMRA or DABCYL, and the progress of hybridization is followed over time using fluorescence resonance energy transfer (FRET). . The result is shown in FIG.

  In hybridization using only the target nucleic acid and the partial double-stranded nucleic acid probe, the hybridization rate decreased as the GC content of the double-stranded site increased. This was thought to be due to the difference in stability between GC base pairs and AT base pairs. Next, when betaine (trimethylglycine) having an effect of lowering the stability of GC base pairs was added so that the final concentration was 5 M, the dependence on the GC content was suppressed, but the reaction rate was remarkably reduced. By performing hybridization in the presence of α-PLL-g-Dex together with betaine, it is possible to accelerate the reaction and improve the discrimination ability between mismatches and full matches while suppressing the dependency on the GC content.

[Example 2] GC content and hybridization rate of single-stranded site of partial double-stranded nucleic acid probe Target nucleic acid and oligodeoxynucleotide (ODN) for nucleic acid probe were chemically synthesized and used after HPLC purification. . The oligodeoxynucleotide was dissolved in PBS buffer (10 mM sodium phosphate, 0.5 mM EDTA, 0.15 M NaCl, pH 7.2). Further, both the target nucleic acid and the partial double-stranded nucleic acid probe were prepared by mixing equal amounts of + and − strand oligonucleotides, heating at 90 ° C. for 1 minute, and gradually cooling and annealing (FIG. 3). Partial double-stranded nucleic acid probes P-s0, P-s40, and P-s80 have GC contents of single-stranded sites of 0%, 40%, and 80%, respectively. Hybridization was performed by adding the target nucleic acid (final concentration: 60 nM) to the partial double-stranded nucleic acid probe (final concentration: 12 nM). Furthermore, hybridization was performed under the condition of adding a cationic graft copolymer (α-PLL-g-Dex) and betaine (trimethylglycine) disclosed in Japanese Published Patent Application 2001-78769. The 5 'and 3' ends of oligodeoxynucleotides for nucleic acid probes are labeled with a fluorescent dye such as TAMRA or DABCYL, and the progress of hybridization is followed over time using fluorescence resonance energy transfer (FRET). . The result is shown in FIG.

  As the GC content of the single-stranded site of the partial double-stranded nucleic acid probe increases, the stability of hybridization with the target nucleic acid increases and the hybridization rate increases, but the ability to discriminate between mismatches and full matches decreases. Next, betaine (betaine), which has the effect of reducing the stability of GC base pairs, is added so that the final concentration is 5 M. The discrimination ability between mismatches and full matches when the GC content is high is improved. Decreased significantly. On the other hand, α-PLL-g-Dex coexists with betaine to perform hybridization, thereby improving the discrimination ability between mismatches and full matches while suppressing the dependency on the GC content rate and increasing the hybridization rate. Indicates that

  The hybridization solution can improve the speed and accuracy of nucleic acid hybridization, and can be applied to diagnoses such as single nucleotide polymorphisms and genome typing.

FIG. 1 shows the sequences of the target nucleic acid and the partial double-stranded nucleic acid probe. FIG. 2 shows the results of hybridization using partial double-stranded nucleic acid probes having different GC contents at the double-stranded sites in the presence of betaine or α-PLL-g-Dex. Hybridization is detected by an increase in fluorescence due to FRET. In the hybridization between the target nucleic acid and the partial double-stranded nucleic acid probe, ■ indicates a full match and ▲ indicates a mismatch. FIG. 3 shows the sequences of the target nucleic acid and the partial double-stranded nucleic acid probe. FIG. 4 shows the results of hybridization using partial double-stranded nucleic acid probes having different GC contents at the double-stranded sites in the presence of betaine or α-PLL-g-Dex. Hybridization is detected by an increase in fluorescence due to FRET. In the hybridization between the target nucleic acid and the partial double-stranded nucleic acid probe, ■ indicates a full match and ▲ indicates a mismatch.

Claims (10)

A solution for use in hybridization of a nucleic acid, the solution for nucleic acid hybridization comprising a nucleic acid melting temperature adjusting agent and a cationic polymer in the solution. The solution for nucleic acid hybridization according to claim 1, wherein the melting temperature adjusting agent contains at least one of DMSO, proline, betaine, glycerol, formamide, trimethylamine N-oxide, and tetraalkylammonium salt. The solution for nucleic acid hybridization according to claim 1 or 2, wherein the cationic polymer is a copolymer composed of a polymer chain having a cationic group and a hydrophilic polymer chain. 4. The nucleic acid hybridization solution according to claim 3, wherein the polymer chain having a cationic group is polylysine or polyallylamine. The solution for nucleic acid hybridization according to claim 3 or 4, wherein the hydrophilic polymer chain is dextran or polyethylene glycol. The nucleic acid hybridization solution according to any one of claims 1 to 5, wherein the cationic polymer contains at least one of α-PLL-g-Dex, ε-PLL-g-Dex, and PAA-g-Dex. A hybridization method comprising hybridizing a sample nucleic acid and a nucleic acid probe in the nucleic acid hybridization solution according to claim 1. A method for detecting a mismatch of a sample nucleic acid in the nucleic acid hybridization solution according to any one of claims 1 to 6, wherein (a) a sample nucleic acid and a nucleic acid probe are hybridized to form a hybrid. Measuring the rate or rate; (b) measuring the rate or rate at which a reference nucleic acid and a nucleic acid probe are hybridized to form a fully matched hybrid; and (c) the rate or rate of (a) and (b) A method of detecting the sequence of a sample nucleic acid, comprising comparing the rate or rate and (d) determining that there is a mismatch in the sequence of the single-stranded nucleic acid molecule of the sample when the rate or rate of (a) is low. A method for detecting a full match of a sample nucleic acid in the nucleic acid hybridization solution according to any one of claims 1 to 6, wherein (a) a sample nucleic acid and a nucleic acid probe are hybridized to form a hybrid. Measuring the rate or rate; (b) measuring the rate or rate at which the reference nucleic acid and nucleic acid probe are hybridized to form mismatched hybrids; and (c) the rate or rate of (a) and (b) A method of detecting the sequence of a sample nucleic acid, comprising comparing the rate or rate and determining that the sequence of the nucleic acid molecule of the sample is a full match when the rate or rate of (d) (a) is high. The method according to claim 7, wherein the nucleic acid probe is a partial double-stranded nucleic acid molecule.