JP2006020514A - Method for identifying base polymorphism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting base polymorphism clearly and simply, capable of obtaining a result rapidly and easily in a good reproducibility by suppressing a non-specific bonding reaction as low. <P>SOLUTION: This method for detecting the base polymorphism contained in a specimen solution by performing an amplifying reaction by using a specific nucleic acid sequence-amplifying oligonucleotide in a specimen solution containing a specific nucleic acid sequence and at the same time with or after the amplification reaction, bringing the above in contact with a labeled oligonucleotide complementary bonding with the base polymorphic part of the nucleic acid sequence and detecting the labeled oligonucleotide bonded with the specific nucleic acid sequence, or detecting the amplified nucleic acid sequences through the labeled oligonucleotide is characterized by presenting an oligonucleotide having a sequence with at least one position different from the labeled oligonucleotide complementary bonding with the base polymorphic part, and having a higher Tm value than that of the labeled oligonucleotide complementary bonding with the base polymorphic part. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for identifying a base polymorphism using an oligonucleotide that binds complementarily to a polymorphic site. The present invention is particularly useful for diagnosis of genetic diseases, nucleotide polymorphism analysis, and the like.

  In the present invention, the nucleotide polymorphism means having a nucleotide sequence different from the wild type. The nucleotide polymorphism of a gene plays an important role as a cause of inter-individual variation in the occurrence of side effects and treatment failures in drug metabolism, and is also known as a cause of individual differences such as basic metabolism known as constitution. In addition, they also serve as genetic markers for a number of diseases. Elucidation of these mutations is therefore clinically important and routine phenotyping is particularly recommended for psychiatric patients and volunteers in clinical research (Gram and Brsen, European Consensus Conference on Pharmacogenetics. Commission of the European Communities, Luxembourg, 87-96 (1990); Balant et al., Eur. J. Clin. Pharmacol. 36, 551-554 (1989)). In addition, a nucleic acid sequence analysis method for detecting each genotype following identification of the causative mutant gene is desired.

  As a conventional nucleic acid sequence analysis technique, for example, there is a nucleic acid sequencing method (sequencing method). Nucleic acid sequencing can detect and identify nucleotide polymorphisms contained in nucleic acid sequences, but requires a great deal of effort to prepare template nucleic acids, DNA polymerase reaction, polyacrylamide gel electrophoresis, analysis of nucleic acid sequences, etc. And time is needed. Further, labor can be saved by using a recent automatic sequencer, but there is a problem that an expensive apparatus is required.

  On the other hand, there are various known genetic diseases caused by point mutations in genes, and some of them know which part of the gene and how point mutations cause genetic diseases. Not a few.

  As a method for detecting such an expected point mutation, detection of a point mutation of a gene using a gene amplification method such as a PCR (polymerase chain reaction) method (see, for example, Patent Document 1 or 2) has been conventionally used. The method is known. A gene fragment amplified by the PCR method is treated with a restriction enzyme that cleaves a specific nucleic acid sequence, and a method of judging by the size of the resulting fragment (PCR-RFLP) method is used. Similarly, as a detection method using the PCR method, an Alelle specific amplification method using the specificity of the primer used is used. In this method, as one of the pair of oligonucleotides used in the gene amplification method, a wild-type oligonucleotide that is completely complementary to the end region of the wild-type gene amplification region and the mutant-type gene amplification A mutant oligonucleotide that is completely complementary to the end region of the region is used. Mutant oligonucleotides have nucleotides complementary to the predicted point mutation at the 3 'end. Such wild-type and mutant-type oligonucleotides are separately used to subject the sample gene to the gene amplification method.

  If the sample gene is wild-type, nucleic acid amplification occurs when a wild-type oligonucleotide is used, but if a mutant-type oligonucleotide is used, the 3 ′ end of the oligonucleotide corresponds to the sample gene. Since it is not complementary to the nucleotide (mismatch), no extension reaction occurs, and no nucleic acid amplification occurs. On the other hand, if the sample gene is a mutant type, conversely, amplification does not occur when the wild-type oligonucleotide is used, and amplification occurs when the mutant-type oligonucleotide is used. Therefore, by examining whether amplification occurs when using each oligonucleotide, it is possible to determine whether the sample gene is a wild type or a mutant type, thereby identifying a point mutation in the sample gene. Can do. As a method to check whether amplification has occurred at this time, the amplified product is subjected to agarose gel electrophoresis, stained with a nucleic acid-specific binding fluorescent reagent such as ethidium bromide, and then irradiated with UV to detect the presence of amplified nucleic acid. it can. Other methods include Southern blotting, in which amplified nucleic acid is immobilized on a nylon membrane and detected using a labeled probe, sandwich hybridization in which a detection probe is used after capturing with a supplementary probe immobilized on an individual carrier. Laws have been developed.

  In addition, a nucleic acid amplification method is performed so as to include a gene polymorphic site, and after the reaction, a complex of a gene fragment containing the polymorphic site and a labeled oligonucleotide that binds complementarily to the polymorphic site is formed, and the template contained in the sample When the sequence is a wild type sequence, a label that binds complementarily to a complex with a labeled oligonucleotide that binds complementarily to the wild type sequence, and when the template sequence contained in the sample is a mutant type sequence, In the method of forming a complex with an oligonucleotide, in order to reduce non-specific binding reaction, a labeled oligonucleotide having a sequence complementary to one polymorphic site is complemented simultaneously with the other polymorphic site. A method for identifying a base polymorphism in which an unlabeled oligonucleotide having a typical sequence coexists has been reported (for example, see Non-Patent Document 1).

  However, in this method, an unlabeled oligonucleotide having a sequence complementary to one polymorphic site added at the same time is several times to several tens of the labeled oligonucleotide having a sequence complementary to the other polymorphic site. It was necessary to add twice as much amount, and adjustment of reaction conditions and problems remained economically.

Japanese Examined Patent Publication No. 4-67960 Japanese Patent Publication No. 4-67957 Human Mutation Vol.22, 166, 2003

  Although it seems that the amplified nucleic acid can be detected by the method as described above and the polymorphism can be easily identified, in practice, the operation is complicated, and in order to analyze a large amount of samples quickly, Requires labor or needs to build a large lab automation system.

  For example, according to the electrophoresis method, it is necessary to separately detect the wild type and the polymorphic type, and it is difficult to accurately quantify the amount of nucleic acid from the electrophoretic image. Further, Southern blotting and sandwich hybridization require a hybridization reaction with a probe, and the conditions must be strictly adjusted. Furthermore, a process for removing excess probes is required, and the operation is very complicated.

  An object of the present invention is to provide a method and a reagent therefor that can solve the above-described problems and detect a polymorphism in a nucleic acid sequence clearly and reproducibly.

  In view of the above circumstances, the present inventors, as a result of diligent research, used the oligonucleotide for amplifying a specific nucleic acid sequence in a sample solution containing a specific nucleic acid sequence as compared with the conventional method described above, and Alternatively, after the amplification reaction, a labeled oligonucleotide that binds complementarily to the nucleotide polymorphism site of the nucleic acid sequence (hereinafter sometimes referred to as a detection oligonucleotide) is contacted, and the labeled oligonucleotide is bound to a specific nucleic acid sequence. In the method for identifying a nucleotide polymorphism contained in a sample solution by detecting a nucleotide sequence, it has a sequence that differs from a labeled oligonucleotide that binds complementarily to the nucleotide polymorphism site at least one location, and the Tm value is the nucleotide polymorphism site. Coexist with a higher oligonucleotide (hereinafter sometimes referred to as a competing oligonucleotide) than a labeled oligonucleotide that binds complementarily. Found methods for identifying nucleotide polymorphism, wherein Rukoto, and completed the present invention.

That is, the present invention has the following configuration.
[1] Using the oligonucleotide for amplifying a specific nucleic acid sequence in a sample solution containing a specific nucleic acid sequence, a label that binds complementarily to the nucleotide polymorphism site of the nucleic acid sequence simultaneously with and / or after the amplification reaction is used. The nucleotide polymorphisms contained in the sample solution by detecting the labeled oligonucleotide bound to the specific nucleic acid sequence or detecting the amplified nucleic acid sequence via the labeled oligonucleotide. In this method, an oligonucleotide having a sequence different from that of a labeled oligonucleotide complementary to the nucleotide polymorphism site and having a Tm value higher than that of the labeled oligonucleotide complementary to the nucleotide polymorphism site is allowed to coexist. A nucleotide polymorphism identification method characterized by the above.

[2] A method for identifying a nucleotide polymorphism, wherein the oligonucleotide for amplifying a specific nucleic acid sequence is labeled in advance.

[3] A method for identifying a nucleotide polymorphism, wherein the label is any one selected from the group consisting of biotin, digoxigenin, antigen, antibody, fluorescent substance, luminophore, and enzyme.

[4] A method for identifying a nucleotide polymorphism, wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCA, TMA, LAMP and ICAN.

[5] having at least one sequence different from at least one oligonucleotide for nucleic acid sequence amplification, polymerase, dNTPs, labeled oligonucleotide that binds complementarily to the polymorphic site, and labeled oligonucleotide that binds complementarily to the polymorphic site; A kit for measuring nucleotide polymorphism, which has an oligonucleotide whose Tm value is higher than a labeled oligonucleotide that binds complementarily to the polymorphic site.

  The present invention provides a method capable of clearly and easily detecting a base polymorphism in a sample nucleic acid. In the method of the present invention, the non-specific binding reaction is suppressed as compared with the conventional methods, and the results can be obtained quickly and easily with good reproducibility.

Hereinafter, the present invention will be described in detail.
The chromosome or fragment thereof containing a specific nucleotide polymorphism site contained in the sample is not particularly limited as long as it is a target nucleic acid containing a nucleotide polymorphism site bearing information of the target gene. Examples of the target nucleic acid include Alu sequences, exons, introns and promoters of genes encoding proteins. More specifically, genes related to various diseases including genetic diseases, drug metabolism, lifestyle-related diseases (hypertension, diabetes, etc.) can be mentioned. For example, an ACE (Angiotensin IConverting Enzyme) gene is mentioned as high blood pressure.

  In the present invention, the nucleic acid sequence may be simply referred to as a nucleic acid. Mutant nucleic acids are those in which at least one of wild-type nucleic acids, preferably one nucleotide is point-mutated and replaced with other nucleotides, or inserted or deleted sequences in a part of wild-type nucleic acid, etc. It has been elucidated which part of the nucleotide is mutated. It has been elucidated that the constitution is different depending on such base polymorphism, and the method of the present invention is a method for examining whether or not a nucleic acid in a sample has such an expected mutation. is there.

  In the present invention, the specific nucleic acid sequence amplification oligonucleotide is an oligonucleotide having a sequence complementary to a chromosome containing a nucleotide polymorphism site of interest or a fragment thereof, and is a known amplification method such as PCR, NASBA, There is no particular limitation as long as it can be used for the LCR, SDA, RCA, TMA, LAMP and ICAN methods.

  As the labeled oligonucleotide that binds complementarily to the polymorphic site used in the present invention, those labeled with an oligonucleotide complementary to each polymorphic site can be preferably used. The binding site is not particularly limited.

  The length of the oligonucleotide in the present invention is 7 to 25 bases, preferably 11 to 20 bases.

  In the present invention, the specific nucleic acid sequence amplification oligonucleotide and the labeled oligonucleotide that binds complementarily to the polymorphic site and the labeled oligonucleotide that binds complementary to the polymorphic site have a sequence that differs by at least one place. , Oligonucleotides with higher Tm values than labeled oligonucleotides that bind complementary to the polymorphic site can be allowed to act separately or simultaneously.

  In the present invention, the method for extending the oligonucleotide for amplifying a specific nucleic acid sequence can be basically performed using a conventional method. Usually, a target nucleic acid is used as a template by allowing four types of deoxynucleoside triphosphates (dNTP) and DNA polymerase to act on a chromosome or fragment thereof containing a specific nucleotide polymorphism site denatured into a single strand. The oligonucleotide is extended.

  The extension reaction can be performed according to the method described in Molecular Cloning, A Laboratory Manual (Sambrook et al., 1989). Further, in the method for detecting a base polymorphism depending on whether or not the oligonucleotide is extended, if the target nucleic acid does not contain a sufficient amount for detection, a nucleic acid fragment containing the polymorphic sequence in advance is It can also be amplified by the amplification reaction shown.

  In the present invention, a method for amplifying a chromosome or fragment containing a specific nucleotide polymorphism site can also be basically performed using a conventional method, and usually a specific nucleotide polymorphism denatured into a single strand. By making 4 types of deoxynucleoside triphosphates (dNTPs), DNA polymerase, and oligonucleotides for amplifying specific nucleic acid sequences act on a chromosome containing the site or a fragment thereof, the sequence between the oligonucleotides using the target nucleic acid as a template can be changed. Amplified.

  Examples of nucleic acid amplification methods include PCR, NASBA (Nucleic acid sequence-based amplification method; Nature 350, 91 (1991)), LCR (International Publication No. 89/12696, JP-A-2-2934), SDA (Strand Displacement Amplification: Nucleic acid research, Volume 16, 1691 (1992)), RCA (International Publication No. 90/1069), TMA (Transcription mediated amplification method; J. Clin. Microbiol. Volume 31, 3270 (1993)), LAMP (loop-mediated isothermal amplification method: J Clin Microbiol. 42: 1,956 (2004)), ICAN (isothermal and chimeric primer-initiated amplification of nucleic acids: Kekkaku. 78, 533 (2003)).

In particular, the PCR method repeats a cycle consisting of three steps of denaturation, annealing, and extension in the presence of a sample nucleic acid, four types of deoxynucleoside triphosphates, a pair of oligonucleotides, and a heat-resistant DNA polymerase, and thereby the above paired pair. In this method, the region of the sample nucleic acid sandwiched between the oligonucleotides is exponentially amplified. That is, the sample nucleic acid is denatured in the denaturation step, each oligonucleotide is hybridized with a region on the complementary single-stranded sample nucleic acid in the subsequent annealing step, and each oligonucleotide is the starting point in the subsequent extension step. As described above, a DNA strand complementary to each single-stranded sample nucleic acid serving as a template is extended by the action of DNA polymerase to form double-stranded DNA. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of oligonucleotides is theoretically amplified ²ⁿ times. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis. Therefore, if a gene amplification method is used, it is possible to detect even a very small amount of sample nucleic acid that could not be detected in the past (one molecule is acceptable), which is a very widely used technique recently. .

  In the method using the nucleic acid amplification method as described above, a complex of a gene fragment containing a polymorphic site and a labeled oligonucleotide that binds complementarily to the polymorphic site is formed after the reaction, and the template sequence contained in the sample Is a wild type sequence, a labeled oligonucleotide that binds complementarily to the wild type sequence, and a labeled oligo that binds complementarily to the mutant sequence when the template sequence contained in the sample is a mutant sequence. Forms a complex with the nucleotide. However, the labeled oligonucleotide that binds complementarily to the wild type sequence partially binds to the mutant type sequence, and as a result, it is determined as a heterozygous of the wild type sequence and the mutant type sequence, which is known as a so-called non-specific binding reaction. . In order to solve this problem, a nucleotide sequence in which a labeled oligonucleotide having a sequence complementary to one polymorphic site and an unlabeled oligonucleotide having a sequence complementary to the other polymorphic site coexist with each other. A type identification method has been reported (Human Mutation Vol. 22, 166, 2003).

However, in this method, an unlabeled oligonucleotide having a sequence complementary to one polymorphic site added at the same time is several times to several tens of the labeled oligonucleotide having a sequence complementary to the other polymorphic site. It was necessary to add twice as much amount, and adjustment of reaction conditions and problems remained economically.
In the method of the present invention, a labeled oligonucleotide having a sequence complementary to one polymorphic site and an unlabeled oligonucleotide having a sequence complementary to the other polymorphic site and complementary to the polymorphic site By adding an oligonucleotide having a Tm value higher than that of a labeled oligonucleotide having a sequence, the amount of unlabeled oligonucleotide having a sequence complementary to the other polymorphic site can be reduced. It was confirmed that the specific binding reaction was dramatically improved.

  The difference in Tm value is preferably in the range of 2 to 40 ° C, more preferably 3 to 30 ° C, still more preferably 4 to 25 ° C, and particularly preferably 5 to 20 ° C. If the difference in Tm is less than 2 ° C., the characteristics of the present application may not be obtained. If it exceeds 40 ° C., competing oligonucleotides may not be separated during the PCR reaction, and the amplification reaction may not proceed.

  Tm can be adjusted by a method such as changing the length of the oligonucleotide or shifting the position.

  The Tm value is a temperature at which 50% of the double-stranded DNA molecules that have formed a hybrid are separated from each other. The calculation method is not particularly limited as long as it is a known method. What is calculated | required by either of the GC% method needs to satisfy | fill the characteristic of this invention. By comparing the Tm values calculated by any one of these methods, the probe sequence for detection and the probe sequence to compete with the probe are determined. Note that a particularly preferable Tm value is calculated by the nearest neighbor method.

  The amount of the competitive oligonucleotide added is preferably 0.2 to 10-fold mol, more preferably 0.3 to 5-fold mol, still more preferably 0.4 to 3-fold mol, and particularly preferably 0.5 to mol of the detection oligonucleotide. It is about 2 times mole. The lower limit also depends on the amount of amplification product, and can be increased up to 1-fold mol when a non-specific binding reaction is observed.

  The label used in the present invention is not limited to its use as long as it does not interfere with the detection of the nucleic acid sequence, but is preferably a magnetic substance, electron carrier, antigen, antibody, fluorescent substance, luminophore. And from the group consisting of the enzymes avidin, biotin, digokesigenin.

  As a means for detecting the formed complex, a means for capturing the amplified gene fragment, and a known label capable of detecting a label bound to an oligonucleotide having a sequence complementary to the polymorphic site are known. The method is not particularly limited, but the amplified gene fragment is immobilized on a solid phase by immobilizing an oligonucleotide capable of capturing the amplified fragment on a polystyrene solid support, nylon membrane, or glass fiber filter. And a method of detecting a labeled body. As another method, amplification is performed using a specific nucleic acid sequence amplification oligonucleotide pre-labeled with biotin, etc., the amplified gene fragment is immobilized on a solid phase on which avidin is immobilized, Examples of detection methods

Kit In the present invention, the kit includes at least a specific nucleic acid sequence amplification oligonucleotide, a labeled oligonucleotide having a sequence complementary to the polymorphic site, and an unlabeled oligonucleotide having a sequence complementary to the other polymorphic site. It includes oligonucleotides, polymerases, and dNTPs having a higher Tm value than labeled oligonucleotides that are present and have a sequence complementary to the polymorphic site.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1
Detection of nucleotide polymorphism (677C → T) of Methylenetetrahydrofolatereductase (MTHFR) gene (1) Synthesis of oligonucleotide to detect 677th polymorphism of Methylenetetrahydrofolatereductase gene Using DNA synthesizer type 392 manufactured by PerkinElmer, phosphoramidite method Thus, oligonucleotides having the base sequences shown in SEQ ID NOs: 1 to 6 (hereinafter referred to as oligos 1 to 6) were synthesized. The synthesis was carried out according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, it was commissioned to a DNA synthesis consignment company (Japan Bioservice Co., Ltd., Sawaddy Co., Ltd., GENSET KK, Amersham Pharmacia Biotech Co., Ltd., etc.).
Oligo 1 is the sense strand and is used as an oligonucleotide for amplification reaction in combination with oligo 2. Oligo 3 is a wild-type detection oligonucleotide, and oligo 5 is a mutant-type detection oligonucleotide. Oligo 4 is an oligonucleotide complementary to a mutant nucleic acid sequence used for wild-type detection, and is used in combination with oligo 3. Oligo 6 is an oligonucleotide complementary to the wild-type nucleic acid sequence used for mutation detection. Oligo 2 used in combination with oligo 5 is labeled with biotin at the 5 ′ end, and oligo 3 and 5 are labeled with FITC at the 3 ′ end. Labeled by. Oligo 4 and 6 are modified at the 3 'end with a phosphate group.

(2) Analysis of Methylenetetrahydrofolatereductase gene polymorphism by PCR method Amplification reaction by PCR method Using a DNA solution extracted from human leukocytes by phenol-chloroform method as a sample, the following reagents were added, and human Methylenetetrahydrofolatereductase (MTHFR) under the following conditions: ) The nucleotide polymorphism (677C → T) of the gene was analyzed.

The 25μl solution containing the following reagents reagents were prepared.
Reagent for detecting wild-type sequence Taq DNA polymerase reaction solution Oligo 1 and 2 (oligo 2 is labeled with biotin at the 5 ′ end) 5 pmol,
Oligo 3 (3 ′ end labeled with FITC) and oligo 4 each 5 pmol,
X10 buffer 2.5 μl,
2.5 μl of 2 mM dNTP,
1.5 μl of 25 mM MgCl ₂ ,
Taq DNA polymerase 1.3U,
Extracted DNA solution 100ng

Mutant Sequence Detection Reagent Taq DNA Polymerase Reaction Solution Oligo 1 and 2 (In Oligo 2, the 5 ′ end is labeled with biotin) 5 pmol,
Oligo 5 (3 ′ end labeled with FITC) and oligo 6 each 5 pmol,
X10 buffer 2.5 μl,
2.5 μl of 2 mM dNTP,
1.5 μl of 25 mM MgCl ₂ ,
Taq DNA polymerase 1.3U
Extracted DNA solution 100ng

Amplification conditions 95 ° C, 5 minutes, 95 ° C, 30 seconds,
67 ° C for 30 seconds,
72 ° C, 30 seconds (40 cycles)
72 ℃, 2 minutes,
95 ℃, 3 minutes,
15 ° C for 15 minutes.

(3) Detection Using Porous Filter 20 μl of the amplification reaction solution was added to 30 μl of a POD-labeled anti-FITC antibody (manufactured by DAKO Cytomation) and reacted at room temperature for 5 minutes. As a result, the FITC-labeled oligonucleotide bound to the amplified MTHFR gene fragment is bound to the POD-labeled anti-FITC antibody. When this reaction solution is added to a porous glass filter to which avidin is bound, the amplified MTHFR gene fragment is captured on the filter. Next, after sequentially adding a cleaning solution and a POD substrate solution from the top, color development on the filter surface was visually confirmed. In addition, the glass fiber nonwoven fabric filter which the avidin couple | bonded was performed according to Example 1 of Unexamined-Japanese-Patent No. 2004-156985.

Example 2
Detection of nucleotide polymorphism (681G → A) of Cytochrome P450 2C19 (CYP2C19) gene (1) Synthesis of oligonucleotide for detecting the 681st polymorphism of CYP2C19 gene Using DNA synthesizer type 392 made by PerkinElmer, phosphoramidite By the method, oligonucleotides having the base sequences shown in SEQ ID NOs: 7 to 12 (hereinafter referred to as oligos 7 to 12) were synthesized. The synthesis was carried out according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, it was commissioned to a DNA synthesis consignment company (Japan Bioservice Co., Ltd., Sawaddy Co., Ltd., Proligo, Sigma Genosys, etc.)
Oligo 7 is the sense strand and is used as an oligonucleotide for amplification reaction in combination with oligo 8. Oligo 9 is a wild-type detection oligonucleotide, and oligo 11 is a mutation-type detection oligonucleotide. Oligo 10 is an oligonucleotide complementary to a mutant nucleic acid sequence used for wild-type detection, and is used in combination with oligo 9. Oligo 12 is an oligonucleotide complementary to a wild-type nucleic acid sequence used for mutation detection, and is used in combination with oligo 11. Oligo 8 is labeled with biotin at the 5 ′ end, and oligos 9 and 11 are labeled with FITC at the 3 ′ end. In addition, oligos 10 and 12 are modified at the 3 ′ end with a phosphate group.

(2) Analysis of CYP2C19 gene polymorphism by PCR method Amplification reaction by PCR method Using a DNA solution extracted from human leukocytes by the phenol-chloroform method as a sample, the following reagents were added, and the human CYP2C19 gene was The nucleotide polymorphism (681G → A) was analyzed.

The 25μl solution containing the following reagents reagents were prepared.
Reagent for detecting wild type sequence Taq DNA polymerase reaction solution Oligo 7 and 8 (oligo 8 is labeled with biotin at 5 ′ end) 5 pmol,
Oligo 9 (3 ′ end labeled with FITC) and Oligo 10 each 5 pmol,
X10 buffer 2.5 μl,
2.5 μl of 2 mM dNTP,
1.5 μl of 25 mM MgCl ₂ ,
Taq DNA polymerase 1.3U,
Extracted DNA solution 100ng

Mutant Sequence Detection Reagent Taq DNA Polymerase Reaction Solution Oligo 7 and 8 (Oligo 8 has 5 ′ end labeled with biotin) 5 pmol,
Oligo 11 (3 ′ end labeled with FITC) Oligo 12 5 pmol each
X10 buffer 2.5 μl,
2.5 μl of 2 mM dNTP,
₂ μl of 25 mM MgCl ₂
Taq DNA polymerase 1.3U,
Extracted DNA solution 100ng

Amplification conditions 95 ° C, 5 minutes, 95 ° C, 30 seconds,
67 ° C for 30 seconds,
72 ° C, 30 seconds (40 cycles)
72 ℃, 2 minutes,
95 ℃, 3 minutes,
15 ° C for 15 minutes.

(3) Detection Using Porous Filter 20 μl of the amplification reaction solution was added to 30 μl of a POD-labeled anti-FITC antibody (manufactured by DAKO Cytomation) and reacted at room temperature for 5 minutes. As a result, the FITC-labeled oligonucleotide bound to the amplified CYP2C19 gene fragment and the POD-labeled anti-FITC antibody bind to each other. When this reaction solution is added to a porous filter to which avidin is bound, the amplified CYP2C19 gene fragment is captured on the filter. Next, after sequentially adding a cleaning solution and a POD substrate solution from the top, color development on the filter surface was visually confirmed.

  As described above, the oligonucleotide for amplifying a specific nucleic acid sequence is used in a sample solution containing a specific nucleic acid sequence, and simultaneously and / or after the amplification reaction, it binds complementarily to the nucleotide polymorphism site of the nucleic acid sequence. A label that complementarily binds to a nucleotide polymorphism site in a method of identifying a nucleotide polymorphism contained in a sample solution by contacting a labeled oligonucleotide and detecting the labeled oligonucleotide bound to a specific nucleic acid sequence The genotype can be clearly and easily determined easily and rapidly by coexisting with an oligonucleotide having a sequence different from that of the oligonucleotide and having a Tm value higher than that of the labeled oligonucleotide that binds complementarily to the nucleotide polymorphism site. I was able to.

In addition, Tm of the oligonucleotide of an Example is as follows.
(1) (2) (3)
Oligo 3 tgcgggagccgattt 62.9 ℃ 48 ℃ 56.2 ℃
Oligo 4 gtctgcgggagtcgatttc 64.4 ℃ 60 ℃ 62.3 ℃
Oligo 5 tgcgggagtcgattt 56.2 ℃ 46 ℃ 53.4 ℃
Oligo 6 gtctgcgggagccgatttc 69.7 ℃ 62 ℃ 64.5 ℃
Oligo 9 tcccgggaac 35.7 ℃ 34 ℃ 43.6 ℃
Oligo 10 atttcccaggaaccc 59.9 ℃ 46 ℃ 53.4 ℃
Oligo 11 ttcccaggaac 30.8 ℃ 34 ℃ 41.8 ℃
Oligo 12 atttcccgggaaccc 59.9 ℃ 48 ℃ 56.2 ℃

1) Nearest neighbor method
Tm = 1000ΔH / (− 10.8 + ΔS + Rln (Ct / 4))
−273.15 + 16.6 log [Na +]
ΔH: Total proximity enthalpy change in hybrid −10.8: ΔS (initiation)
ΔS: total proximity entropy change in hybrid R: gas constant Ct: total molar concentration of oligo Na +: sodium concentration 2) Wallace method Tm = 2 × (number of bases of A, T) + 4 × (number of bases of G, C) )
3) GC% method Tm = 81.5 + 16.6log [Na +] + 41 (XG + XC) -500
/L-0.62F
Na + ... Na + molarity [mol / l]
XG, XC: molar fraction of G and C L: length of the oligonucleotide forming the double strand F: molar concentration of formamide

  As described above, the present invention provides a method capable of clearly and easily detecting a base polymorphism in a sample nucleic acid. In the method of the present invention, the non-specific binding reaction was suppressed to a low level as compared with the conventional methods, and a result with quick and easy reproducibility was obtained.

Claims (5)

  Using the oligonucleotide for amplifying a specific nucleic acid sequence in a sample solution containing a specific nucleic acid sequence, a labeled oligonucleotide that binds complementarily to the nucleotide polymorphic site of the nucleic acid sequence simultaneously with and / or after the amplification reaction In a method for identifying a nucleotide polymorphism contained in a sample solution by detecting a labeled oligonucleotide bound to a specific nucleic acid sequence or detecting a nucleic acid sequence amplified via the labeled oligonucleotide. , Characterized in that an oligonucleotide having a sequence different from that of a labeled oligonucleotide that binds complementary to a nucleotide polymorphism site and having a Tm value higher than that of a labeled oligonucleotide that binds complementary to a nucleotide polymorphism site coexists And a method for identifying a nucleotide polymorphism.   The method according to claim 1, wherein the oligonucleotide for amplifying the specific nucleic acid sequence is labeled in advance.   The method according to claim 1 or 2, wherein the label is any one selected from the group consisting of biotin, digoxigenin, antigen, antibody, fluorescent substance, luminophore, magnetic substance, electron carrier and enzyme. 2. The method according to claim 1, wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCA, TMA, LAMP and ICAN. At least one nucleotide sequence amplification oligonucleotide, polymerase, dNTPs, a labeled oligonucleotide that binds complementarily to the polymorphic site, a labeled oligonucleotide that binds complementarily to the polymorphic site, and has a Tm value of A nucleotide polymorphism measuring kit characterized by having a higher oligonucleotide than a labeled oligonucleotide that binds complementarily to the polymorphic site