JP5504676B2 - Genotype identification method - Google Patents

Description

  The present invention relates to a method for identifying genotypes such as gene polymorphisms and somatic mutations, and a kit used in the method. More specifically, the present invention relates to a method for determining the nucleotide sequence of a nucleic acid using competitive hybridization utilizing strand recombination reaction. The present invention relates to a method for improving the identification accuracy of a method for identifying a difference and a kit used in the method.

  Information on the human genome continues to increase due to the decoding of the human genome, especially the international hapmap project that creates SNP (Single Nucleotide Polymorphism) maps. Furthermore, the relationship between the obtained genome information and the individual's constitution is found, the difference in the individual constitution is grasped at the genetic level, and the diagnosis, treatment, prevention and drug administration according to the individual characteristics are enabled. Research aimed at the realization of "medical care according to individual genetic information" (custom-made medicine) is being developed on a large scale all over the world. The difference in gene here means a difference in the base sequence of the genome of an individual, and the main difference is a single base difference (SNP). Recently, it has been found that the difference in the number of times a short base sequence is repeated (copy number) (Copy Number variation: CNV) has spread throughout the genome, and the relationship between this CNV difference and disease has also been pointed out. ing.

  Here, in order to grasp the difference at the genetic level of an individual, it becomes necessary to examine the genotype of each individual. For example, it is assumed that a certain SNP has three types of genotypes AA, AG, and GG. A represents adenine and G represents a guanine base. This SNP is an example in which the position of the genome may be adenine or guanine. Therefore, the test for identifying the genotype of the SNP will determine which of these three genotypes. That is, it may be determined whether A is 0 or 100, G is 0 or 100, or A and G are 50 and 50. As described above, detection of germ cell mutations such as SNP can be said to be almost qualitative detection, and various methods that are relatively easy and simple have been put to practical use.

  On the other hand, in cancer cells, mutations occur at the level of somatic cells, and the mutations are considered to trigger cancer and lead to abnormal growth. Accordingly, a specific gene mutation may be observed in a specific type of cancer cell, and it is also possible to detect a cancer cell using the mutation as an index. However, cancer cells are rich in diversity, and it is not always easy to identify cancer cells with one type of mutation.

  In recent pharmacotherapy, drugs targeting specific molecules (proteins, etc.) in the living body have been developed, and those with low side effects and high effects have been found. These are called molecularly targeted drugs and are actively being developed mainly in the field of cancer treatment. Very recently, it has become clear that these molecularly targeted drugs cannot exert the effect of the drug when a mutation occurs in a protein downstream of signal transduction of the targeted molecule. In this case, it has become possible to predict the effect of the drug by examining the mutation of the gene encoding the protein in which the mutation has occurred, and a new area of customized medicine different from SNP detection has been opened. It's getting on.

  Most of the mutations characteristic of cancer cells or the resistance to molecular target drugs described here are somatic mutations. In the case of germline mutations described above, a common mutation is seen in all cells, whereas in somatic mutations, mutations are found only in the cells that have mutated, and cells that are not mutated (usually normal cells) ) Shows no mutation. Therefore, normally, in the specimen (sample to be tested), mutated cells and normal cells are mixed, and depending on the abundance ratio of these cells, mutated and normal genes exist. Will do. In other words, if most of the sample is normal cells and some mutant cells are included, it is necessary to detect a few mutant genes present in many normal genes, and this is the detection of mutations in germ cells. The difference is that it makes it more difficult to detect genetic mutations in somatic cells.

  There are two main methods for detecting gene mutations in somatic cells. One is a method of distinguishing a normal gene and a mutant gene at the stage of gene amplification, and specifically, a method of specifically amplifying only the mutant gene.

For example, the most sensitive method is a method called “mutant-enriched PCR” in which only a normal gene is cleaved with a restriction enzyme and only a mutant gene that has not been cleaved is amplified. (For example, refer nonpatent literature 1.). In this way, by repeating the reaction to amplify the mutant gene is to be able to detect the mutant gene of one molecule of normal gene 10 ⁶ molecule (e.g., Non-Patent Document 2 see.). Although this method is excellent in terms of such high sensitivity, the operation is very complicated and is not a method applicable to general diagnosis.
In addition, in a primer extension reaction such as PCR, a method of amplifying by distinguishing a single base difference has been developed. This method is a method called “ARMS (amplification restitution structure system)” (for example, see Non-patent Document 3), “ASPCR (allele specific PCR)” (for example, see Non-Patent Document 4), or the like. is there. This method is relatively sensitive, requires no operations other than the general PCR amplification reaction, can perform all of the reaction in a closed system, is very simple, and carries over PCR. It is an excellent method without contamination. However, it can be said that when a normal gene is amplified by mistake in single nucleotide identification, the normal gene is also amplified in the subsequent amplification reaction in the same manner as the amplification of the mutant gene, so that the risk of false positive is high. When this method is used, it is necessary to strictly control the reaction conditions, that is, the reaction temperature, the salt concentration, etc., and the template amount must be exactly the same (for example, refer to Non-Patent Document 5). It is unsuitable for clinical examinations that examine a large number of specimens and diagnoses that require high accuracy.

Another method for detecting somatic cell gene mutation is a method in which a mutated gene and a normal gene are amplified simultaneously, and then the mutated gene and the normal gene are distinguished and detected. As a method for distinguishing and detecting the amplified mutant gene and the normal gene, there are a method utilizing electrophoresis, various methods utilizing hybridization, and the like (for example, see Non-Patent Document 5). However, in most methods, it is difficult to accurately detect a small amount of mutant genes contained in a large amount of normal genes. For example, a dideoxy sequencing method is known as a gold standard for detecting mutant genes. The dideoxy sequencing method can detect mutant genes with relatively high sensitivity, but when mutant genes and normal genes coexist, the detection sensitivity of mutant genes is about 10%, which is very sensitive. It cannot be detected. In addition, it has been reported that the pyro sequencing method can increase the detection sensitivity to about 5% and is superior to the dideoxy sequencing method (see, for example, Non-Patent Document 6).
In addition, a method has been developed in which a sequence containing a mutation is amplified by PCR, a melting curve of the double-stranded DNA of the product is obtained, and the ratio of the mutant gene is obtained from the difference between the melting curves of the mutant gene and the normal gene. Even with this method, it is said that mutant genes contained in normal genes can be detected up to about 5% (see, for example, Non-Patent Document 7).

  In addition, a PCR-PHFA method using a recombination reaction (strand displacement reaction) between two strands having the same base sequence has been developed. The PCR-PHF method can distinguish each strand if the base sequence is exactly the same between a sample (double-stranded nucleic acid) whose genotype is to be identified and a standard double-stranded nucleic acid with a known sequence. The strand recombination (strand substitution) occurs, but even if there is a difference in even one base, the strands having completely complementary base sequences preferentially form two strands, so the sample and standard This is a mutation detection method utilizing the fact that no recombination occurs between double-stranded nucleic acids. It has been reported that by using this PCR-PHFA method, a mutated gene can be detected from an actual specimen with a high sensitivity of about 1% (see, for example, Non-Patent Document 8). As described above, the PCR-PHFA method is a method having high detection sensitivity and excellent reproducibility. However, the operation is somewhat complicated, and carry-over contamination is also a problem. In order to solve these problems, several improved methods have been proposed.

  For example, Patent Document 1 discloses a method using fluorescence resonance energy transfer as an improved method of the PCR-PHFA method. In the PCR-PHFA method for accurately measuring a minute amount of a mutant gene with high sensitivity, it is necessary to detect strand recombination between two double-stranded nucleic acids having the same sequence. In many cases, the nucleic acid is unlabeled, and a standard nucleic acid with a known sequence for causing strand recombination is labeled. In the method described in Patent Document 1, a fluorescent substance is bound to the vicinity of the 5 'end of one strand of a standard nucleic acid and labeled, and the vicinity of the 3' end of the other strand is labeled with another fluorescent substance. When the recombination reaction does not occur and the standard nucleic acid is the original double strand, fluorescence resonance energy transfer between two different fluorescent substances is observed. On the other hand, when a strand recombination reaction occurs between the sample double-stranded nucleic acid, fluorescence resonance energy transfer is not observed. Therefore, the degree of strand recombination can be measured by measuring the degree of this fluorescence resonance energy transfer.

Specifically, for example, when a case where a certain position (mutation site to be identified) of a certain nucleic acid sequence is adenine is distinguished from a case where it is guanine, the base including the position is adenine ( Prepare a standard double-stranded nucleic acid which is thymine in the complementary strand. Further, the standard double-stranded nucleic acid is labeled with a fluorescent substance X on one strand and a fluorescent substance Y capable of energy transfer with the fluorescent substance X on the other strand. That is, in the standard double-stranded nucleic acid, since two fluorescent substances are close to each other, the fluorescence resonance energy transfer occurs as it is.
On the other hand, a nucleic acid derived from a sample is prepared by amplifying the nucleic acid by a nucleic acid amplification reaction so as to have exactly the same length as a standard double-stranded nucleic acid. The obtained sample-derived double-stranded nucleic acid and standard double-stranded nucleic acid are mixed, heat is applied to denature the double-stranded, and then the temperature is gradually lowered to form a double-stranded again. At this time, when all the mutation sites of the sample-derived double-stranded nucleic acid are the same adenine as the standard double-stranded nucleic acid, a chain recombination reaction occurs between the sample-derived double-stranded nucleic acid and the standard double-stranded nucleic acid. . Theoretically, if the ratio of the number of molecules of the sample-derived double-stranded nucleic acid and the standard double-stranded nucleic acid is 1: 1, the probability of recombination is ½, and the probability of returning to the original double-stranded nucleic acid is also possible. The degree of fluorescence resonance energy transfer is also halved. When all the mutation sites of the sample-derived double-stranded nucleic acid are guanine different from the standard double-stranded nucleic acid, the strand recombination reaction does not occur, and therefore the degree of fluorescence resonance energy transfer does not change. With this, it becomes possible to detect whether the target base to be detected (identified) in the sample is adenine or guanine. The degree of recombination can be increased by increasing the ratio of sample-derived double stranded nucleic acid to standard double stranded nucleic acid. For example, if the ratio of the sample-derived double-stranded nucleic acid to the standard double-stranded nucleic acid is 20: 1, the recombination ratio is 20/21, that is, the probability that the standard double-stranded nucleic acid returns to the original double-stranded is 1. / 21, and the change in fluorescence resonance energy transfer becomes large and detection becomes easy.

JP 2003-174882 A

Chen, 1 other, Analytical biochemistry, 1991, 195, 51-56. Jacobson, 1 other, Oncogene, 1994, Vol. 9, pages 553-563. Newton, 7 others, Nucleic acids research, 1989, 17th, pp. 2503-2516. Wu, 3 others, Proceedings of the National Academy of Sciences of the United States of the United States of Sciences of the United States of Sciences of the United States of America), 1989, 86, 2757-2760. Nolau, 1 other, Clincal Chemistry, 1997, 43, 1114-1128. Ogino, 9 others, The Journal of Molecular Diagnostics, 2005, Vol. 7, pages 413-421. Krypuy, 4 others, BMC Cancer, 2006, volume 6, page 295. Tada, 7 others, Clinica Chimica Acta, 2002, volume 324, page 105.

In the conventional PCR-PHFA method, the base sequence identification accuracy in the chain recombination reaction is not sufficient, and the difference in 1-2 bases cannot be discriminated, and the chain recombination reaction occurs between different nucleic acid strands. As a result, false positives were sometimes produced.
Although the method described in Patent Document 1 uses fluorescence resonance energy transfer and is a good method with high sensitivity and relatively simple operation, it may still give false positives. The accuracy was not enough. There is also no method for preventing variation in each measurement of fluorescence.
Thus, none of the known PCR-PHFA methods are actually methods for detecting mutant genes with high sensitivity.

On the other hand, the progress of gene detection technology in recent years has been remarkable, and methods for simultaneously detecting many gene expressions and mutations combining microfabrication technology and fluorescence detection methods have been developed. Mutant genes that can be combined with these technologies Therefore, highly sensitive detection is desired.
Under such circumstances, the present invention provides a method capable of improving the accuracy of identifying differences in base sequences in a genotype identification method using the PCR-PHFA method, and a kit suitable for the method. With the goal.

  As a result of diligent research to solve the above-mentioned problems, the present inventor has obtained bases by substituting all or part of bases of standard double-stranded nucleic acids with known sequences for causing strand recombination with nucleotide analogs. The present inventors have found that the accuracy of discriminating sequence differences can be improved and completed the present invention. Furthermore, the present inventor may be able to improve the accuracy of discriminating the difference in base sequence by modifying a portion other than the mutation site in the standard double-stranded nucleic acid to a base sequence that is non-complementary to the base sequence of the natural gene. The headline and the present invention were completed.

That is, the present invention
(1) A method for identifying a genotype in a gene mutation, comprising a step of amplifying a region containing a mutation site in a gene contained in a sample by a nucleic acid amplification reaction to prepare a sample double-stranded nucleic acid, A standard double-stranded nucleic acid whose mutation site is a specific genotype and labeled with a labeling substance is mixed with the sample double-stranded nucleic acid to perform a competitive strand recombination reaction. Identifying the identity of the standard double-stranded nucleic acid and the sample double-stranded nucleic acid by measuring the degree of occurrence of strand recombination with the double-stranded nucleic acid, and Ri least one base nucleotide analogs der mutation sites of the standard double-stranded nucleic acid, the only mutation site of a standard double-stranded nucleic acid is constituted by nucleotide analogs, said nucleotide analogue is locked nucleus Method of identifying the genotype characterized by acid (LNA) Der Rukoto,
(2) the identification method (1) Symbol placement genotype, characterized in that one of the mutation site of two nucleic acid strands that constitute the standard double-stranded nucleic acid is also constituted by nucleotide analogs,

( 3 ) Of the two nucleic acid strands constituting the standard double-stranded nucleic acid, the 3 ′ end of one strand is a first labeling substance, and the 5 ′ end of the other strand is a second labeling substance. Each of the first labeling substance and the second labeling substance is labeled with each other and is capable of energy transfer with each other, and the degree of energy change due to energy transfer between the first labeling substance and the second labeling substance is measured. And measuring the degree of strand recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid in the identification step. The genotype of (1) or (2) Identification method,
( 4 ) At least one of the first labeling substance and the second labeling substance is a fluorescent substance, and the competitive strand recombination reaction in the identification step includes the standard double-stranded nucleic acid and the sample double-stranded nucleic acid. The temperature of the reaction solution is gradually decreased from a high temperature, and ± 3 to the temperature at which the average rate of change of the fluorescence intensity of the first labeling substance or the second labeling substance with respect to the temperature is maximized. The method for identifying a genotype according to ( 3 ) above, wherein the rate of temperature decrease in a temperature range of 5 ° C. is lower than that in the case of a temperature outside the temperature range,
( 5 ) At least one of the first labeling substance and the second labeling substance is a fluorescent substance, and the competitive strand recombination reaction in the identification step includes the standard double-stranded nucleic acid and the sample double-stranded nucleic acid. The reaction solution is measured by gradually decreasing the temperature of the reaction solution from a high temperature and measuring the degree of strand recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid. The genotype identification method according to the above ( 3 ) or ( 4 ), characterized in that the measurement is based on the amount of change in fluorescence intensity due to a decrease in temperature;
( 6 ) At least one of the first labeling substance and the second labeling substance is a fluorescent substance, and the competitive strand recombination reaction in the identification step includes the standard double-stranded nucleic acid and the sample double-stranded nucleic acid. The reaction solution is measured by gradually decreasing the temperature of the reaction solution from a high temperature and measuring the degree of strand recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid. Measured based on a ratio between the amount of change in fluorescence intensity due to temperature drop and the amount of change in fluorescence intensity due to temperature drop of a control reaction solution that does not contain the sample double-stranded nucleic acid but contains the standard double-stranded nucleic acid. The method for identifying a genotype according to ( 3 ) or ( 4 ),
( 7 ) A kit used for identifying a genotype by the genotype identification method described in ( 3 ) to ( 6 ) above, wherein a nucleic acid amplification reagent for preparing a sample double-stranded nucleic acid and each other Two kinds of labeling substances capable of energy transfer, a reagent for introducing a labeling substance into the 3 ′ end of the nucleic acid chain, a reagent for introducing a labeling substance into the 5 ′ end of the nucleic acid chain, and a mutation site A standard double-stranded nucleic acid of a specific genotype , wherein at least one mutation site in the standard double-stranded nucleic acid is a nucleotide analog, and only the mutation site in the standard double-stranded nucleic acid is a nucleotide analog is constituted by, genotype identification kit said nucleotide analog is characterized locked nucleic acid (LNA) der Rukoto,
Is to provide.

Other,
(A) A method for identifying a genotype of a gene mutation, the region containing the mutation site in the gene contained in the sample is amplified by a nucleic acid amplification reaction, preparing a double-stranded nucleic acid sample, wherein the mutation site A standard double-stranded nucleic acid having a specific genotype is mixed with the sample double-stranded nucleic acid to perform a competitive strand recombination reaction, and strand recombination is performed between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid. Determining the identity of the standard double-stranded nucleic acid and the sample double-stranded nucleic acid by measuring the degree of occurrence, and other than the mutation site in the standard double-stranded nucleic acid A genotype characterized in that a site consisting of 1 base, or a continuous or non-contiguous site of 2 or more bases is modified to a base that is non-complementary to the natural gene containing the gene mutation Identification method,
( B ) The site in the sample double-stranded nucleic acid corresponding to the modified site in the standard double-stranded nucleic acid is modified to the same base sequence as that of the standard double-stranded nucleic acid. A method for identifying a genotype as described in ( a ) above,
( C ) The base species of the modified site in the standard double-stranded nucleic acid is adenine, thymine, or uracil, and the base species of the site corresponding to the site in the gene is guanine or cytosine A method for identifying the genotype according to ( a ) or ( b ),
( D ) Modification in which the nucleic acid amplification step modifies a site in the sample double-stranded nucleic acid corresponding to the modified site in the standard double-stranded nucleic acid to the same base sequence as the standard double-stranded nucleic acid. A method of identifying a genotype as described in ( a ) or ( b ) above, which is a step of preparing a sample double-stranded nucleic acid by performing a polymerase chain reaction (PCR) using a primer having a position;
( E ) The method for identifying a genotype as described in ( d ) above, wherein the modified part is located at a position 2 bases or more away from the 3 ′ end of the primer,
There is also.

In the present specification, the genotype identification method according to (1) is referred to as a first identification method, and the genotype identification method according to ( a ) is referred to as a second identification method .

The method for identifying genotypes of the present invention has greatly improved the accuracy of identifying nucleic acid base sequences, so that not only germ cell mutations such as SNPs but also somatic cells that were difficult to identify by conventional PCR-PHFA methods Mutations can also be identified with high accuracy.
Moreover, the genotype identification method of the present invention can be performed more easily by using the genotype identification kit of the present invention.

It is the figure which showed typically an example of the fluorescence behavior of the donor labeling substance in case a genotype differs. It is the figure which showed typically an example of the fluorescence behavior of the acceptor label | marker substance in case a genotype differs. Prepare PCR products (sample double-stranded nucleic acid), standard double-stranded nucleic acid (match), standard double-stranded nucleic acid (mismatch A), and standard double-stranded nucleic acid (mismatch B) in the same manner as in FIG. It is the figure which showed (DELTA) F of the donor labeling substance at the time of performing a target strand recombination reaction. In Example 1, sample double-stranded nucleic acid (G12D), standard double-stranded nucleic acid (G12D), standard double-stranded nucleic acid (G12D (LNA)), standard double-stranded nucleic acid (G12S_2), or standard double-stranded It is the figure which mixed the nucleic acid (G12S_2 (LNA)) and showed the result of having performed the strand recombination reaction. In Example 1, sample double-stranded nucleic acid (G12S_2), standard double-stranded nucleic acid (G12D), standard double-stranded nucleic acid (G12D (LNA)), standard double-stranded nucleic acid (G12S_2), or standard double-stranded It is the figure which mixed the nucleic acid (G12S_2 (LNA)) and showed the result of having performed the strand recombination reaction. In Example 1, it is the figure which showed (DELTA) FAM calculated | required by mixing sample double stranded nucleic acid (G12D) and each standard double stranded nucleic acid, and performing strand recombination reaction. In Example 1, it is the figure which showed (DELTA) FAM calculated | required by mixing sample double stranded nucleic acid (G12S_2) and each standard double stranded nucleic acid, and performing strand recombination reaction.

  In the present invention, the gene mutation means a difference in the base sequence of a gene existing between individuals of the same organism species, and the mutation site means a different site in the base sequence. Specifically, a difference in the base sequence is caused by substitution, deletion, or insertion of one or more bases in the base sequence. That is, in the present invention, gene mutation includes congenital mutation such as gene polymorphism such as SNP and microsatellite polymorphism in addition to acquired mutation such as somatic mutation.

  In the present invention, the nucleotide analog is a non-natural nucleotide and has the same function as deoxyribonucleotide (DNA) or ribonucleotide (RNA), which are natural nucleotides. That is, a nucleotide analog is a modification of the base or sugar skeleton of a natural nucleotide, can form a chain by a phosphodiester bond in the same way as a nucleotide, and is a primer formed using a nucleotide analog The probe and probe can be used for PCR and hybridization in the same manner as primers and probes formed using only nucleotides. Examples of such nucleotide analogs include PNA (polyamide nucleotide derivatives), LNA (BNA), ENA (2'-O, 4'-C-Ethylene-bridged nucleic acids), and complexes thereof. Here, PNA is obtained by substituting a main chain composed of phosphoric acid and pentose of DNA or RNA with a polyamide chain. LNA (BNA) is an RNA analog having a structure in which a 2′-position oxygen atom and a 4′-position carbon atom of a ribonucleoside are methylene-bridged.

  In the genotype identification method of the present invention, the standard double-stranded nucleic acid is a double-stranded nucleic acid with a known base sequence that undergoes recombination competitively with the double-stranded nucleic acid derived from the sample to be identified in the PCR-PHFA method. Means nucleic acid. Specifically, the standard double-stranded nucleic acid is a double-stranded nucleic acid that is a partial region including a mutation site of a target gene and that includes the same base sequence as the sequence whose mutation site is a specific genotype. When a strand recombination reaction occurs between the standard double-stranded nucleic acid and the sample-derived double-stranded nucleic acid, the gene contained in the sample has the same genotype as the standard double-stranded nucleic acid. If no strand recombination reaction occurs, it can be identified as a genotype different from that of the standard double-stranded nucleic acid.

  In the first identification method of the present invention, when a genotype in gene mutation is identified using the PCR-PHFA method, at least one mutation site base is used as a standard double-stranded nucleic acid used for competitive strand recombination reaction. This is a method for improving the discrimination accuracy of a base sequence in a competitive strand recombination reaction by using a modified nucleotide analog. Although it is not clear why the nucleotide sequence identification accuracy can be improved by introducing a nucleotide analog at the mutation site of the standard double-stranded nucleic acid, the affinity between the nucleic acid strands of the standard double-stranded nucleic acid at the mutation site is increased. As a result, it is assumed that the strand recombination reaction between the standard double-stranded nucleic acid and the nucleic acid strand having a different base sequence is remarkably suppressed.

  For example, when an oligonucleotide containing LNA anneals to an oligonucleotide made of DNA, the conformation of the double-stranded nucleic acid changes. For example, an oligonucleotide containing an LNA every 3 'nucleotide changes the double helix from a B structure to an A structure. This improves the stacking of double-stranded nucleic acids and increases the stability. That is, when the base pair between the two nucleic acid strands of the standard double-stranded nucleic acid is only DNA-DNA at the mutation site, the case where the standard double-stranded nucleic acid is LNA-DNA or LNA-LNA. In the competitive strand recombination reaction, the nucleic acid strands of the standard double-stranded nucleic acid hybridize preferentially over the nucleic acid strand of the sample-derived double-stranded nucleic acid. Thereby, when the base sequence of the sample-derived double-stranded nucleic acid is different from that of the standard double-stranded nucleic acid, the standard double-stranded nucleic acid is remarkably suppressed from being recombined with the sample-derived nucleic acid strand by mistake. be able to.

  In general, modification with LNA is intended to significantly increase the Tm value, and modification is performed on an oligonucleotide of about 20 mer. In contrast, in the PCR-PHFA method, a PCR product is often used as a sample double-stranded nucleic acid to be identified. For this reason, the standard double-stranded nucleic acid used for the competitive strand recombination reaction is about 40 to 50 bp. Is expected to be required. Furthermore, it has been reported that the increase in Tm value due to LNA modification tends to decrease in an oligonucleotide length-dependent manner (Yong et al., Nucleic acids research, 2006, 34, 8). : See e61). In other words, modification of introducing a LNA into a relatively long oligonucleotide of 40 to 50 bp, influence on hybridization efficiency by modifying a double-stranded nucleic acid by LNA, and further, a double-stranded nucleic acid modified by LNA It has not been carried out in the past to use the PCR-PHFA method, and its effect was unknown. Then, by using a double-stranded nucleic acid whose mutation site is LNA-modified as a double-stranded nucleic acid with a known base sequence to be used for competitive strand recombination reaction in the PCR-PHFA method, a nucleic acid with improved discrimination accuracy and a different base sequence The present inventors have found for the first time that the probability that a strand recombination reaction will occur erroneously with a strand is greatly reduced.

  In the first identification method of the present invention, the nucleotide analog introduced into the standard double-stranded nucleic acid can increase the affinity between the nucleic acid strands of the standard double-stranded nucleic acid as compared with the case where only the natural nucleotide is used. It is not particularly limited as long as it is an analog, and any of the nucleotide analogs listed above may be used, and modified versions of these nucleotide analogs may be used. In the present invention, it is preferable to use LNA because the effect of improving the identification accuracy is more excellent.

  In the standard double-stranded nucleic acid, the site to be modified to a nucleotide analog only needs to contain a mutation site, but it is preferable to modify only the mutation site. This is because the base sequence identification accuracy can be further improved. When the mutation site is composed of two or more bases, or when there are a plurality of mutation sites in one standard double-stranded nucleic acid, at least one base among the bases constituting these mutation sites May be modified to a nucleotide analog. Of course, all of the mutation sites may be modified to nucleotide analogues.

  In addition, a nucleotide analog may be introduced only into the mutation site of one of the two nucleic acid strands constituting the standard double-stranded nucleic acid, or a nucleotide analog may be introduced into the mutation site of both nucleic acid strands. Also good.

  A standard double-stranded nucleic acid having such a nucleotide analog modification site can be prepared by, for example, known chemical synthesis. Examples of the chemical synthesis method include triester method and phosphorous acid method. For example, a large amount of single-stranded DNA is prepared using an ordinary automatic synthesizer (APPLIED BIOSSYSTEMS 392, etc.) using a liquid phase method or a solid phase synthesis method using an insoluble carrier, and then annealed. By doing so, double-stranded DNA can be prepared. When synthesizing a single strand, a nucleotide analog can be introduced.

  Specifically, the first identification method of the present invention includes a nucleic acid amplification step of amplifying a region containing a mutation site in a gene contained in a sample by a nucleic acid amplification reaction to prepare a sample double-stranded nucleic acid, and the mutation A standard double-stranded nucleic acid of a specific genotype is mixed with the sample double-stranded nucleic acid to perform a competitive strand recombination reaction, and a strand recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid. And a discriminating step for identifying the identity between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid by measuring the degree of occurrence.

  Examples of the sample used in the first identification method of the present invention include pathogens such as bacteria and viruses, blood separated from living bodies such as humans, saliva, tissue lesions, and excreta such as manure. It is done. Furthermore, when performing a prenatal diagnosis, fetal cells existing in amniotic fluid or a part of a dividing egg cell in a test tube can be used as a specimen. In addition, these samples are concentrated as a sediment directly or if necessary by centrifugation, etc., and then subjected to cell destruction treatment such as enzyme treatment, heat treatment, surfactant treatment, ultrasonic treatment, or a combination thereof. Those previously applied can be used. In this case, the cell destruction treatment is performed for the purpose of revealing the nucleic acid derived from the target tissue. In addition, the specific method of the cell destruction treatment may be performed according to a known method described in a literature such as PCR Protocols Academic Press Inc. (p14, p352, p352 (1990)). it can. Further, the total amount of nucleic acid in the sample is preferably about 5 to 50 ng, but sufficient amplification is possible even with 5 ng or less.

  Examples of mutation sites to be identified in the first identification method of the present invention include cancer-related genes, genes related to genetic diseases, viral genes, bacterial genes, and genes showing polymorphisms called disease risk factors. What exists is listed. Examples of cancer-related genes include k-ras gene, N-ras gene, p53 gene, BRCA1 gene, BRCA2 gene, or APC gene. Examples of genes related to genetic diseases include genes reported to be associated with various inborn errors of metabolism. Examples of viral genes and bacterial genes include genes such as hepatitis C virus and hepatitis B virus. Examples of genes showing polymorphism include genes having different nucleotide sequences that are not directly related to the cause of illness, such as genes related to HLA (Human Leukocyte Antigen) and blood groups, And genes that are considered to be related to the onset of diabetes and the like. Most of these genes are present on the host chromosome, but may be encoded by mitochondrial genes.

  In the present invention, first, as a nucleic acid amplification step, a region containing a mutation site in a gene contained in a sample is amplified by a nucleic acid amplification reaction to prepare a sample double-stranded nucleic acid. The nucleic acid amplification reaction is not particularly limited as long as it can amplify a region containing a mutation site as a double-stranded nucleic acid. The PCR method, LCR (Ligase chain Reaction) method, 3SR (Self-stained Sequence) It can be used by appropriately selecting from known nucleic acid amplification reactions such as replication (Replication) method and SDA (Strand Displacement Amplification) method (Manak, DNA Probes 2nd Edition p255-291, Stockton Press (1993)). In the present invention, the PCR method is particularly suitable.

  For example, a sample double-stranded nucleic acid can be prepared by designing a primer so as to sandwich an amplification region including a mutation site and repeatedly performing a primer extension reaction using a polymerase. Reagents such as dNTP and polymerase used in this extension reaction can be appropriately selected from reagents usually used for nucleic acid amplification. For example, polymerases include E. coli. coli DNA polymerase I, E. coli. Any DNA polymerase such as Klenow fragment of E. coli DNA polymerase I or T4 DNA polymerase can be used, but it is particularly preferable to use a thermostable DNA polymerase such as Taq DNA polymerase, Tth DNA polymerase or Vent DNA polymerase. There is no need to add a new enzyme for each cycle, the cycle can be automatically repeated, and the annealing temperature can be set to 50-60 ° C. The gene amplification reaction can be carried out quickly and specifically (see JP-A-1-314965 and JP-A-1-252300 for details). For specific methods such as reaction conditions for carrying out this elongation reaction, Experimental Medicine Vol. 8 No. 9 (Yodosha, (1990)), PCR Technology Stockton Press (PCR Technology Stockton press) ) (1989).

  A sample double-stranded nucleic acid is a double-stranded nucleic acid obtained by nucleic acid amplification of a region in a gene including a mutation site that is completely identical to a standard double-stranded nucleic acid. Thus, since the sample double-stranded nucleic acid and the standard double-stranded nucleic acid differ only in the mutation site, the discrimination accuracy when the mutation site is only one base can be further improved.

  Next, as a discriminating step, the standard double-stranded nucleic acid is mixed with the sample double-stranded nucleic acid to perform a competitive strand recombination reaction, and the degree of strand recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid. By measuring the identity of the standard double-stranded nucleic acid and the sample double-stranded nucleic acid.

  Competitive strand recombination is a competitive reaction that occurs between a double-stranded nucleic acid having a homologous base sequence and a single-stranded nucleic acid, or between a double-stranded nucleic acid having a homologous base sequence and a double-stranded nucleic acid. This is a nucleic acid strand displacement reaction (competitive hybridization), which can be performed by mixing and denaturing a standard double-stranded nucleic acid and a sample double-stranded nucleic acid, followed by annealing.

  As a method for denaturing the standard double-stranded nucleic acid and the sample double-stranded nucleic acid, a method using heating or a method using alkali is preferable. In this invention, since it is simple, it is preferable to denature by heating. Specifically, the double-stranded nucleic acid can be denatured by heating to 90 to 100 ° C., preferably 95 to 100 ° C. for a certain time.

  When annealing the denatured standard double-stranded nucleic acid and the sample double-stranded nucleic acid, it is preferable to prepare the salt concentration in the reaction solution to be optimum. The optimum salt concentration generally depends on the chain length. Generally, in hybridization, SSC (20 × SSC: 3M sodium chloride, 0.3M sodium citrate) or SSPE (20 × SSPE: 3.6M sodium chloride, 0.2M sodium phosphate, 2mM EDTA) is used. In the identification method of the present invention, these solutions can be diluted to a suitable concentration and used. Moreover, organic solvents, such as a dimethyl sulfoxide (DMSO) and a dimethylformamide (DMF), can also be added as needed.

  When denatured by heating, annealing is performed by lowering the temperature of the reaction solution from a high temperature (generally, the denaturation temperature, for example, any temperature in the range of 90 to 100 ° C.). A competitive strand recombination reaction can be performed. Conditions such as the temperature drop rate of the reaction solution and the temperature of the reaction solution at the end of the reaction can be appropriately set according to the chain length and base sequence of the standard double-stranded nucleic acid and the sample double-stranded nucleic acid. In the temperature range near Tm (Tm ± 3 to 5 ° C.) of the standard double-stranded nucleic acid, the temperature decrease of the reaction solution is slow (eg, at a rate of 0.1 ° C./min to 0.3 ° C./min). The probability that single strands having non-complementary base sequences will hybridize is reduced, and competitive strand recombination can be carried out with high accuracy.

  The degree of strand recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid can be measured by labeling any nucleic acid with a labeling substance and using the label as an indicator. For example, one of the two nucleic acid strands constituting the standard double-stranded nucleic acid is labeled with a certain labeling substance, and the other strand is labeled with another labeling substance. In this case, when the chain recombination reaction has not occurred, the two kinds of labeling substances are all detected from the same molecule. On the other hand, when a chain recombination reaction occurs, there are molecules that can detect only one of the two types of labeling substances. Therefore, the extent to which recombination occurred between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid by detecting with which labeling substance each molecule of the double-stranded nucleic acid in the reaction solution is labeled. Can be measured. Note that the sample double-stranded nucleic acid may be similarly labeled instead of the standard double-stranded nucleic acid. Further, one nucleic acid strand of the standard double-stranded nucleic acid is labeled with a certain labeling substance, and one nucleic acid strand of the sample double-stranded nucleic acid that can undergo a strand recombination reaction with this labeled nucleic acid chain is labeled with another labeling substance. May be.

  As the labeling substance, either a non-radioactive substance or a radioactive substance may be used, but a non-radioactive substance is preferably used. Non-radioactive labeling substances include fluorescent substances [for example, fluorescein derivatives (fluorescein isothiocyanate, etc.), rhodamine and derivatives thereof (tetramethylrhodamine isothiocyanate, etc.)], chemiluminescent substances (for example, Acridine, etc.). Further, the labeling substance can be indirectly detected by using a substance that specifically binds to the labeling substance. Examples of such a labeling substance include biotin, a ligand, a specific nucleic acid, or a protein hapten. In the case of biotin, the avidin or streptavidin that specifically binds to this substance is specifically bound to the labeling substance, and in the case of a hapten, the antibody that specifically binds to this is the ligand. When the receptor is a specific nucleic acid or protein, a nucleic acid that specifically binds to the receptor, a nucleic acid binding protein, a protein having an affinity for the specific protein, or the like can be used. As the hapten, a compound having 2,4-dinitrophenyl group or digoxigenin can be used, and biotin or a fluorescent substance can also be used as a hapten. Any of these labeling substances may be used alone or in combination of a plurality of kinds if necessary (see JP-A-59-93099, JP-A-59-148798, JP-A-59-204200). )).

  In addition, when one of the two types of labeling substances is a substance that can bind to a solid phase, a standard double-stranded nucleic acid and a sample can be obtained by performing a widely used solid-liquid separation operation. The degree to which strand recombination has occurred between the double-stranded nucleic acid can be measured. For example, one strand of a standard double-stranded nucleic acid is labeled with a labeling substance A, the other strand is labeled with a labeling substance B that can bind to a solid phase alone, and the labeling substance B binds to the reaction solution after the strand recombination reaction. Contact with possible solid phase alone. Thereafter, the labeling substance A in the double-stranded nucleic acid bound to the solid phase carrier is measured. When the strand recombination reaction occurs, the ratio of the double-stranded nucleic acid labeled with the labeling substance A in the double-stranded nucleic acid bound to the solid phase carrier decreases.

  In particular, in the present invention, two kinds of labeling substances capable of transferring energy to each other (for example, a donor labeling substance that generates fluorescence by excitation and an acceptor labeling substance that absorbs the fluorescence) are used, and between these labeling substances. It is preferable to measure the degree of strand recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid, using the degree of energy change due to energy transfer as an index.

  The energy transfer between the labeling substances in the present invention means that at least two kinds of labeling substances, that is, a donor labeling substance that generates energy and an acceptor labeling substance that absorbs energy generated from the donor labeling substance are in proximity to each other. In some cases, it refers to the transfer of energy from a donor labeling substance to an acceptor labeling substance. For example, when the two kinds of labeling substances are fluorescent substances, the acceptor labeling substance absorbs the fluorescence generated by exciting the donor labeling substance and measures the fluorescence emitted by the acceptor labeling substance, or the donor labeling substance is excited. The quenching of the donor labeling substance caused by the absorption of the fluorescence generated by the acceptor labeling substance can be measured (PCR Methods and applications 4,357-362 (1995), Nature Biotechnology 16, 49-53 (1998)). Note that energy transfer may occur even if there is no overlap between the fluorescence wavelength of the donor labeling substance and the absorption wavelength of the acceptor labeling substance. Such energy transfer is also included in the present invention. The acceptor labeling substance may be a quencher. As such, the quencher includes, for example, dubcyl and black hole.

  Specifically, as a standard double-stranded nucleic acid, among the two nucleic acid strands constituting one, the 3 ′ end of one strand is labeled with the first labeling substance, and the 5 ′ end of the other strand is the first. A labeling substance labeled with a second labeling substance capable of energy transfer with each other is used. Either the first labeling substance or the second labeling substance may be a donor labeling substance. In this standard double-stranded nucleic acid, energy transfer occurs because the first labeling substance and the second labeling substance are close to each other. On the other hand, when a competitive strand recombination reaction occurs between the sample double-stranded nucleic acid, energy transfer is caused because the first labeled substance and the second labeled substance are separated in the double-stranded nucleic acid in which strand recombination has occurred. Does not occur, and the proportion of double-stranded nucleic acids in which energy transfer occurs in the reaction solution decreases. Therefore, by measuring the energy emitted from the first labeling substance or the second labeling substance (fluorescence intensity in the case of a fluorescent substance), the degree of energy change due to energy transfer is measured, and the standard double-stranded nucleic acid and The degree to which strand recombination has occurred with the sample double-stranded nucleic acid can be measured.

  Compared to nucleic acid strands with different genotypes (different mutation base sequences), nucleic acid strands with the same genotype (having completely complementary base sequences) are more preferentially double-stranded. Form. For this reason, by measuring the degree of energy change due to energy transfer between the labeled substances, that is, the degree of energy transfer change caused or lost by the strand displacement reaction, by using an arbitrary detector. Whether the genotype of the mutation site of the gene contained in the sample is the same as the standard double-stranded nucleic acid, and the ratio of the same genotype as the standard double-stranded nucleic acid contained in the sample Can be detected. For example, when fluorescence energy transfer is used for detection, the presence or absence of a gene having the same genotype as that of a standard double-stranded nucleic acid is measured by measuring a fluorescence spectrum of a specific wavelength with a spectrofluorometer, a fluorescence plate reader, or the like. And the ratio can be easily detected.

The standard double-stranded nucleic acid is not labeled. Of the two nucleic acid strands constituting the sample double-stranded nucleic acid, the 3 ′ end of one strand is labeled with the first labeling substance, and the 5 ′ end of the other strand is labeled. A part labeled with a second labeling substance may be used. In this case, as in the case of labeling the standard double-stranded nucleic acid, the energy transfer does not occur in the double-stranded nucleic acid in which the strand recombination has occurred, and the ratio of the double-stranded nucleic acid in which the energy transfer in the reaction solution has occurred. Decrease.
In addition, a sample double-stranded nucleic acid in which the 3 ′ end (or 5 ′ end) of one nucleic acid strand of a standard double-stranded nucleic acid is labeled with a first labeling substance and a recombination reaction can occur with the labeled nucleic acid strand. One of the nucleic acid strands (complementary strand of the nucleic acid strand labeled with the first labeling substance) 5 'end (or 3' end) labeled with the second labeling substance may be used. In this case, energy transfer occurs in the double-stranded nucleic acid in which strand recombination has occurred, and the ratio of the double-stranded nucleic acid in which the energy transfer has occurred in the reaction solution increases.

  Thus, by measuring the degree of energy change due to energy transfer between the labeled substances, the degree of strand recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid is measured, The identity between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid can be quickly and easily identified without requiring complicated work such as separation work. In addition, by introducing both labeling substances into the 3 ′ end and the 5 ′ end adjacent to each other, it is possible to accurately and surely capture the degree of strand displacement, and the standard double-stranded nucleic acid or Even if the sample double-stranded nucleic acid is a long-chain gene fragment, the degree of substitution of the complementary strand can always be measured accurately and reliably with good sensitivity, and the genotype identity can be accurately and stably identified. Can do. In particular, since it is a simple method that does not require a conventional complicated solid-liquid separation operation, it is possible to automate and meet the demands of the frontline medical site.

The labeling substance that can be used as the first labeling substance or the second labeling substance is not particularly limited as long as it can transfer energy in the state of being close to each other, but among them, a fluorescent substance and a delayed fluorescent substance are preferable. A chemiluminescent substance, a bioluminescent substance, etc. can also be used. As a combination of such labeling substances, fluorescein and its derivatives (for example, fluorescein isothiocyanate) and rhodamine and its derivatives (for example, tetramethylrhodamine isothiocyanate, tetramethylrhodamine-5- (and-6) hexanoic acid) Etc.), a combination of fluorescein and dabsyl, and the like, and any combination can be selected from these (Nisotopic DNA Probe Techniques. Academic Press (1992)). In addition, it may be a combination of molecules in which thermal energy is released when close to each other. Examples of combinations of such labeling substances include Alexa Fluor (registered trademark) 488 (manufactured by Invitrogen), ATTO 488 (manufactured by ATTO-TEC GmbH), Alexa Fluor (registered trademark) 594 (manufactured by Invitrogen), and ROX ( Examples thereof include a combination of 1 selected from the group consisting of Carboxy-X-rhodamine) and BHQ (registered trademark, Black hole quencher) -1 or BHQ (registered trademark) -2.
In addition, since guanine has the ability to quench when FAM adjoins (Nucleic acids Research 2002, vol.30.no.9 2089-2195), you may utilize this. For example, when the 3 ′ end of one strand of a standard double-stranded nucleic acid is labeled with FAM, and the base at the 5 ′ end of the other strand is guanine, the other strand is labeled as a labeling substance. It is not necessary to label with.

  As a method for introducing the first labeling substance or the second labeling substance into the standard double-stranded nucleic acid or the sample double-stranded nucleic acid, a general method for introducing a label into a nucleic acid can be employed. For example, a method of directly introducing a labeling substance into a nucleic acid (Biotechniques 24, 484-489 (1998)), a method of introducing a labeling substance-bound mononucleotide by a DNA polymerase reaction or an RNA polymerase reaction (Science 238, 336-3341). (1987)), a method of introducing a PCR reaction using a primer into which a labeling substance has been introduced (PCR Methods and Applications 2, 34-40 (1992)) and the like.

  The position where the labeling substance is introduced into the standard double-stranded nucleic acid or the sample double-stranded nucleic acid is a position where energy transfer occurs or disappears by the strand displacement reaction, that is, the 3 ′ end and / or the 5 ′ end of the nucleic acid strand. Need to be. Specifically, in the present invention, the 5 ′ end and the 3 ′ end indicate ranges within 30 bases from the 5 ′ end and 3 ′ end of the nucleic acid strand, respectively, but are closer if both labeling substances are close. Since energy transfer is likely to occur, it is preferably within 10 bases from each end, and most preferably 5 ′ end and 3 ′ end. Here, if a large number of labeling substances are introduced into the base portion that hybridizes with the complementary strand, substitution of about one base may not be detected, so it is preferable to introduce it only at the end portion of each nucleic acid strand. For example, one of two kinds of labeling substances is introduced into the 5 ′ end (3 ′ end) of one nucleic acid strand and the 3 ′ end (5 ′ end) of the other nucleic acid strand complementary thereto By introducing the other labeling substance into the two, both nucleic acid strands cause energy transfer or disappear by the strand displacement reaction without affecting the hybridization reaction.

  Specifically, in order to prepare a nucleic acid chain having a label at the 5 ′ end, a method in which a linker having a labeling substance introduced at the 5 ′ end is bonded to an arbitrary nucleic acid chain by ligase (Nucleic Acids Res. 25). 922-923 (1997)), or a method of performing a PCR reaction using a primer having a labeling substance introduced at the 5 ′ end (PCR Methods and Applications 2, 34-40 (1992)).

  On the other hand, in order to prepare a nucleic acid chain having a label at the 3 ′ end, as in the case of introducing a labeling substance at the 5 ′ end, a linker having a labeling substance introduced at the 3 ′ end and an arbitrary nucleic acid There is a method of joining the strands with ligase. In addition, when the nucleic acid chain is RNA instead of DNA, or the 3 ′ end of DNA is RNA, the sugar (ribose) part of the RNA at the end is selectively opened, and the resulting aldehyde group It can also be labeled using.

  Furthermore, mononucleotide triphosphates into which a labeling substance has been introduced can also be introduced into the 3 'end of a nucleic acid chain by the action of terminal deoxynucleotidyl transferase (Biotechniques 15, 486-496 (1993)).

  When the standard double-stranded nucleic acid is a relatively short nucleic acid strand of 100 bases or less, a labeled nucleic acid can also be prepared by direct chemical synthesis (Nucleic Acids Res. 16, 2659-2669 (1988), Bioconjug. Chem. 3, 85-87 (1992)).

  Measurement of the degree of energy change due to energy transfer between labeling substances is generally performed by measuring the fluorescence emitted from the labeling substance. This fluorescence measurement can measure a large number of specimens at the same time. In many cases, a so-called real-time PCR apparatus or the like capable of controlling temperature is used. However, in such an apparatus, the accuracy of fluorescence measurement for each detection is not necessarily high, and in many cases results in large variations. In addition, variation in the amount of the standard double-stranded nucleic acid to be added can be a factor that greatly affects the measurement accuracy. In an apparatus for carrying out the reaction in a very small amount, there is a concern that such variations in measurement and variations in the amount of standard substance added will become even greater. Therefore, when performing quantitative measurement, it is preferable to correct the variation between these measurements.

  In general, as a method for correcting the variation between measurements, there is a method using a standard substance that clearly shows the same result between measurements. For example, it is possible to add a fluorescent substance different from both the first labeling substance and the second labeling substance to each reaction solution and correct the target fluorescence measurement value based on the value of the fluorescent substance. is there. However, such a correction method is indirect and cannot correct variations in the amount of standard double-stranded nucleic acid added.

  In addition, in the detection method using fluorescence resonance energy transfer, generally, a method of correcting the variation between measurements by calculating the ratio of the fluorescence values of both the donor fluorescent substance and the acceptor fluorescent substance is performed. It has been broken. That is, it is a method of measuring both the fluorescence generated by exciting the donor and the fluorescence of the acceptor excited by the energy transfer from the donor and determining the ratio. Therefore, the present inventor determined the ratio between the fluorescence value of the donor labeling substance and the fluorescence value of the acceptor labeling substance when measuring the degree of energy change due to the energy transfer between the labeling substances in the genotype identification method of the present invention. However, it was not possible to achieve satisfactory results. This is because the fluorescence value of the donor labeling substance after the strand recombination reaction is very small, and the fluorescence measurement in such a state is likely to vary greatly. Therefore, the fluorescence value of the donor labeling substance and the acceptor labeling substance It is considered that the ratio with the fluorescence value also causes a very large variation. The donor labeling substance has a small fluorescence value because the energy transfer occurs when the standard double-stranded nucleic acid after denaturation returns to the original double-stranded nucleic acid without causing the strand recombination reaction. This is because most of the fluorescence of the light becomes very weak light emission as a result of energy transfer to the acceptor labeling substance.

  Furthermore, in the genotype identification method of the present invention, the type of labeling substance, the type of genotype, the type of base sequence of standard double-stranded nucleic acid or sample double-stranded nucleic acid, the position of the mutation site in the standard double-stranded nucleic acid The present inventors have found that there is a problem that the change in the fluorescence value of the labeling substance, particularly the behavior of the change in the fluorescence value of the donor labeling substance, is affected by the above. FIG. 1 is a diagram schematically showing an example of the fluorescence behavior of the donor labeling substance when the genotype is different, and FIG. 2 is a schematic example of the fluorescence behavior of the acceptor labeling substance when the genotype is different. It is the figure shown in. First, for a gene mutation having three genotypes, a PCR product that is one genotype is used as a sample double-stranded nucleic acid, and a standard double-stranded nucleic acid (match) having the same genotype as the PCR product, A standard double-stranded nucleic acid having a genotype different from the PCR product (mismatch A) and a standard double-stranded nucleic acid having a genotype different from each other (mismatch B) are prepared, and each standard double-stranded nucleic acid is constituted. Of the two nucleic acid strands, the 3 ′ end of one strand was labeled with a donor labeling substance, and the 5 ′ end of the other strand was labeled with an acceptor labeling substance. After mixing and denaturing each sample double-stranded nucleic acid and each standard double-stranded nucleic acid, the temperature of the reaction solution was lowered from 95 ° C. to 35 ° C., and the fluorescence intensity at each temperature was measured. The fluorescence value of the donor labeling substance differs greatly between the nucleic acid (mismatch A) and the standard double-stranded nucleic acid (mismatch B). The fluorescence intensity of the standard double-stranded nucleic acid (mismatch B) is particularly high at the end point (35 ° C). The fluorescence intensity of the standard double-stranded nucleic acid (mismatch A) is significantly larger than that of the standard double-stranded nucleic acid (match). Thus, the fluorescence intensity may be affected by the type of base of the genotype. In the genotype identification method of the present invention, the ratio between the fluorescence value of the donor labeling substance and the fluorescence value of the acceptor labeling substance is obtained. In some cases, this does not lead to correction of data variation.

  Therefore, as a result of further studies, the present inventor has found that not only the fluorescence intensity after the chain recombination reaction (end point) but also the amount of change in the fluorescence intensity due to the temperature drop of the reaction solution, that is, the state of a single strand by denaturation It was found that the variation between measurements can be well corrected by correcting based on the amount of change ΔF (fluorescence) between the fluorescence intensity in and the fluorescence intensity in the double-stranded nucleic acid state after annealing. ΔF may be a change amount of the donor labeling substance or a change amount of the acceptor labeling substance. Specifically, ΔF of the donor labeling substance can be obtained by the following formula (1). Similarly, ΔF of the acceptor labeling substance can be obtained by the following formula (2). In the following formulas (1) and (2), “F [start-point]” is the fluorescence intensity at the temperature at the start of the temperature drop of the reaction solution, and “F [end-point]” is the end of the temperature drop of the reaction solution. It means the fluorescence intensity at the temperature at the time.

  In addition, ΔF can be obtained by the following formula (3) for any of the donor labeling substance and the acceptor labeling substance. In the following formula (3), “F [max]” means the highest fluorescence intensity in the temperature-dependent fluorescence behavior from the start to the end of the temperature drop of the reaction solution, and “F [min]” It means the lowest fluorescence intensity in the temperature-dependent fluorescence behavior.

  FIG. 3 shows PCR products (sample double-stranded nucleic acid), standard double-stranded nucleic acid (match), standard double-stranded nucleic acid (mismatch A), and standard double-stranded nucleic acid (mismatch B) in the same manner as FIG. It is the figure which showed (DELTA) F of the donor labeling substance calculated | required based on the said Formula (1) at the time of preparing and performing competitive strand recombination reaction. In FIG. 3, the ΔF of the standard double-stranded nucleic acid (match) has a negative value because of the change in the fluorescent substance due to the temperature-dependent pH change of the Tris buffer or the temperature dependence of the fluorescent substance. is there. In particular, when fluoracein is used as the donor labeling substance, the behavior is such that ΔF becomes a negative value. That is, the ΔF value when the genotype is different from the sample double-stranded nucleic acid (in the case of mismatch) is large, and the ΔF value when the sample double-stranded nucleic acid and the genotype are the same (in the case of match) is minus 0 A value close to. Conversely, when the ΔF value of the acceptor labeling substance is used as an index, the ΔF value when the genotype is the same as that of the sample double-stranded nucleic acid is large, and the ΔF value when the genotype is different from the sample double-stranded nucleic acid is The value is close to 0 from minus. The genotype can be identified by calculating these ΔF values.

  When the standard double-stranded nucleic acid after denaturation returns to the original double-stranded nucleic acid without the strand recombination reaction, the fluorescence of the donor labeling substance after the strand recombination reaction is weak, and the acceptor in the single-stranded state Although the fluorescence of the labeling substance is weak, the variation in ΔF is not as great as the ratio between the fluorescence value of the donor labeling substance and the fluorescence value of the acceptor labeling substance, and it was found that the variation between measurements can be corrected well.

  Although the measured value of the fluorescence intensity tends to vary depending on the type of the measuring device, it is difficult to correct the variation between the measuring devices by the correction using ΔF. Therefore, the present inventor prepared a control reaction solution that does not contain a double-stranded nucleic acid that is not labeled with a labeling substance among the sample double-stranded nucleic acid and the standard double-stranded nucleic acid, and the fluorescence due to the temperature drop of the control reaction solution. It has been found that the variation in the measurement equipment can be further reduced by using the intensity change ΔF. This is because ΔF of the control reaction solution serves as an external standard at the time of fluorescence measurement by referring to each measurement or each measurement device.

  That is, when a standard double-stranded nucleic acid is labeled with a labeling substance, a sample containing no standard double-stranded nucleic acid but containing a standard double-stranded nucleic acid is used as a control reaction solution, and ΔF of the reaction solution is compared with ΔF of the control reaction solution. By measuring the ratio, the degree to which strand recombination has occurred between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid is measured. Specifically, this ratio can be obtained by the following equation (4). In the formula (4), “ΔF [reaction solution]” means the fluorescence change amount ΔF of the reaction solution, and “ΔF [control reaction solution]” means the fluorescence change amount ΔF of the control reaction solution.

  The degree of fluorescence resonance energy transfer of only the labeled double-stranded nucleic acid, that is, ΔF [control reaction solution] is assumed to be 100%, and the degree of recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid can be determined. it can. When standard double-stranded nucleic acid and sample double-stranded nucleic acid are mixed and subjected to strand recombination reaction, if the INDEX value is close to 100%, this indicates that strand recombination has not occurred, and sample double strand The genotype of the nucleic acid is identified as different from the standard double stranded nucleic acid. On the other hand, when the INDEX value is close to 0%, it indicates that strand recombination has occurred, and the genotype of the sample double-stranded nucleic acid is identified as being the same as that of the standard double-stranded nucleic acid.

  In addition, as described above, in the strand recombination reaction, it is important to slowly lower the temperature of the reaction solution, but the problem that the strand recombination reaction takes a very long time by lowering the cooling rate. There is also. Therefore, when measuring the degree of energy change due to energy transfer between the labeled substances to measure the degree of strand recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid, labeling is performed in advance. The melting curve of the double-stranded nucleic acid is drawn, and the average change rate with respect to temperature (dF / dT; change amount of fluorescence intensity with respect to temperature) of the first labeled substance or the second labeled substance is obtained, and this average change rate In the temperature range where the temperature is large, the temperature drop rate of the reaction solution is made sufficiently small, and in other temperature ranges, the temperature drop rate is higher, so that the strand recombination reaction can be performed in a short time without impairing the discrimination accuracy in the strand recombination reaction. be able to.

  Specifically, only in a temperature range of ± 3 to 5 ° C. of the temperature at which the average rate of change of the fluorescence intensity with respect to the temperature is maximized, a sufficiently low cooling rate, for example, 0.1 ° C./min to 0.3 ° C. Minute, more preferably a very slow temperature decrease rate of 0.1 ° C./minute, and a faster temperature decrease rate in other temperature ranges. By grasping the temperature in the vicinity of the phase transition of the double-stranded nucleic acid labeled in advance, efficient measurement can be performed, which leads to shortening of the measurement time.

  In addition, if the temperature is lowered at a high speed even near the phase transition, even if the genotypes of the sample double-stranded nucleic acid and the standard double-stranded nucleic acid are different, a strand recombination reaction may occur by mistake, resulting in a mismatch. Response, that is, in the case of fluorescent PCR-PHFA, the degree of fluorescence resonance energy transfer becomes smaller than the original, which may lead to false positives.

  In the second identification method of the present invention, when a genotype in gene mutation is identified using the PCR-PHFA method, the standard double-stranded nucleic acid used for the competitive strand recombination reaction is other than the mutation site, Competitive strand recombination by using a partial region consisting of bases, or a continuous or non-contiguous partial region of 2 or more bases modified to a base that is non-complementary to the natural gene containing the gene mutation This is a method for improving the base sequence identification accuracy in the reaction. The reason why the identification accuracy of the base sequence can be improved by providing a standard double-stranded nucleic acid with a site modified to a base (base sequence) that is non-complementary to the natural gene is not clear. Regardless of the genotype, the affinity between the sample double-stranded nucleic acid and the standard double-stranded nucleic acid is reduced, and as a result, the recombination reaction between the standard double-stranded nucleic acid and the nucleic acid strand having a different base sequence is remarkably suppressed. It is guessed that

  In the present invention and the specification of the present application, the “natural gene” means a gene that an organism has without being artificially modified. That is, in the second identification method of the present invention, two standards that are modified in the base species of the region other than the mutation site (that is, the region having the same nucleotide sequence in all genotypes of the gene mutation to be identified). Use strand nucleic acid.

  For example, in the case of a natural gene, when there is a site that forms a base pair with a relatively strong force, such as a GC-rich region (a region having a high content of guanine or cytosine), in the vicinity of the mutation site, Due to the high affinity of this site, it is difficult to recognize a difference of one base, and even nucleic acid strands having different genotypes at the mutation site are likely to hybridize. Therefore, by changing the base type of the site forming the base pair with such a strong force to a non-complementary base type, the affinity between the single-stranded nucleic acids of the site decreases, When the genotype of a sample-derived double-stranded nucleic acid is different from that of a standard double-stranded nucleic acid, the standard double-stranded nucleic acid can be remarkably suppressed from being recombined with the sample-derived nucleic acid strand by mistake. it can.

  In the present invention, in particular, a site that is guanine or cytosine in a natural gene is modified to adenine or thymine (uracil in the case of forming an RNA strand or a DNA-RNA chimeric strand) in a standard double-stranded nucleic acid. Is preferred.

  Further, the site for modifying the base species in the standard double-stranded nucleic acid (hereinafter referred to as the base species-modified site) is not particularly limited as long as it is other than the mutation site, and the standard double-stranded nucleic acid or the sample double-stranded nucleic acid is not limited. It can be determined appropriately in consideration of the type of the base sequence. The base type modification site may be a partial region consisting of one base or a continuous partial region of two or more bases. Further, the number of base species modification sites contained in one standard double-stranded nucleic acid may be one, or two or more. For example, a plurality of base species modification sites consisting of one base or two or more bases may be present discontinuously in a standard double-stranded nucleic acid.

  A standard double-stranded nucleic acid having a base species modification site can be prepared by known chemical synthesis in the same manner as a standard double-stranded nucleic acid having a nucleotide analog modification site. In addition, it can be prepared by using a nucleic acid amplification reaction generally used for introducing a mutation into a nucleic acid. For example, a standard double-stranded nucleic acid having a base type modification site is prepared by performing PCR using a nucleic acid derived from a natural gene as a template and using a primer or the like whose base type modification site is modified to a desired base type. Can do. Furthermore, the nucleic acid amplification reaction product is incorporated into a vector selected from a plasmid vector, a phage vector, or a chimeric vector of a plasmid and a phage, and introduced into any proliferative host such as bacteria such as E. coli and Bacillus subtilis or yeast. It can also be prepared in large quantities (gene cloning).

  Specifically, the second identification method of the present invention includes a nucleic acid amplification step of amplifying a region containing a mutation site in a gene contained in a sample by a nucleic acid amplification reaction to prepare a sample double-stranded nucleic acid, and the mutation A standard double-stranded nucleic acid of a specific genotype is mixed with the sample double-stranded nucleic acid to perform a competitive strand recombination reaction, and a strand recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid. And a discriminating step for identifying the identity between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid by measuring the degree of occurrence. The nucleic acid amplification step and the identification step in the second identification method of the present invention can be performed in the same manner as in the first identification method of the present invention described above.

  In the second identification method of the present invention, the site of the sample double-stranded nucleic acid corresponding to the base type modification site in the standard double-stranded nucleic acid is also modified to the same base sequence as that of the standard double-stranded nucleic acid. It is preferable. This is because the identification accuracy can be further improved by reducing the affinity between the nucleic acid strands of the sample double-stranded nucleic acid as well as the standard double-stranded nucleic acid at a site other than the mutation site.

  In this way, the sample double-stranded nucleic acid in which the base species corresponding to the base species-modified site in the standard double-stranded nucleic acid has been modified is used to introduce mutations into the nucleic acid in the same manner as the standard double-stranded nucleic acid. It can be prepared by using a commonly used nucleic acid amplification reaction. Specifically, in the nucleic acid amplification step, it is prepared by performing PCR using a primer having a modified site obtained by modifying the base species corresponding to the base species modified site to the same base sequence as the standard double-stranded nucleic acid. be able to. In addition, it is preferable that the said modification location is located in the place 2 bases or more away from the 3 'end of the primer used for nucleic acid amplification of a sample double stranded nucleic acid.

  Furthermore, the second identification method of the present invention may be combined with the first identification method of the present invention. That is, as a standard double-stranded nucleic acid, a mutation site is modified with a nucleotide analog, and a site other than the mutation site is modified to a non-complementary base (preferably, a site that is a guanine or cytosine in a natural gene is adenine, By changing to thymine or uracil), the genotype identification accuracy can be further improved.

  The gene identification method of the present invention has very good genotype identification accuracy, and not only germ cell mutations such as SNP but also somatic mutations observed in cancer cells etc. with sufficient accuracy. It is possible to identify.

The gene identification method of the present invention is useful in clinical examinations and the like because of its high identification accuracy.
For example, K-ras is a signal transduction system protein and a proto-oncogene. It has been reported that mutations have occurred in the K-ras gene in many cancer cells. In particular, mutations involving amino acid substitutions are remarkably observed in codons 12 and 13 of the K-ras gene, and it is known that 13 types of mutation patterns exist. Recently, it has been clarified one after another that EGFR antibody drugs (cetuximab, panitumumab), etc., which are anticancer agents, cannot exert their efficacy in patients with mutations in the K-ras gene. Such anticancer drug treatment requires not only side effects but also high costs. Therefore, it has been proposed as a part of custom-made medicine to test K-ras mutation before treatment and to select and treat only effective patients.

  Also, cetuximab, which is an EGFR antibody drug, is used as a colorectal cancer therapeutic drug. The annual incidence of colorectal cancer was just under 100,000, and the number of deaths in 2005 was 40,800. There is a tendency for the EGFR antibody drug manufacturer to estimate that 40,000 colorectal cancer patients will be the target of EGFR antibody drug treatment in 4 years, due to the increasing trend of eating habits in the West. If the estimate is correct, the K-ras inspection market is expected to exceed 400 million yen in four years in Japan alone.

  However, the conventional identification method has a problem that it is difficult to identify somatic mutations with sufficient accuracy, and there are many false positives. Since the gene identification method of the present invention can identify somatic mutations with very high accuracy, it can be expected not only to improve accuracy in clinical examinations but also to reduce medical costs.

  According to the first identification method of the present invention, the genotype identification kit of the present invention identifies the genotype of a gene mutation contained in a sample or detects the proportion of a specific genotype. A nucleic acid amplification reagent for preparing a sample double-stranded nucleic acid, two kinds of labeling substances capable of energy transfer with each other, and a labeling substance at the 3 ′ end of the nucleic acid chain And a reagent for introducing a labeling substance into the 5 ′ end of the nucleic acid strand. In addition, the kit may be combined with a cell destruction reagent for sample pretreatment, a reagent for detecting the label of the labeling substance, or the like. Thus, genotype discrimination can be performed more easily and in a short time by using a kit necessary for the first identification method of the present invention.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not limited to the following Example.

[Example 1]
The identification accuracy of the first identification method of the present invention was examined using the gene mutations of codon 12 and codon 13 of the oncogene K-ras as the mutation site to be identified. The standard double-stranded nucleic acid and the sample double-stranded nucleic acid to be used were prepared by a conventional chemical synthesis method.
First, standard double-stranded nucleic acids of the genotypes shown in Table 1 were prepared for the genotypes of the gene mutations of codon 12 and codon 13, respectively. Each standard double-stranded nucleic acid was labeled with FAM (Glen Research) at one 5 ′ end and Alexa labeled (manufactured by Invitrogen) at the other 3 ′ end. The standard double-stranded nucleic acid of each genotype can be prepared by PCR by designing primers, but in this example, the chemically synthesized two nucleic acid strands are hybridized one by one. It was prepared by. Table 1 shows the sequences of chemically synthesized nucleic acid strands for each genotype.
In Table 1, codons 12 and 13 are underlined, and mutation sites are shown in lower case. “Wild” indicates the wild type, “6-FAM” indicates the FAM label, “Ale594” indicates the Alexa label, and the numbers in the right column indicate the corresponding sequence numbers in the sequence listing.

  Among the standard double-stranded nucleic acids listed in Table 1, those obtained by modifying the mutation sites of the standard double-stranded nucleic acids of genotypes G12D and G12S_2 [standard double-stranded nucleic acids (G12D (LNA)) and standard double-stranded nucleic acids ( G12S_2 (LNA))] was also produced. Furthermore, double-stranded nucleic acids having a genotype of G12D or G12S_2 were prepared as sample double-stranded nucleic acids [sample double-stranded nucleic acid (G12D) and sample double-stranded nucleic acid (G12S_2)]. In addition, each sample double-stranded nucleic acid was also prepared by hybridizing the chemically synthesized two nucleic acid strands one by one in the same manner as the standard double-stranded nucleic acid. Table 2 shows the sequences of chemically synthesized nucleic acid chains for each genotype. In Table 2, codons 12 and 13 are underlined, and mutation sites are shown in lower case. The numbers in the right column indicate the corresponding sequence numbers in the sequence listing.

  First, a reaction solution in which any one of the standard double-stranded nucleic acids shown in Table 1 and any sample double-stranded nucleic acid shown in Table 2 was mixed was prepared to have the composition shown in Table 3. Each prepared reaction solution is set in a fluorescence intensity (fluorescence resonance energy transfer) measuring device, denatured by holding at 95 ° C. for 5 minutes, and then at each temperature in the temperature range of 85 ° C. to 75 ° C. for 3 minutes. The temperature was lowered while being held, further lowered from 75 ° C. to 35 ° C., and held at 35 ° C. for 5 minutes. In addition, the apparatus of ABI7900 was used for the fluorescence intensity measuring apparatus. Further, the amount of change ΔFAM in the fluorescence intensity of FAM was determined by the following formula (5). In Formula (5), “FAM [95 ° C.]” is the fluorescence intensity immediately before the temperature of the reaction solution drops from 95 ° C., and “FAM [35 ° C.]” is the fluorescence intensity at the end point.

FIG. 4 shows a sample double-stranded nucleic acid (G12D), a standard double-stranded nucleic acid (G12D), a standard double-stranded nucleic acid (G12D (LNA)), a standard double-stranded nucleic acid (G12S_2), or a standard double-stranded nucleic acid. It is the figure which showed the result of having mixed (G12S_2 (LNA)) and performing chain recombination reaction. Since FAM is a donor-labeled molecule, theoretically, in the case of a standard double-stranded nucleic acid (G12S_2) and a standard double-stranded nucleic acid (G12S_2 (LNA)) that differ in genotype from the sample double-stranded nucleic acid, the chain No recombination reaction occurs, fluorescence resonance energy transfer occurs, and the fluorescence intensity of FAM decreases. On the other hand, in the case of a standard double-stranded nucleic acid (G12D) and a standard double-stranded nucleic acid (G12D (LNA)) that have the same genotype as the sample double-stranded nucleic acid, a chain recombination reaction occurs, so the fluorescence intensity of FAM Does not change much.
However, in actuality, in the standard double-stranded nucleic acid (G12S_2), the fluorescence behavior of the donor (FAM) is not inflected, that is, no quenching phenomenon is observed, and the mutation site cannot be recognized and the strand recombination reaction is erroneously performed. Indicates what happened. In contrast, in the standard double-stranded nucleic acid (G12S_2 (LNA)) in which the mutation site was modified with LNA, it was observed that FAM was quenched in a temperature-dependent manner. These results suggest that the mismatch discrimination ability was significantly improved by LNA modification.

  FIG. 5 shows a sample double-stranded nucleic acid (G12S_2), a standard double-stranded nucleic acid (G12D), a standard double-stranded nucleic acid (G12D (LNA)), a standard double-stranded nucleic acid (G12S_2), or a standard double-stranded nucleic acid. It is the figure which showed the result of having mixed (G12S_2 (LNA)) and performing chain recombination reaction. As in FIG. 4, the standard double-stranded nucleic acid (G12D) has no quenching phenomenon despite the fact that the genotype differs from that of the sample double-stranded nucleic acid. A recombination reaction was taking place. On the other hand, in the standard double-stranded nucleic acid (G12D (LNA)) in which the mutation site was modified with LNA, it was observed that FAM was quenched in a temperature-dependent manner. By doing so, it was observed that the nucleic acid discrimination in the chain recombination reaction was improved.

Further, the sample double-stranded nucleic acid (G12D) and each standard double-stranded nucleic acid were mixed to perform a strand recombination reaction. Based on this result, ΔFAM of each reaction solution was calculated and compared. The ΔFAM of each reaction solution is shown in FIG. Theoretically, in the standard double-stranded nucleic acid (G12D) and the standard double-stranded nucleic acid (G12D (LNA)) that have the same genotype as the sample double-stranded nucleic acid, a strand recombination reaction occurs, and ΔFAM is close to 0. However, in other genotypes, chain recombination reaction does not occur and ΔFAM increases.
Actually, however, ΔFAM was small in the standard double-stranded nucleic acid (G12S_2), and it became a false positive. On the other hand, in the standard double-stranded nucleic acid modified with LNA (G12S_2 (LNA)), ΔFAM was large and negative.

  FIG. 7 is a diagram showing ΔFAM obtained by mixing a sample double-stranded nucleic acid (G12S_2) and each standard double-stranded nucleic acid and performing a strand recombination reaction. As a result, when the genotype of the standard double-stranded nucleic acid was G12D, wild type, G12A, or G12V, the value of ΔFAM was small and the result was false positive. On the other hand, even with the same genotype, ΔFAM was large and negative for the standard double-stranded nucleic acid modified with LNA (G12D (LNA)).

  That is, from these results, it has been clarified that, in the PCR-PHFA method, by modifying the standard double-stranded nucleic acid, particularly the mutation site, with LNA, mismatch discrimination can be greatly improved and the probability of false positive can be reduced. . In addition, with this modification, no false negative (a strand recombination reaction does not occur despite the same genotype as the standard double-stranded nucleic acid) was not observed.

  In K-ras test, it is very important not to give false positives. This is because a highly effective colorectal cancer molecular target drug or the like is not administered depending on the determination. In that sense, the genotype identification method of the present invention is very important when considering somatic mutation detection by the PCR-PHFA method.

  Since the genotype identification method of the present invention has very good genotype identification accuracy, it can be used in the field of clinical examination, particularly in the field of somatic mutation examination.

Claims (7)

A method for identifying a genotype in a genetic mutation, comprising:
A nucleic acid amplification step of amplifying a region containing a mutation site in a gene contained in a sample by a nucleic acid amplification reaction to prepare a sample double-stranded nucleic acid;
A standard double-stranded nucleic acid whose mutation site is a specific genotype and labeled with a labeling substance is mixed with the sample double-stranded nucleic acid to perform a competitive strand recombination reaction, An identifying step of identifying the identity between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid by measuring the degree of strand recombination between the sample double-stranded nucleic acid and
Have
And, Ri least one base nucleotide analogs der mutation sites of the standard double-stranded nucleic acid,
Only the mutation site in the standard double-stranded nucleic acid is composed of nucleotide analogs,
Method of identifying genotypes said nucleotide analog is characterized locked nucleic acid (LNA) der Rukoto.   2. The method for identifying a genotype according to claim 1, wherein any mutation site of the two nucleic acid strands constituting the standard double-stranded nucleic acid is constituted by a nucleotide analog. Of the two nucleic acid strands constituting the standard double-stranded nucleic acid, the 3 ′ end of one strand is labeled with the first labeling substance, and the 5 ′ end of the other strand is labeled with the second labeling substance. And
The first labeling substance and the second labeling substance are substances capable of transferring energy to each other,
By measuring the degree of energy change due to energy transfer between the first labeling substance and the second labeling substance, strand recombination occurred between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid in the discrimination step. The method for identifying a genotype according to claim 1 or 2, wherein the degree is measured. At least one of the first labeling substance and the second labeling substance is a fluorescent substance;
The competitive strand recombination reaction in the identification step is performed by gradually lowering the temperature of the reaction solution containing the standard double-stranded nucleic acid and the sample double-stranded nucleic acid from a high temperature, The rate of temperature decrease in the temperature range of ± 3 to 5 ° C. of the temperature at which the average rate of change in the fluorescence intensity of the 1st labeling substance or the second labeling substance with respect to the temperature is maximum is smaller than in the case of the temperature outside the temperature range 4. The method for identifying a genotype according to claim 3 . At least one of the first labeling substance and the second labeling substance is a fluorescent substance;
The competitive strand recombination reaction in the identification step is performed by gradually lowering the temperature of the reaction solution containing the standard double-stranded nucleic acid and the sample double-stranded nucleic acid from a high temperature,
The measurement to the extent that strand recombination has occurred between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid is measured based on the amount of change in fluorescence intensity due to the temperature drop of the reaction solution. 5. A method for identifying a genotype according to 3 or 4 . At least one of the first labeling substance and the second labeling substance is a fluorescent substance;
The competitive strand recombination reaction in the identification step is performed by gradually lowering the temperature of the reaction solution containing the standard double-stranded nucleic acid and the sample double-stranded nucleic acid from a high temperature,
In addition, the measurement of the degree of strand recombination between the standard double-stranded nucleic acid and the sample double-stranded nucleic acid does not include the amount of change in fluorescence intensity due to the temperature drop of the reaction solution and the sample double-stranded nucleic acid. The genotype identification method according to claim 3 or 4 , wherein the measurement is performed based on a ratio with a change amount of fluorescence intensity due to a temperature drop of a control reaction solution containing the standard double-stranded nucleic acid. A kit used to identify the genotype by the identification method of the genotype as claimed in claim 3-6,
A nucleic acid amplification reagent for preparing a sample double-stranded nucleic acid, two kinds of labeling substances capable of transferring energy to each other, a reagent for introducing a labeling substance at the 3 ′ end of the nucleic acid chain, and 5 ′ of the nucleic acid chain A reagent for introducing a labeling substance at the end , and a standard double-stranded nucleic acid whose mutation site is a specific genotype ,
The base of at least one mutation site of the standard double-stranded nucleic acid is a nucleotide analog;
Only the mutation site in the standard double-stranded nucleic acid is composed of nucleotide analogs,
Genotype identification kit said nucleotide analog is characterized locked nucleic acid (LNA) der Rukoto.

Images