Method for detecting mutant gene

Abstract

The purpose of the present invention is to provide a method for detecting a mutant gene that is capable of comprehensively and exhaustively measuring a gene mutation rate while keeping cost down. In a method for detecting a mutant gene according to the present invention, a base extension reaction is carried out using each of a first dye set and a second dye set, the first dye set is configured such that one of AT is not fluorescently labeled and the other is fluorescently labeled and GC are fluorescently labeled, the second dye set is configured such that one of GC is not fluorescently labeled and the other is fluorescently labeled and AT are fluorescently labeled, and the first dye set and the second dye set are both configured so that the base extension reaction terminates a base that is fluorescently labeled, not at a base that is not fluorescently labeled (see fig. 6).

Description

Mutant gene detection method

[0001] The present invention relates to a method for detecting a mutant gene in a DNA sample.

[0002] It has become clear that of the approximately 20,000 genes, only a few hundred are oncogenes or tumor suppressor genes. Gene mutations that cause cancer in humans are still being discovered, but it is predicted that the number will ultimately remain at around a few hundred. In fact, the OncoGuide NCC Oncopanel for next-generation sequencers, jointly developed by the National Cancer Center Research Institute and Sysmex, which was covered by insurance in 2019, contains 124 genes. Furthermore, the Foundation-one CDx, developed by Foundation Medicine in the United States, contains 324 genes. These developments demonstrate the advancement of cancer diagnosis by detecting the behavior of a limited number of genes, a few hundred in number. Among these genes, point mutations are particularly important. This is because cancer develops and progresses through the accumulation of random point mutations.

[0003] However, these tests are inevitably expensive because they use a method called next-generation sequencing (NGS), which decodes genes in a massive parallel manner. In fact, the cost of the two cancer gene panel tests mentioned above is 560,000 yen, which is a heavy burden for the average patient.

[0004] We want to detect genetic mutations with high sensitivity, multiplexing, and low cost. For example, high sensitivity means ≤0.1% MT / WT (mutant / wild type), multiplexing means 30 mutations, and low cost means 10,000 yen per measurement. NGS requires approximately 600,000 yen per measurement, which does not meet the low-cost requirement. ddPCR (Droplet Digital PCR) can multiplex at most 10 genes per measurement, which does not meet the multiplex requirement. qPCR (quantitative PCR) has a sensitivity of ≤1%, which does not meet the high sensitivity requirement.

[0005] SNaPshot, which uses a capillary sequencer, is a technology that enables multiplexed mutation detection while satisfying low cost requirements. SNaPshot employs a method in which a primer is hybridized immediately before the mutated base of an amplified DNA template, incorporating a fluorescent ddNTP (dideoxynucleotide triphosphate) that has been fluorescently labeled in advance. This is called the single-base extension method, and comprehensive mutation detection can be achieved by multiplexing primers for the extension reaction. However, a drawback is the low sensitivity, with a mutation detection rate of approximately 5%.

[0006] TrimGen employs a method called the Shifted Termination Assay. As shown in Figure 1 of Non-Patent Document 1, this method is characterized by the following points. While Single Base Extension adds only one base to a primer hybridized to a DNA template, in Shifted Terminator Assay, if the corresponding base mutation is present, the base extension reaction stops at that point, and if not, the extension reaction proceeds. Similarly, in Figure 1 of Non-Patent Document 2, the primer is hybridized to the template, and then the base extension reaction is carried out. In the TrimGen system, if the template is wild-type, the base extension reaction stops at the first 1 bp and no color development occurs. If the template is mutant-type, the base extension reaction continues, resulting in an extension of 2 bp or more and color development. This allows the wild-type and mutant types to be distinguished. That is, by introducing a base length difference between the wild type and the mutant type, it is possible to distinguish between the two. As shown on page 5 of Non-Patent Document 3, more base extension reactions occur in the wild type than in the mutant type. This makes it easier to separate the peaks of the mutant and wild type when their respective reaction products are electrophoresed.

[0007] Patent Document 1 also describes a primer extension reaction, and describes a method in which a base extension reaction is carried out using a combination of four types of dNTPs and one type of ddNTP.

[0008] Patent Document 2 describes an example of fluorescent labeling. 0074 of the same document describes an example in which, for example, the same label is attached to A and G, a second label is attached to T, and a third label is attached to C, thereby distinguishing between A and G. 0074 of the same document further describes an example in which, for example, a first label is attached to A and G and a second label is attached to T and C, and then the labeling configuration is reversed to attach the first label to A and C and the second label to G and T.

[0009] US6824980B2 Special Table No. 2011-515102

[0010] A new technology for mutation detection, Ann NY Acad Sci. 2004 Jun:1022:257-62.A colorimetric method for detection of K-ras codon 12 point mutations in DNA extracted from tissue and peripheral blood in pancreatic disorders, Biochem Genet, 2010 Aug;48(7-8):577-89Trimgen Mutector PIK3CA User Manual V1.5

[0011] TrimGen's Multi Base Extension method can introduce a base length difference of 1-2 bases between MT and WT. This improves the detection sensitivity for mutation rates. Specifically, it is possible to detect mutations up to 1%. However, of the four types of bases contained in TrimGen's extension reaction reagent, only one base is an intact dNTP not labeled with a fluorescent dye, and the remaining three are presumably fluorescently labeled ddNTPs. Therefore, the types of bases that can introduce base differences between MT and WT are limited to two types: the base in question and its complementary base, making it difficult to comprehensively perform reactions for multiple different mutant genes. In other words, multiplexing is considered difficult. Therefore, the TrimGen method makes it difficult to comprehensively measure gene mutation rates. Non-Patent Documents 2 and 3 and Patent Document 1 are considered to have similar issues.

[0012] The present invention has been made in consideration of the above-mentioned problems, and aims to provide a mutant gene detection method that can comprehensively and exhaustively measure gene mutation rates while keeping costs down.

[0013] The mutant gene detection method according to the present invention carries out a base extension reaction using a first dye set and a second dye set, respectively, wherein the first dye set is configured so that one of the ATs is not fluorescently labeled but the other is fluorescently labeled, and GC is fluorescently labeled; the second dye set is configured so that one of the GCs is not fluorescently labeled but the other is fluorescently labeled, and AT is fluorescently labeled; and both the first dye set and the second dye set are configured so that the base extension reaction does not terminate at bases that are not fluorescently labeled, but terminates at bases that are fluorescently labeled.

[0014] The mutant gene detection method according to the present invention makes it possible to measure the mutation rate of genes comprehensively and exhaustively while suppressing costs. Other objects, configurations, advantages, etc. of the present invention will become clear from the description of the following embodiments.

[0015] FIG. 1 is a schematic diagram illustrating a mutant gene detection method according to embodiment 1. FIG. 2 is a schematic diagram illustrating a mutant gene detection method according to embodiment 2. FIG. 3 is a schematic diagram illustrating a mutant gene detection method according to embodiment 2. FIG. 4 is a schematic diagram illustrating a mutant gene detection method according to embodiment 2. FIG. 5 is a schematic diagram illustrating a mutant gene detection method according to embodiment 3. FIG. 6 is a schematic diagram illustrating a mutant gene detection method according to embodiment 4. FIG. 7 is a schematic diagram illustrating a mutant gene detection method according to embodiment 6. FIG. 8 is a schematic diagram illustrating a mutant gene detection method according to embodiment 7. FIG. 9 is a schematic diagram illustrating a mutant gene detection method according to embodiment 9.

[0016] First Embodiment FIG. 1 is a schematic diagram illustrating a mutant gene detection method according to a first embodiment of the present invention. In this method, multiplex PCR is first performed simultaneously in a single tube for multiple point mutations, and then fluorescent labeling called Single Base Extension is performed on the amplified target mutant gene fragments. These DNA fragments are then electrophoresed in a capillary DNA sequencer. Wild-type peaks and mutant peaks for multiple target genes are measured in the obtained electropherogram. For each of these multiple peaks, wild-type and mutant signals are measured, allowing the mutation ratio for each target gene to be calculated. The labeling method used in this embodiment is generally called Single Base Extension, and is sold by Thermo Fisher under the reagent name SNaPShot.

[0017] More specifically, multiplex PCR is performed in the same tube for mutation sites 102, 103, 104, and 105 in genomic DNA 101 extracted from a sample, thereby enabling specific amplification of multiple desired target genes.

[0018] Next, a fluorescent labeling process called Single Base Extension is performed on the resulting amplified genes 110, 111, 112, and 113. Specifically, primers 106, 107, 108, and 109 are hybridized to the amplified genes 110, 111, 112, and 113, and the mutation state is detected by hybridizing the primers with polymerase in the presence of four dideoxynucleotides (ddATP, ddGTP, ddTTP, or ddUTP and ddCTP) labeled with different fluorescent dyes. This allows a single-base extension reaction to be performed.

[0019] After the Single Base Extension reaction, unnecessary nucleotides and fluorescent dyes are purified and removed using an ultrafiltration column or the like, and the reaction product is then subjected to electrophoresis using a capillary DNA sequencer. The electropherogram obtained thereby shows wild-type peak 114 and mutation-derived peak 118 for target gene 1, wild-type peak 115 and mutation-derived peak 119 for target gene 2, wild-type peak 116 and mutation-derived peak 120 for target gene 3, and wild-type peak 117 and mutation-derived peak 121 for target gene n. The number of target genes can be increased to n=30, n=100, or n=300.

[0020] 2A to 2D are schematic diagrams illustrating a mutant gene detection method according to a second embodiment of the present invention. Here, the structure of the GNAS gene in a base extension reaction for fluorescent labeling is described. The mutation site is a mutation at R843, c. 2609. In other words, the wild-type sequence, which is adenine A, changes to thymine T, guanine G, and cytosine C in other different mutants. Specifically, a wild-type DNA double strand is represented by sense strand 201 and antisense strand 202. Sense strand 201 contains a site where mutations frequently occur, called a hotspot. Sense strand 201 and antisense strand 202 are wild-type. Therefore, the sequence of the hotspot in sense strand 201 is adenine A 203, and the sequence in antisense strand 202 is thymine T 204.

[0021] On the other hand, MT1 will be described as an example containing a mutation as one of the three types of mutations. In MT1, the mutation is c. 2609A>T. Specifically, the DNA double strand of mutant MT1 is shown as sense strand 211 and antisense strand 212. There is a location in sense strand 211 where mutations frequently occur, called a hotspot. Sense strand 211 and antisense strand 212 are mutant types. The sequence of the hotspot in sense strand 211 is thymine T213, and the sequence in antisense strand 212 is adenine A214.

[0022] Similarly, the DNA double strands of mutant MT2 are shown as sense strand 221 and antisense strand 222. The sequence of the hotspot in sense strand 221 is guanine G223, and the sequence in antisense strand 222 is cytosine C224. Similarly, the DNA double strands of mutant MT3 are shown as sense strand 231 and antisense strand 232. The sequence of the hotspot in sense strand 231 is cytosine C233, and the sequence in antisense strand 222 is guanine G234.

[0023] The base extension reaction for fluorescent labeling will be explained using the sense strand 205 and antisense strand 207, which are wild-type PCR products. Antisense primer 206 and sense primer 208 can be hybridized to the sense strand 205 and antisense strand 207, respectively. The antisense primer 206 and sense primer 208 are hybridized exactly one base before the hot spot, respectively. This allows the presence or absence and state of mutation to be confirmed from the fluorescent dye incorporated during base extension.

[0024] Similarly, the mutant PCR product MT1 will be explained using sense strand 215 and antisense strand 217. Antisense primer 216 and sense primer 218 can be hybridized to sense strand 215 and antisense strand 217, respectively. Antisense primer 216 and sense primer 218 are hybridized exactly one base before the hot spot, respectively. This allows the presence or absence and state of a mutation to be confirmed from the fluorescent dye incorporated during base elongation.

[0025] Similarly, the mutant PCR product MT2 will be explained using sense strand 225 and antisense strand 227. Antisense primer 226 and sense primer 228 can be hybridized to sense strand 225 and antisense strand 227, respectively. Antisense primer 226 and sense primer 228 are hybridized exactly one base before the hot spot, respectively. This allows the presence or absence and state of a mutation to be confirmed based on the fluorescent dye incorporated during base elongation.

[0026] Similarly, the mutant PCR product MT3 will be explained using sense strand 235 and antisense strand 237. Antisense primer 236 and sense primer 238 can be hybridized to sense strand 235 and antisense strand 237, respectively. Antisense primer 236 and sense primer 238 are hybridized exactly one base before the hot spot, respectively. This allows the presence or absence and state of a mutation to be confirmed from the fluorescent dye incorporated during base elongation.

[0027] <Embodiment 3> Figures 3A and 3B are schematic diagrams illustrating a mutant gene detection method according to embodiment 3 of the present invention. In this embodiment, the Shifted Termination Assay adopted by TrimGen, Inc., is described, rather than the conventional single-base extension reaction. While Single Base Extension adds only one base to a primer hybridized to a DNA template, in the Shifted Terminator Assay, three of the four bases are fluorescently labeled ddNTPs, and the remaining nucleotide is an unlabeled dNTP. More specifically, this embodiment is characterized in that ddATP is labeled with a black fluorescent dye, ddGTP with a red fluorescent dye, and ddCTP with a blue fluorescent dye, as shown in the table. For thymine, intact, fluorescently unlabeled dTTP is used. A distinctive feature of dNTPs is that when a ddNTP is incorporated into a DNA chain during base elongation, the base elongation stops, whereas when a dNTP is incorporated, the base elongation does not stop and the next base continues to be incorporated.

[0028] There are sense strands 301, 302, 303, and 304 corresponding to the wild-type and mutant forms of GNAS R843. The hot spots in each sense strand are adenine 309, thymine 310, guanine 311, and cytosine 312. In addition, antisense primers 305, 306, 307, and 308 having sequences complementary to these sense strands 301, 302, 303, and 304 can be hybridized. Here, the antisense primers 305, 306, 307, and 308 have the same sequence. The 3' ends of the antisense primers 305, 306, 307, and 308 are positioned exactly one base before the complementary base of the hot spot.

[0029] To these primers, the aforementioned Dye Set containing ddATP labeled with a black fluorescent dye, ddGTP labeled with a red fluorescent dye, and ddCTP labeled with a blue fluorescent dye, and containing intact dTTP, and polymerase are added. The polymerase binds to the 3' ends of antisense primers 305, 306, 307, and 308 and incorporates bases that are relative to the hot spot and undergo Brownian motion in the solution.

[0030] Specifically, the antisense primer 306 incorporates adenine 314, which is the complementary base of the mutated thymine 310. In this case, the adenine is ddATP labeled with a black fluorescent dye, so the extension reaction stops upon incorporation of ddATP. In other words, no further base extension occurs.

[0031] Similarly, antisense primer 307 incorporates cytosine C315, which is the complementary base of mutated guanine 311. Since cytosine is ddCTP labeled with a blue fluorescent dye, the extension reaction terminates upon incorporation of ddCTP. In other words, no further base extension occurs.

[0032] Similarly, antisense primer 308 incorporates guanine G316, which is the complementary base of mutated cytosine 312. Since guanine is ddGTP labeled with a red fluorescent dye, the extension reaction terminates upon incorporation of ddGTP. In other words, no further base extension occurs.

[0033] The behavior of the extension reaction for antisense primer 305 differs from that of antisense primers 306 to 308. Since sense strand 301 is wild-type to begin with, the hot spot is also wild-type adenine 309. Thymine T, the complementary base of adenine 309, is intact and unlabeled dTTP. Therefore, the base extension reaction does not terminate upon incorporation of dTTP. Therefore, the next base is incorporated immediately after incorporation of dTTP. In this case, the corresponding base is guanine 313. Since guanine 313 is ddGTP labeled with a blue fluorescent dye, the base extension reaction terminates upon incorporation of guanine 313.

[0034] As a result of the reaction, antisense primer 305 differs from the other antisense primers 306, 307, and 308 in that the base length of antisense primer 305 is one base longer than the other antisense primers. This one-base longer base length has the effect of allowing the peaks of both to be separated without overlapping in capillary electrophoresis, as described below. In other words, it has the advantage of allowing the wild-type peak and the mutant-type peak to be measured separately. This advantage is more pronounced the lower the mutant-type ratio. Specifically, it is advantageous when the MT / WT ratio is low, such as 0.1%, in terms of low mutation. When the MT / WT ratio is 0.1%, the WT peak signal is approximately 1,000 times stronger than the MT peak signal.

[0035] One of the major challenges facing capillary DNA sequencers is pull-up and pull-down. These are noise components that arise when the pre-measured fluorescent dye spectrum during calibration does not completely match the fluorescence spectrum from the sample electrophoresed. It is generally known that pull-up and pull-down occur 3% of the time within ±1 base just below the peak signal, and this problem has not been fundamentally resolved to date. To detect mutations of 3% or less, it is necessary to measure the minute mutation signal at a different time or position from the large wild-type signal. Otherwise, the minute mutation signal will be mixed in with the pull-up or pull-down, making detection difficult. One way to avoid this is the method described in this embodiment, in which base extension is performed using three types of fluorescently labeled ddNTPs and one type of dNTP.

[0036] <Embodiment 4> Figure 4 is a schematic diagram illustrating a mutant gene detection method according to embodiment 4 of the present invention. Embodiment 4 is a more detailed explanation of embodiment 3. In embodiment 4, the shifted termination assay adopted by TrimGen, rather than the conventional single-base extension reaction, will be explained. Here again, the wild-type WT and mutant MT of GNAS R843 will be explained as examples.

[0037] An antisense primer 352 complementary to the sense strand 351 is hybridized. A polymerase 353 binds to the 3' end of the antisense primer 352. The polymerase 353 incorporates a base complementary to the sense strand 351 into the 3' end of the antisense primer 352. The solution contains ddGTP 355 labeled with a red fluorescent dye, ddATP 356 labeled with a black fluorescent dye, ddCTP 357 labeled with a blue fluorescent dye, and intact dUTP or dTTP 354. These fluorescently labeled ddNTPs and unlabeled dNTPs undergo Brownian motion in the solution, repeatedly colliding with and dissociating from surrounding molecules.

[0038] The sense strand 360 has a wild-type sequence at the mutation site. Polymerase 362 binds to the 3' end of antisense primer 361 hybridized with sense strand 360. The wild-type mutation site is adenine A, and its complementary base is thymine T. In this case, the corresponding thymine is unlabeled thymine dTTP 363, which polymerase 362 adds to the 3' end of antisense primer 361. Since the added thymine is intact, unlabeled thymine dTTP 363, the base extension reaction is not inhibited and proceeds as is. Therefore, polymerase 362 adds the next complementary base pair, ddGTP 364 labeled with a red fluorescent dye, to the 3' end of antisense primer 361. This reaction adds two bases to the original antisense primer 361.

[0039] Sense strand 370 has a mutant sequence at the mutation site. Polymerase 372 binds to the 3' end of antisense primer 371 hybridized with sense strand 370. The mutation site of the mutant is thymine T, and its complementary base is adenine A. Therefore, the base added to the primer by polymerase 372 is adenine A. In this case, the corresponding adenine is ddATP 374 labeled with a black fluorescent dye, and polymerase 372 adds ddATP 374 labeled with a black fluorescent dye to the 3' end of antisense primer 371. Finally, ddATP 378 labeled with a black fluorescent dye binds to the 3' end of antisense primer 371. Because the incorporated base is ddATP, the base extension reaction is inhibited. Therefore, polymerase 372 can no longer incorporate the next complementary base pair, and the extension reaction stops. As a result of this reaction, one base was added to the original antisense primer 376.

[0040] After the above extension reaction, the 3' end of wild-type primer 380 is ddGTP364 labeled with a red fluorescent dye, and the 3' end of mutant-type primer 381 is ddATP374 labeled with a black fluorescent dye. After the extension reaction, the base length difference between wild-type primer 380 and mutant-type primer 381 is one base. DNA fragments with a one-base difference can be separated as fragments using a conventional capillary DNA sequencer.

[0041] In a typical SNaPshot, the base lengths of the wild-type primer and the mutant primer are identical during a single-base extension reaction. Therefore, for example, when a mutant primer and a wild-type primer are electrophoresed, they appear as peaks at approximately the same position and time. In particular, when the ratio of mutant primer to wild-type primer is low, for example, 1% or 0.1%, the mutant primer will be present directly below the extremely strong signal from the wild-type primer. Meanwhile, in capillary sequencer analysis, false signals known as pull-up and pull-down are known to occur. These signals represent approximately 3% of the parent signal peak. Pull-up and pull-down are caused by a slight discrepancy between the pre-measured fluorescence spectrum of each fluorescent dye and the actual measured fluorescence spectrum. Because it is difficult to perfectly match these spectra, pull-up and pull-down remain an unresolved issue even in the measurement technology of capillary DNA sequencers that has been around since the 1990s.

[0042] One solution to avoiding the pull-up and pull-down problems is to separate the emission positions of the wild-type and mutant peaks. In other words, this involves changing the migration speeds of the two. To achieve this, fluorescent dyes with different mobilities can be used. Another possible approach is to improve the conventional single-base extension reaction and modify the reaction system so that multiple base extension reactions proceed. Embodiment 4 employs the latter approach, and its advantage is that it can introduce base differences between the mutant and wild-type samples without changing the existing fluorescent dyes. In contrast, the former approach requires searching for fluorescent dyes with different mobilities, which is costly and time-consuming.

[0043] 5 is a schematic diagram illustrating a mutant gene detection method according to a fifth embodiment of the present invention. In the fifth embodiment, a shifted termination assay that is assumed to be employed by TrimGen, instead of the conventional single-base extension reaction, will be described.

[0044] Multiplex PCR is performed for multiple DNA targets in one tube 401. The specific number of DNA targets is two or more, and in many cases is 20, 30, 100, or even as many as 500.

[0045] Next, the fluorescent labeling reaction proceeds via a base extension reaction. A first DyeSet 430 is used for the fluorescent labeling reaction. The first DyeSet 430 is composed of ddTTP (or ddUTP) labeled with a black fluorescent dye, ddGTP labeled with a blue fluorescent dye, ddCTP labeled with a red fluorescent dye, and dATP not labeled with a fluorescent dye. (a) Target DNA fragments 402, 404, 406, and 408; (b) sense primer 403, antisense primer 405, antisense primer 407, and antisense primer 409, which hybridize complementarily with the sense strand or antisense strand of the target DNA fragment, respectively; and (c) polymerase are coexisted in the reaction solution.

[0046] The wild-type base sequence in the hotspot region of the target DNA fragment 402, where mutant genes frequently occur, is adenine in the sense strand and thymine in the antisense strand. In contrast, if an extension reaction of two or more bases is desired in the wild-type sequence, the sense primer 403 can be hybridized to the antisense strand. Since the hotspot of the antisense strand is thymine, adenine, a base complementary to thymine, is incorporated at the 3' end of the sense primer 403. Because this adenine is dATP that is not labeled with the fluorescent dye in the first DyeSet, the extension reaction is not inhibited, and the extension reaction of the second base proceeds.

[0047] If a mutation occurs in the hot spot of the antisense strand, the corresponding base will be any of adenine, guanine, and cytosine bases other than thymine. Regardless of the base, since all of the corresponding nucleotides in the first DyeSet are ddNTPs labeled with fluorescent dyes, the base extension reaction is completed with one base, and no extension reaction of two or more bases occurs. In other words, in the above configuration, two-base extension can be selectively generated in the wild type, and one-base extension can be selectively generated in the mutant type. Therefore, a base length difference of one or more bases can be reliably introduced between the wild type and the mutant type.

[0048] When this antisense strand is subjected to capillary DNA electrophoresis, the wild-type peak 421 and the mutant-type peak 422, which is one or more bases shorter than the wild-type peak 421, appear in an electropherogram separated and without overlapping. Even when the amount of mutation is minute and the mutant-type peak 422 is extremely small, the mutant-type peak 422 is not affected by the pull-up or pull-down of the large wild-type peak 421, making it possible to detect a low-mutation gene. Note that detection of a low-mutation gene here means detection of a mutant gene when the amount of the mutant-type gene is 1%, 0.1%, 0.01%, or less, for example, when the amount of the wild-type gene is 100%. The same applies hereinafter.

[0049] In the target DNA fragment 402, the wild-type base sequence in the sense strand is adenine. When an antisense primer is hybridized to this and a base extension reaction is carried out, the base incorporated is thymine, which is complementary to adenine. Since thymine is ddTNP labeled with a black fluorescent dye in the first DyeSet, in this case, an extension reaction of two or more bases cannot proceed in the wild-type base sequence. As can be seen from this example, in order to generate base extension of two or more bases in the wild-type base sequence, care must be taken when selecting whether the hybridizing primer is a sense primer or an antisense primer.

[0050] The sense primer 403 and the antisense primers 405, 407, and 409 are all different in primer length. Specifically, it is desirable that the difference in length between these primers be at least three bases. It is possible to introduce differences of 5, 10, 20, or more bases. It is desirable to introduce a base length difference of at least two bases for each different allele. This is done so that the fluorescent signals emitted from each gene target due to the different primer lengths can be detected in a separated state without overlapping during electrophoresis.

[0051] The wild-type base sequence in the hotspot region of the target DNA fragment 406 where mutant genes frequently occur is thymine in the sense strand and adenine in the antisense strand. To generate a base extension reaction of two or more bases in a wild-type mutation, an antisense primer 407 can be hybridized to the sense strand. Since the hotspot of the sense strand is thymine, adenine, a base complementary to thymine, is incorporated into the 3' end of the antisense primer 407. Since the adenine corresponds to dATP in the first DyeSet 430 that is not labeled with a fluorescent dye, the extension reaction is not inhibited, and the extension reaction of the second base occurs.

[0052] When this sense strand is subjected to capillary DNA electrophoresis, the wild-type peak 425 and the mutant-type peak 426, which is one or more bases shorter than the wild-type peak 425, appear in an electropherogram in a separated form without overlapping. Even if the mutation is minute and the mutant-type peak 426 is extremely small, the mutant-type peak 426 is not affected by the pull-up or pull-down of the large wild-type peak 425, making it possible to detect a low-mutation gene.

[0053] Consider the case where a sense primer is hybridized to the antisense strand of the target DNA fragment 406 and an extension reaction occurs. Since the hot spot in the wild-type antisense strand is adenine, thymine, a base complementary to adenine, is incorporated. Since the thymine in the first DyeSet 430 is ddTTP labeled with a black fluorescent dye, the extension reaction is inhibited and no extension reaction of the second base occurs. Therefore, when using the set of nucleotides contained in the first DyeSet 430, it is not possible to introduce a base length difference between the wild-type and mutant types in the sense primer.

[0054] Only when the mutation in the antisense strand is thymine, the corresponding complementary base becomes adenine, and a base length difference can be introduced relative to the wild type. If the mutation in the antisense strand is other than thymine, a base length difference cannot be introduced. In order to selectively generate a base extension of two or more bases for a wild type mutation, it is necessary to select a sense primer or antisense primer so that a nucleotide corresponding to a dNTP not labeled with a fluorescent dye is incorporated during primer extension. Failure to do so may result in a case where a long base chain cannot be introduced between the wild type and the mutant. When the reaction product after base extension is subjected to capillary DNA electrophoresis in a state where a long base chain cannot be introduced, the following situation may occur, for example.

[0055] In the electropherogram, the wild-type peak 423 and the mutant-type peak 424, which has the same base length, appear overlapping and difficult to separate. If the mutation is minute and the mutant-type peak 424 is extremely small, the mutant-type peak 424 will be affected by the pull-up and pull-down of the huge wild-type peak 423, making it impossible to detect the low-mutation gene. The same is true for the wild-type peak 427 and the mutant-type peak 428.

[0056] The problems of the fifth embodiment are described below. The fifth embodiment has a problem in detecting a mutation represented by the target DNA fragment 404. The wild-type base sequence in the hotspot region of the target DNA fragment 404 is guanine in the sense strand and cytosine in the antisense strand. In contrast, when it is desired to generate a base extension reaction of two or more bases in a wild-type mutation, it is conceivable to first hybridize an antisense primer 405 to the sense strand.

[0057] Since the hot spot of the sense strand is guanine, cytosine, a base complementary to guanine, is incorporated into the 3' end of the antisense primer 405. Since the dATP in the first DyeSet 430 is not labeled with a fluorescent dye, cytosine is required to generate an extension reaction of two or more bases in the wild type. However, since the cytosine in the first DyeSet 430 is ddCTP labeled with a red fluorescent dye, the extension reaction stops when the ddCTP labeled with the red fluorescent dye is incorporated into the 3' end of the primer.

[0058] In this case, since the wild-type DNA fragment cannot undergo an extension reaction of two or more bases, it is possible to hybridize a sense primer to the antisense strand. However, since the hot spot of the antisense strand is cytosine, when the sense primer is hybridized, guanine, a base complementary to cytosine, is incorporated. Since the guanine in the first DyeSet 430 is ddGTP labeled with a blue fluorescent dye, the base extension reaction is terminated. Therefore, as long as the first DyeSet 430 is used in the target DNA fragment 404, an extension reaction of two or more bases cannot occur for the primer hybridized to the wild-type DNA fragment.

[0059] When the reaction product after base extension, which cannot introduce a long base chain, is subjected to capillary DNA electrophoresis, the wild-type peak 423 and the mutant-type peak 424, which has the same base length, appear overlapping in the electropherogram and are difficult to separate. If the mutation is minute and the mutant-type peak 424 is extremely small, the mutant-type peak 424 will be affected by the pull-up and pull-down of the huge wild-type peak 423, making it impossible to detect the low-mutation gene.

[0060] Similarly, a case where an extension reaction of two or more bases cannot occur for a wild-type mutation will be described using target DNA fragment 408. The wild-type base sequence in the hotspot region of target DNA fragment 408 is cytosine in the sense strand and guanine in the antisense strand. On the other hand, if an extension reaction of two or more bases is desired for a wild-type mutation, it is possible to first hybridize antisense primer 409 to the sense strand.

[0061] Since the hot spot of the sense strand is cytosine, guanine, a base complementary to cytosine, is incorporated. Since the guanine in the first DyeSet 430 is ddGTP labeled with a fluorescent dye, the base extension reaction stops when ddGTP is incorporated. Therefore, an extension reaction of two or more bases cannot occur for the wild type.

[0062] Therefore, it is possible to hybridize a sense primer to the antisense strand. However, since the hot spot of the antisense strand is guanine, when a sense primer is hybridized, a cyanine base, which is complementary to guanine, is incorporated. Since the cyanine in the first DyeSet 430 is ddCTP labeled with a red fluorescent dye, the base extension reaction will be terminated. Therefore, as long as the first DyeSet 430 is used in the target DNA fragment 408, an extension reaction of two or more bases cannot occur for the primer hybridized to the wild-type.

[0063] As is clear from the above description, the fifth embodiment cannot reliably introduce a base length difference between the wild type and the mutant type. Unless the base complementary to the base to be detected matches the dNTP in the first DyeSet 430, the change in base length cannot be reflected in the extension reaction, resulting in a base length difference that cannot be separated by electrophoresis.

[0064] For simplicity, the above explanation has been given with reference to an example in which an extension reaction of two or more bases occurs for the wild type, and the extension reaction of only one base is stopped for the mutant type. Conversely, when an extension reaction of two or more bases occurs for the mutant type, only one type of mutation exists, and only the mutation having a base species complementary to a fluorescently labeled dNTP in its sequence can detect the wild type and the mutant type with high sensitivity. When there are two or more types of mutations, the other mutations, except for the mutation having a base species complementary to a fluorescently labeled dNTP in its sequence, incorporate fluorescently labeled ddNTP, so the reaction is completed by the extension of only one base. Similarly, the wild type is also completed by the extension of only one base. Therefore, when there are two or more types of mutations, the other mutations, except for the mutation having a base species complementary to a fluorescently labeled dNTP in its sequence, have the same base length as the wild type, making it difficult to separate the peaks in capillary electrophoresis without overlapping each other.

[0065] Even when there is only one type of mutation, base extension occurs in the mutation only when the base type of the dNTP in the first DyeSet 430 happens to be complementary to the base sequence of the mutation. When multiple target DNA fragments are the measurement targets, the base types of the mutations are random, making comprehensive and systematic detection of mutant bases difficult. Therefore, it is desirable to target the wild type when generating multiple base extension reactions of two or more bases. This is the reason why the wild type has been described as the target of extension reactions of two or more bases.

[0066] Non-Patent Documents 1 to 3 do not describe the selection of sense primers or antisense primers as shown in Figure 5. Furthermore, when it is desired to detect G or C, these documents require a separate dye set (a set in which G or C corresponds to dNTP) different from the first DyeSet. Therefore, it should be noted that these documents are thought to have problems in such cases.

[0067] 6 is a schematic diagram illustrating a mutant gene detection method according to a sixth embodiment of the present invention. The sixth embodiment overcomes the problems of the fifth embodiment. Specifically, it is possible to introduce a base length difference between any wild type and any mutant type. This provides a comprehensive mutation detection technology.

[0068] In embodiment 6, a first DyeSet and a second DyeSet are used in combination. The first DyeSet is composed of ddATP labeled with a black fluorescent dye, ddGTP labeled with a blue fluorescent dye, ddCTP labeled with a red fluorescent dye, and dATP not labeled with a fluorescent dye. The second DyeSet is composed of ddATP labeled with a blue fluorescent dye, ddTTP labeled with a black fluorescent dye, ddCTP labeled with a red fluorescent dye, and dGTP not labeled with a fluorescent dye.

[0069] In the first DyeSet and the second DyeSet, the dNTP not labeled with a fluorescent dye is either adenine or thymine in the first DyeSet, and either guanine or cytosine in the second DyeSet. In other words, one nucleotide from each of the two groups that form complementary base pairs, adenine and thymine (A-T) or guanine and cytosine (G-C), is used as the dNTP. By using the first DyeSet and the second DyeSet in combination and selecting and combining either a sense primer or an antisense primer as the type of primer for the hotspot, it becomes possible to introduce a base length difference in the extension reaction product for any mutation.

[0070] A PCR product that has undergone multiplex PCR is produced in the sample tube 701. In multiplex PCR, PCR is performed on multiple DNA targets in one sample tube 701.

[0071] Next, the process proceeds to an extension reaction in which fluorescent labeling is performed. The PCR product in sample tube 701 is divided or dispensed in the required amount into sample tubes 702 and 703. The PCR product in sample tube 702 and the PCR product in sample tube 703 contain multiple DNA targets at the same concentration, and are in the same solution state.

[0072] First, the extension reaction that proceeds in the sample tube 702 will be described. The target DNA fragment 704 has a thymine at the hot spot of the antisense strand. A sense primer 705 is hybridized to the antisense strand of the DNA fragment 704. The sense primer 705 hybridizes one base before the hot spot of the antisense strand of the DNA fragment 704. The wild-type base in the antisense strand is thymine. When the extension reaction proceeds, adenine, which is the complementary base of thymine, is incorporated as the wild-type base at the 3' end of the sense primer 705. Since the adenine in the first DyeSet is intact dATP that is not fluorescently labeled, the incorporated dATP does not stop the extension reaction, and base extension proceeds. On the other hand, since all bases incorporated in the mutant antisense strand are ddNTPs, extension is completed with only one base. Therefore, a difference in base length of one or more bases can be introduced between the wild type and the mutant type in the target DNA fragment 704.

[0073] A similar reaction can be performed on target DNA fragment 710. The difference from target DNA fragment 704 is that target DNA fragment 704 uses sense primer 705, while target DNA fragment 710 uses antisense primer 712. This is because the complementary strand of thymine, which is the hotspot of the antisense strand of target DNA fragment 710, is adenine. In other words, by hybridizing antisense primer 712 to target DNA fragment 710, the wild-type extension reaction can be extended to two or more bases. On the other hand, in the mutant type, the base species incorporated at the 3' end of antisense primer 712 is either ddTTP, ddGTP, or ddCTP, so the base extension reaction terminates upon incorporation of one base. Therefore, by employing antisense primer 712 and the first DyeSet, the mutant type is limited to one-base extension, while the wild-type can proceed with an extension reaction of two or more bases. In other words, a base length difference of one or more bases can be introduced between the wild-type and mutant types.

[0074] In the reaction using the first DyeSet, as described above, either a sense primer or an antisense primer is selected so that the dNTP in the first DyeSet matches the base type initially incorporated at the 3' end of the primer at the hotspot. This allows detection of two of the four bases for any hotspot mutation sequence by selecting an appropriate primer.

[0075] The extension reaction using the second DyeSet will be described. In the first DyeSet, a dNTP that is not labeled with a fluorescent dye is assigned to either the complementary base species adenine or thymine. In contrast, the second DyeSet assigns a dNTP that is not labeled with a fluorescent dye to either the complementary base species guanine or cytosine. By combining either the first DyeSet or the second DyeSet with either a sense primer or an antisense primer, it is possible to introduce a base length difference of one base into the extension reaction products generated for all different target DNA fragments. In other words, while there are four types of nucleotides, there are two types of first DyeSet and second DyeSet, and two types of primers, a sense primer and an antisense primer, and by combining the two, four types can be covered.

[0076] These reaction products are subjected to capillary electrophoresis, so it is desirable that each target gene has a different base length and that the elution times as fragments are different. To achieve this, for example, the base length difference between primers is set to 5 bases. For example, if there are 10 target genes, 10 genes can be positioned from 50 bp to 95 bp, every 5 bases. The hot spots in these genes are the base chains of one or more bases between the wild type and mutant type that are introduced as base extension occurs.

[0077] The extension reaction that proceeds in sample tube 703 will now be described. A target DNA fragment 706 identical to target DNA fragment 704 is present in sample tube 703. While sense primer 705 is present in sample tube 702, the corresponding sense primer 705 is not present in sample tube 703. This is because it is the reaction using the first DyeSet, not the reaction using the second DyeSet, that can introduce a base length difference between the wild type and mutant type for the hotspot to be detected.

[0078] Similarly, a sense primer 709 is present in sample tube 703, whereas the corresponding sense primer 709 is not present in sample tube 702. The reason for this is that it is the reaction using the second DyeSet, not the reaction using the first DyeSet, that can introduce a base length difference between the wild type and mutant type for the hotspot to be detected.

[0079] In the sense described above, the first DyeSet and the second DyeSet are complementary to each other. Furthermore, in each reaction using the first DyeSet and the second DyeSet, the sense primer and the antisense primer are complementary to each other. The number of combinations of the first DyeSet and the second DyeSet, and the sense primer and the antisense primer is 2 × 2 = 4, which corresponds to the four types of nucleotides. In other words, by examining the combinations of the first DyeSet and the second DyeSet, and the sense primer and the antisense primer, it is possible, in principle, to comprehensively and exhaustively perform mutation analysis for any base sequence.

[0080] In addition, to efficiently separate the base extension reaction products using capillary electrophoresis, it is necessary to separate the target base sequences, each of which has a hotspot, by shifting them. Even if 10 target genes are prepared, each with a difference in length of one base, if all of the base lengths are standardized to, for example, 50 bases, the signals of 50 bases, 51 bases, and 52 bases will overlap, making it impossible to extract information. Therefore, if the primers that form the basis of the base extension reaction are designed in advance to have lengths that differ by five bases from each other, the state of each hotspot can be displayed on the capillary array every five bases. By further examining the details of the profiles separated every five bases, the wild-type and mutant profiles can be further confirmed, enabling comprehensive and efficient measurement of the mutation rate at each hotspot. More specifically, the primer lengths of sense primer 705, sense primer 709, antisense primer 712, and antisense primer 715 are all different. For example, the base length difference may be in increments of 10 bases, such as 50 bp, 60 bp, 70 bp, and 80 bp. Alternatively, the base length difference may be in increments of 5 bases, such as 50 bp, 55 bp, 60 bp, and 65 bp. Alternatively, when there are a large number of target gene sequences, these base lengths may be arranged in 10 bp increments, for example, from 50 bp to 500 bp. Alternatively, these base lengths may be arranged in 5 bp increments, for example, from 50 bp to 500 bp. Alternatively, these base lengths may be arranged in 3 bp increments, for example, from 50 bp to 500 bp. Furthermore, the difference in primer length does not necessarily have to be constant; the spacing between some primers may be 3 bp, and the spacing between other primers may be 10 bp. The base length difference between these primers can be set sequentially depending on the mutation status of the hotspot to be measured each time.

[0081] In embodiment 6, an optimal extension reaction for detecting each hotspot was performed for n target gene sequences using a first DyeSet and a second DyeSet. After the reaction, the reaction products were mixed and subjected to a single capillary electrophoresis. Peak signals in the electropherogram obtained as a result of capillary electrophoresis include (a) a wild-type signal 721 and a mutant-type signal 722 derived from target DNA fragment 704, (b) a wild-type signal 723 and a mutant-type signal 724 derived from target DNA fragment 708, (c) a wild-type signal 725 and a mutant-type signal 726 derived from target DNA fragment 710, and (d) a wild-type signal 728 and a mutant-type signal 727 derived from target DNA fragment 714.

[0082] The wild-type and mutant signals for each target DNA fragment are separated by at least one base, and their peaks do not overlap. Generally, the lower the mutation rate, the weaker the mutant signal and the stronger the wild-type signal. If the wild-type and mutant signals are close together in this state, the mutant signal becomes difficult to detect due to the pull-up and pull-down effects of the wild-type signal. Even if detection is possible, it becomes difficult to accurately calculate the mutation rate calculated from the wild-type and mutant signal intensities. Detection of low mutation levels is particularly important for the early diagnosis of cancer, as cancer is a disease in which point mutations accumulate through random generation. The earlier point mutation abnormalities can be detected, the more effective treatment can be administered.

[0083] Seventh Embodiment FIG. 7 is a diagram illustrating a mutant gene detection method according to a seventh embodiment of the present invention. As described in the above embodiments, in the first DyeSet, one of the complementary adenine or thymine bases is an intact dNTP that is not labeled with a fluorescent dye, and the other three nucleotides are ddNTPs labeled with different fluorescent dyes. Alternatively, in the first DyeSet, one of the complementary guanine or cytosine bases is an intact dNTP that is not labeled with a fluorescent dye, and the other three nucleotides are ddNTPs labeled with different fluorescent dyes. dNTP1 in the first DyeSet has its complementary nucleotide 1', and similarly, dNTP2 in the second DyeSet has its complementary nucleotide 2'. A characteristic feature here is that the four nucleotides, dNTP1 and its complementary nucleotide 1', and dNTP2 and its complementary nucleotide 2', are always adenine, cytosine, thymine, and guanine. In other words, by considering the above combinations and combinations of primers (sense primer and antisense primer), dNTPs can be selectively incorporated for adenine, cytosine, thymine, and guanine mutations, and one-base extension and two-base extension can be selectively generated.

[0084] There are four combinations of the first and second DyeSets that satisfy the above conditions: 2 x 2. Fig. 7 shows all four combinations of the first and second DyeSets.

[0085] <Embodiment 8> Figure 8 is a schematic diagram illustrating a mutant gene detection method according to embodiment 8 of the present invention. Embodiment 8 is a modification of embodiment 6. Like embodiment 6, an extension reaction is performed using a first DyeSet and a second DyeSet. The difference is that, rather than mixing the reactants after the reaction and performing electrophoresis using a single capillary, the reactants are not mixed, and two electrophoresis runs are performed using a single capillary 821 and a single capillary 822.

[0086] The advantages of using this method compared to embodiment 6 are that it eliminates the need to mix samples and increases the amount of data acquired by performing measurements using two capillaries. In other words, the number of genes that can be measured can be increased by the number of capillaries. For example, if the upper limit of the number of target genes that can be measured with one capillary is 500, using two capillaries can increase the number of target genes to 1,000. In such cases, it is better to adopt two capillaries as a system without forcing the samples to be mixed.

[0087] In the eighth embodiment, in order to distinguishably separate a signal peak from other signal peaks in the fluorescent signal detection, it is sufficient that the lengths of the primers are different from each other within the same capillary. In other words, even if the primer lengths are the same between capillaries, there is no problem in distinguishing the signal peaks from each other, because the signal detection is performed for each capillary.

[0088] <Embodiment 9> Figure 9 is a schematic diagram illustrating a mutant gene detection method according to embodiment 9 of the present invention. Embodiment 9 is a modification of embodiment 6. Like embodiment 6, an extension reaction is performed using a first DyeSet and a second DyeSet. The difference is that it assumes a case in which three types of mutations occur per base, as observed in KRAS codons 12 and 13, for example. "Z" is the base position at which the base extension reaction terminates, and which type of base this is varies depending on the sample.

[0089] A sense primer 906 is hybridized to the antisense strand of the target DNA fragment 904 to perform an extension reaction. Three mutant types and one wild-type type are present at the hotspot, and in this embodiment, only the wild-type type is extended by two bases. Fluorescently labeled ddNTPs are incorporated into the three mutant types, terminating the extension reaction. When this reaction product is electrophoresed, a group of three peaks 952 consisting of MT1, MT2, and MT3 and a wild-type peak 951 that is one base longer than the three peaks are observed. The group of three mutant peaks 952 consisting of MT1, MT2, and MT3 are separated from the wild-type peak 951 and are therefore not affected by the pull-up or pull-down of the wild-type peak 951. Therefore, by dividing each mutant peak by the wild-type peak (i.e., calculating the three mutation rates), an accurate mutation rate can be calculated.

[0090] Similarly, by separating the wild-type peaks 953, 955, and 957 from the three mutant peak groups 954, 956, and 958 of the mutant type by one or more base positions, respectively, it becomes possible to accurately detect multiple mutations in the hotspot.

[0091] The mutation rate can be calculated in the same manner as above even when two types of mutations occur for one base. In this case, there will be one wild-type signal peak and two mutant-type signal peaks, and the mutation rate of each mutation can be calculated by dividing each mutant-type peak by the wild-type peak (i.e., calculating two mutation rates).

[0092] <Regarding Modifications of the Present Invention> The present invention is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present disclosure, and are not necessarily limited to those including all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, or to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace part of the configuration of each embodiment with other configurations.

[0093] For example, the multiplex PCR described in embodiment 1, the reaction using sense primers / antisense primers described in embodiment 2, the method for generating base length differences described in embodiments 3 and 4, and the selection of sense primers / antisense primers described in embodiment 5 can also be used in embodiments 6 to 9.

[0094] In the above embodiments, fluorescent signal detection can be performed using a capillary sequencer. In this case, the wild-type signal peak is very large, and the mutant signal peak is very small compared to the wild-type. It is desirable that the capillary sequencer has a dynamic range that can measure both of these. Specifically, it is desirable that the capillary sequencer has a dynamic range that can measure the signal intensity when a wild-type DNA sample is detected without saturation, and can measure the signal intensity when a mutant DNA sample is detected at an intensity that can be distinguished from noise. Explanation of symbols

[0095] 102, 103, 104, 105... Mutation site 110, 111, 112, 113... Amplified gene 106, 107, 108, 109... Primer 114, 115, 116, 117... Peak derived from wild type 118, 119, 120, 121... Peak derived from mutation 201, 211, 221, 231, 205, 215, 225, 235, 301, 302, 303, 304, 351, 362, 360, 365, 370, 375... Sense strand 202, 212, 222, 232, 207, 217, 227, 237, 305, 306, 307, 308... Antisense strand 208, 218, 228, 238, 403, 905...sense primers 206, 216, 226, 236, 305, 306, 307, 308, 352, 361, 366, 371, 376, 405, 407, 409...antisense primers 203, 214, 309, 314...adenine 204, 213, 310...thymine 223, 234, 311, 313, 316...guanine 224, 233, 312, 315...cytosine 353, 362, 372...polymerase 355, 364, 368...ddGTP labeled with red fluorescent dye 356, 374, 378...ddATP labeled with black fluorescent dye 357: ddCTP labeled with blue fluorescent dye; 354, 363, 367: Intact dUTP or dTTP; 380: Wild-type primer; 381: Mutant primer; 402, 404, 406, 408, 704, 707, 710, 713, 706, 708, 711, 714, 904: DNA fragments; 421, 423, 425, 721, 723, 725, 728: Wild-type peaks; 422, 424, 236, 722, 724, 726, 727: Mutant peaks; 701, 702, 703: Sample tubes; 716, 821, 822: Capillaries; 951, 953, 955, 957: Wild-type peaks. 953, 955, 957, 959...Three mutation peak groups

Claims

1. A method for detecting a mutant gene contained in a DNA sample, comprising: - performing a fluorescence labeling reaction on the DNA sample by proceeding with a base extension reaction using a first dye set; - performing a fluorescence labeling reaction on the DNA sample by proceeding with a base extension reaction using a second dye set; - detecting the mutant gene by performing fluorescence signal detection on the product obtained by the base extension reaction performed using the first dye set and the product obtained by the base extension reaction performed using the second dye set; - wherein the first dye set is a dye set in which either adenine or thymine is not fluorescently labeled and the other is fluorescently labeled, and guanine and cytosine are fluorescently labeled; - the second dye set is a dye set in which either guanine or cytosine is not fluorescently labeled and the other is fluorescently labeled, and adenine and thymine are fluorescently labeled; - both the first dye set and the second dye set are configured such that the base extension reaction does not stop at a base that is not fluorescently labeled and stops at a base that is fluorescently labeled. A method for detecting a mutant gene, characterized by the above.

2. In the step of proceeding with the base extension reaction using the first dye set, select either a sense primer or an antisense primer such that the base species first incorporated from the primer at the detection target site of the DNA sample matches the base species not fluorescently labeled by the first dye set; - In the step of proceeding with the base extension reaction using the second dye set, select either a sense primer or an antisense primer such that the base species first incorporated from the primer at the detection target site of the DNA sample matches the base species not fluorescently labeled by the second dye set. The method for detecting a mutant gene according to claim 1, characterized by the above.

3. In the step of proceeding with the base extension reaction using the first dye set, the base length incorporated between the start of incorporating a base species not fluorescently labeled by the first dye set at the detection target site of the DNA sample until the base extension reaction stops, and the base length incorporated between the start of incorporating a base species fluorescently labeled by the first dye set at the detection target site until the base extension reaction stops, the base length difference between them is 1 base or more, and the base extension reaction is carried out. In the step of proceeding with the base extension reaction using the second dye set, the base length incorporated between the start of incorporating a base species not fluorescently labeled by the second dye set at the detection target site of the DNA sample until the base extension reaction stops, and the base length incorporated between the start of incorporating a base species fluorescently labeled by the second dye set at the detection target site until the base extension reaction stops, the base length difference between them is 1 base or more, and the base extension reaction is carried out. The method for detecting a mutant gene according to claim 1, characterized in that.

4. The base length of the sense primer used in the step of proceeding with the base extension reaction using the first dye set, the base length of the antisense primer used in the step of proceeding with the base extension reaction using the first dye set, the base length of the sense primer used in the step of proceeding with the base extension reaction using the second dye set, the base length of the antisense primer used in the step of proceeding with the base extension reaction using the second dye set are different from each other. The method for detecting a mutant gene according to claim 2, characterized in that.

5. In the step of proceeding with the base extension reaction using the first dye set, the lengths of the respective primers hybridizing to different detection target sites on the DNA sample are different from each other. In the step of proceeding with the base extension reaction using the second dye set, the lengths of the respective primers hybridizing to different detection target sites on the DNA sample are different from each other. The method for detecting a mutant gene according to claim 1, characterized in that.

6. The step of performing the fluorescence signal detection is carried out by introducing the product obtained by the base extension reaction carried out using the first dye set and the product obtained by the base extension reaction carried out using the second dye set into a common capillary. The base length of the primer used in the step of performing the base extension reaction using the first dye set and the base length of the primer used in the step of performing the base extension reaction using the second dye set are different from each other. The method for detecting a mutant gene according to claim 1, characterized in that.

7. In the step of proceeding with the base extension reaction using the first dye set and the step of proceeding with the base extension reaction using the second dye set, the base extension reaction is advanced by performing multiplex PCR on a plurality of target genes. In the step of performing the fluorescence signal detection, the fluorescence signal detection is performed using capillary electrophoresis. The method for detecting a mutant gene according to claim 1, characterized in that.

8. The step of performing the fluorescence signal detection is carried out on the DNA sample in the first capillary by introducing the product obtained by the base extension reaction carried out using the first dye set into the first capillary, and at the same time, the product obtained by the base extension reaction carried out using the second dye set is introduced into the second capillary to perform the detection on the DNA sample in the second capillary. The method for detecting a mutant gene according to claim 1, characterized in that.

9. In the step of proceeding with the base extension reaction using the first dye set, the lengths of the primers hybridizing to different detection target sites on the DNA sample are different from each other. In the step of proceeding with the base extension reaction using the second dye set, the lengths of the primers hybridizing to different detection target sites on the DNA sample are different from each other. The method for detecting a mutant gene according to claim 8, characterized in that.

10. The DNA sample is a sample in which two or more types of mutations can occur for one base, and the mutant gene detection method further includes: a step of respectively obtaining a first fluorescence signal obtained from a base type fluorescently labeled by the first dye set or the second dye set and a second fluorescence signal obtained from a base type not fluorescently labeled; a step of calculating a mutation rate in the DNA sample by dividing a signal peak level of the first fluorescence signal by a signal peak level of the second fluorescence signal. The mutant gene detection method according to claim 1, characterized by having the above.

11. The fluorescence signal detection is performed using a capillary sequencer, and the capillary sequencer has a dynamic range such that the signal intensity when detecting the wild-type DNA sample can be measured without saturation, and the signal intensity when detecting the mutant-type DNA sample can be measured at an intensity distinguishable from noise. The mutant gene detection method according to claim 1, characterized by having the above.

12. Both the first dye set and the second dye set are configured such that base types to be fluorescently labeled are colored with different colors from each other. The mutant gene detection method according to claim 1, characterized by having the above.

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