Quantitative analysis method for gene mutation, quantitative analysis device, and quantitative analysis kit

Abstract

The present invention relates to a quantitative analysis method for a gene mutation, particularly a gene mutation that is present at a low frequency, and a quantitative analysis device and a quantitative analysis kit for a gene mutation based on said method. Specifically, the present invention relates to a quantitative analysis method for a gene mutation, said method characterized by including (a) a step for adding a fixed concentration of an internal standard labeled with a first fluorescent pigment to a test sample, (b) a step for dividing the test sample into the number of varieties of detection base types, or preparing said number of test samples, (c) a step for adding a primer for a single-base extension reaction for detecting a target base sequence to the test samples, (d) a step for performing single-base extension reactions using the test samples divided or prepared per base type, the primer for a single-base extension reaction for detecting a target base sequence, and terminators per base type labeled with a second fluorescent pigment, (e) a step for subjecting the products of the single-base extension reactions per base type to electrophoresis, and (f) a step for measuring the fluorescent intensities of the fluorescent pigments and the electrophoretic mobility, and on the basis of the fluorescent intensities, detecting a wild type and a mutated type of the target base sequence, wherein the proportions of the peak fluorescent intensities of the second fluorescent pigment in the products of the single-base extension reactions relative to the peak fluorescent intensities of the first fluorescent pigment of the internal standard obtained per base type are calculated, and a quantity ratio of the single-base extension reaction products for each base type is quantified from said proportions.

Description

Method for quantitative analysis of gene mutation, quantitative analysis device, and quantitative analysis kit

[0001] The present invention relates to a method for quantitatively analyzing gene mutations, particularly gene mutations that occur at low frequencies, as well as an apparatus and kit for quantitative analysis of gene mutations based on such a method.

[0002] Advances in genetic analysis technology are elucidating the relationship between various diseases and genetic mutations. Somatic mutations, which are acquired genetic mutations resulting from diseases such as cancer, are characterized by the unpredictability of their genomic location and the unpredictability of their prevalence within individuals or tissues. For example, tissue samples from tumors removed from cancer patients contain both cancer cells and normal cells, and even among cancer cells, genetic mutations vary widely. Therefore, the prevalence of cells with a specific mutation at a specific location in a specific gene may be extremely low. Therefore, highly sensitive detection methods are needed to detect acquired genetic mutations resulting from disease. Furthermore, when selecting treatments or therapeutic agents, the prevalence of a specific mutation at a specific location in a target gene may be used as an indicator, not just the presence or absence of the mutation. Therefore, in addition to highly sensitive detection of genetic mutations, quantification of their prevalence is also required.

[0003] One type of genetic mutation is a single nucleotide polymorphism (SNP). For example, Non-Patent Document 1 shows that, using SNaPshot (registered trademark), a single-base extension reaction kit for SNP detection, and fragment analysis using a capillary electrophoresis device, target tumor-derived gene sequences are selectively enriched by multiplex polymerase chain reaction (PCR) in formalin-fixed and paraffin-embedded tissue, and 120 known genetic mutations in 13 cancer genes can be detected. In measuring the KRAS G12D mutation in the human lung cancer A427 cell line and the EGFR T790M mutation in the lung adenocarcinoma cell line NCI-H1975, the detection sensitivity of the mutant type relative to the wild type has been shown to be approximately 3%. The allele frequency that can be detected by the single-base extension reaction of SNaPshot is typically about 5%. This is because, as described in Non-Patent Document 1, there is a large variation in the fluorescence intensity of fluorescent dye-labeled dideoxynucleotide triphosphates (terminators), i.e., ddATP, ddCTP, ddGTP, and ddTTP, which act on the four bases adenine (A), cytosine (C), guanine (G), and thymine (T), and the detection sensitivity is thought to be limited to a few percent due to unnecessary peaks other than the products obtained by the single-base extension reaction.

[0004] In terms of improving the detection sensitivity of genetic mutations, Patent Document 1 reports a method for eliminating the effects of fluorescent signal crosstalk (changes in fluorescent signal intensity caused by interference between multiple fluorescent signals), which can reduce sensitivity when detecting trace amounts of somatic gene polymorphisms. A method has been proposed in which a nucleic acid sample is separated, each reacted with a different base type, electrophoresed in a separate channel for each base type, and genetic mutations are detected for each nucleic acid sample electrophoresed in each of the multiple channels based on waveform data obtained from the labeling signals for each base type (although the specific extent of the improvement in detection sensitivity achieved is not described). Furthermore, in terms of improving quantitative accuracy, Patent Document 2 proposes a method for improving normalization accuracy to obtain information on trace genetic mutations present in a target genetic region with high sensitivity and accuracy. A nucleic acid sample to be analyzed, which has been labeled for each base type, is electrophoresed, the labeling signals for each base type are detected to generate waveform data of the detection intensity, other peak positions are selected for each peak position in the waveform data for each base type, the relative signal intensity of the signal intensity at each peak position to the signal intensity of the other selected peak position is calculated, and the relative signal intensity of the nucleic acid sample to be analyzed and the relative signal intensity of a known nucleic acid sample are compared at each peak position to analyze each base type at the base sequence coordinate position of the nucleic acid sample.

[0005] Furthermore, when detecting trace amounts of genetic mutations, such as when the abundance ratio of mutants to wild-type is less than 1%, one approach is to simply increase the sample concentration. In this case, an internal standard method is used to quantify the mutant gene. The internal standard method requires that the signal intensities of the analyte and internal standard be proportional to their respective concentrations, i.e., that the increase in signal intensity with respect to the concentrations of the analyte and internal standard be linear. Assuming that the linearity of signal intensity is maintained when the sample concentration is increased, this characteristic enables quantitative detection of genetic mutations.

[0006] Patent No. 5723993 (U.S. Patent Application Publication No. 2014 / 0336949) Patent No. 5789720 (U.S. Patent No. 10,274,459)

[0007] Dias-Santagata, D. et al., EMBO Molecular Medicine Vol. 2, pp. 146-158 (2010)

[0008] One gene mutation detection technique is the detection of gene mutations (e.g., SNPs) using single-base extension reaction products via fragment analysis in capillary electrophoresis. However, the detection sensitivity of mutants relative to wild-type DNA is generally limited to a few percent. In particular, when single-base extension reaction products in which four bases are labeled with different fluorescent dyes are electrophoresed in each capillary, signal intensity errors occur during color conversion (fluorescent signal unmixing) for base identification, impairing quantitative accuracy. Accurate quantitative detection of low-frequency gene mutations (<1%) requires ingenious methods for acquiring and processing fluorescent signals. In addition, detecting gene mutations with a sensitivity of <1% requires detecting the fluorescent signals from wild-type and mutant DNA in samples over a wider concentration range (dynamic range) than conventional methods. However, when applying the internal standard method for quantitative detection in actual capillary electrophoresis, while the fluorescent signal intensity increases linearly with concentration in low-concentration samples, the fluorescent signal intensity saturates above a certain concentration.

[0009] As a result of extensive research aimed at solving the above-mentioned problems, we have found that the abundance ratio of gene mutations can be quantified by mixing a fluorescently labeled marker of known base length as an internal standard with the sample before the single-base extension reaction, dividing the sample into portions corresponding to the base species to be detected, and performing electrophoresis in different capillaries for each portion, and comparing the ratio of the fluorescent signal intensity of the peak of the single-base extension reaction product to the fluorescent signal intensity of the peak of the internal standard obtained in each capillary.This is a solution to the above-mentioned problems that can be realized by applying the internal standard method to samples with a wide dynamic range, in that by comparing the ratio of the fluorescent signal intensity of the peak of the single-base extension reaction product to the fluorescent signal intensity of the peak of the internal standard obtained in each capillary, quantification is possible without saturating the fluorescent signal intensity even in samples with a certain concentration or higher.

[0010] Therefore, in one aspect, the present invention provides a method for quantitatively analyzing genetic mutations, comprising the steps of: (a) adding a constant concentration of an internal standard labeled with a first fluorescent dye to a test sample; (b) dividing or preparing the test sample into portions equal to the number of base types to be detected; (c) adding a single-base extension primer for detecting a target base sequence to the test sample; (d) performing a single-base extension reaction using the test sample divided or prepared for each base type, the single-base extension primer for detecting the target base sequence, and a terminator for each base type labeled with a second fluorescent dye; (e) subjecting the products of the single-base extension reaction for each base type to electrophoresis; and (f) measuring the electrophoretic mobility and the fluorescence intensity of the fluorescent dye, and detecting a wild type and a mutant type of the target base sequence based on the fluorescence intensity. The present invention provides a method for quantitatively analyzing gene mutations, characterized by calculating a ratio of the fluorescence intensity of a second fluorescent dye at the peak of the product of the single-base extension reaction to the fluorescence intensity of a first fluorescent dye at the peak of the internal standard obtained for each base type, and quantifying the quantitative ratio of the single-base extension reaction products of each base type from the ratio.

[0011] In another aspect, the present invention provides an apparatus for quantitatively analyzing gene mutations for carrying out the method for quantitatively analyzing gene mutations, comprising: a measurement unit that performs single-base extension reactions, electrophoresis, and measurement of fluorescent signals; a data analysis unit that includes a measurement data storage unit that stores measurement data obtained by the measurement unit and a data processing device; and a control unit, wherein the measurement unit is configured to electrophorese the single-base extension reaction products for each type of base species to be detected and measure the fluorescent signals obtained from each electrophoresis; and the control unit is configured to control the data analysis unit to calculate a ratio of the fluorescent intensity of the fluorescent dye at the peak of the single-base extension reaction product to the fluorescent intensity of the fluorescent dye at the peak of the internal standard obtained for each base species.

[0012] In yet another aspect, the present invention provides a kit for quantitative analysis of gene mutations for carrying out the method for quantitative analysis of gene mutations, comprising: an internal standard at a constant concentration labeled with a first fluorescent dye; a primer for a single-base extension reaction for detecting a target base sequence; and a terminator for a single-base extension reaction labeled with a second fluorescent dye, wherein the terminator is contained in a different container for each base type so as to be used in the single-base extension reaction for each base type.

[0013] This specification includes the disclosure of Japanese Patent Application No. 2023-109354, filed on July 3, 2023, from which this application claims priority.

[0014] According to the present invention, in detecting genetic mutations (SNPs) in single-base extension reaction products using fragment analysis of capillary electrophoresis, genetic mutations present at a frequency of 1% or less can be quantitatively measured, thereby improving the accuracy of diagnosing diseases related to genetic abnormalities, including cancer. Quantitative analysis of genetic mutations with a mutant abundance ratio of 1% or less is possible without requiring special processing of electrophoresis data or hardware modifications, and is achieved solely by modifying the analytical method, primarily related to reagents. Problems, configurations, and effects other than those described above will be clarified in the description of the embodiments below.

[0015] FIG. 1 is an explanatory diagram of capillary electrophoresis fragment analysis for quantitatively measuring the abundance ratio of mutant to wild-type forms of a target base sequence using a single-base extension reaction. FIG. 2 is a schematic diagram showing electric field injection of a sample (measurement specimen) into a capillary electrophoresis device. FIG. 3 is an explanatory diagram of a method for quantitatively analyzing low-frequency gene mutations by capillary electrophoresis over a high dynamic range using a single-base extension reaction. FIG. 4 is an explanatory diagram of an example of an embodiment in which a decrease in sensitivity is prevented when the amount of template DNA (test sample) used in a single-base extension reaction is small by capturing and recovering the template DNA with magnetic beads. FIG. 5 is a flowchart showing an example of the processing procedure in a quantitative analysis device and quantitative analysis kit for low-frequency gene mutations for carrying out the present invention. FIG. 6 is a block diagram showing an example of the functions provided in the quantitative analysis device for gene mutations of the present invention. FIG. 7 is a diagram showing an example of a user interface screen output by the quantitative analysis device for gene mutations of the present invention.

[0016] Hereinafter, an example of an embodiment of the present invention will be described with reference to the drawings.

[0017] FIG. 1 is an explanatory diagram of capillary electrophoresis fragment analysis, which quantitatively measures the abundance ratio of mutant to wild-type target base sequences using a single-base extension reaction. Generally, target base sequence 101 is used as template DNA, and primers 102 of different base lengths (molecular weights) are used for single-base extension reactions, designed to vary the electrophoretic mobility of the resulting products. By polymerase chain reaction (PCR), ddNTPs modified with four colors (four types) of fluorescent dyes 103, whose terminal bases correspond to adenine (A), cytosine (C), guanine (G), and thymine (T), are added to the 3'-end of primer 102 corresponding to the genetic mutation through single-base extension reaction. Double-stranded DNA is converted into single strands by formamide treatment and heat denaturation, and the genetic mutation is identified by fluorescent detection of the fluorescent dye label 103 at the 3'-end of this single-base extension reaction product 104. In this case, by using primers of different base lengths for each target, the electrophoretic mobility of each single-base extension reaction product can be changed.

[0018] Previous academic research has shown that by using primers with different base lengths for each target, more than 20 known gene mutations can be detected with a single electrophoresis run. Furthermore, this technology can detect not only single nucleotide polymorphisms (SNPs), in which only one base is mutated, but also gene sequences of specific lengths depending on the primer design. Furthermore, the same principle can be used to detect insertions (IN) and deletions (DEL), which are types of gene mutations.

[0019] One of the causes of reduced detection sensitivity and quantitative accuracy during fluorescence detection is the influence of fluorescent signal crosstalk from neighboring products when electrophoresed in a capillary, since single-base extension reaction products, each with a different fluorescent dye bound to each of the four bases, are electrophoresed in each capillary (i.e., a single capillary). Furthermore, color conversion (fluorescent signal unmixing) for base species identification also generates errors in fluorescent signal intensity, impairing quantitative accuracy. Therefore, software for analyzing electrophoretic data has been developed to minimize fluorescent signal crosstalk and color conversion errors. The color conversion error is caused by overlapping of the fluorescence spectra of fluorescent dye labels used, such as x-rhodamine (ROX), rhodamine 110 (R110), rhodamine 6G (R6G), tetramethylrhodamine (TAMRA), fluorescein, or its derivatives such as fluorescein isothiocyanate (FITC), Alexa 488, Alexa 532, cy3, cy5, and Texas red. When the excitation wavelength of the laser light installed in the capillary electrophoresis apparatus is limited, it is technically difficult to completely separate the fluorescence spectra of the fluorescent dye labels used for the four types of bases (eliminating overlap). In addition to the fluorescent dye labels used for the four bases, a size standard with a fluorescent dye label attached is used as an internal standard to compensate for electrophoretic mobility and base length. This allows the internal standard to be mixed at a fixed concentration, correcting for electrophoretic mobility and base length, and quantitating the single-base extension reaction product. However, when this internal standard method is applied to actual capillary electrophoresis for quantitative detection, it has been found that while the fluorescent signal intensity increases linearly with concentration in low-concentration samples, the fluorescent signal intensity saturates above a certain concentration. The reason for this is explained in the next section.

[0020] 2 is a schematic diagram showing the electric field injection of a sample (measurement sample) in a capillary electrophoresis apparatus. One end of a capillary 202 filled with a polymer solution 201 is immersed in the sample solution. FIG. 2A shows the state before electric field injection, and FIG. 2B shows the state after electric field injection. In the following explanation, single-base extension reaction products obtained for two types of target base sequences are shown as examples, but similar considerations can be applied to single-base extension reaction products obtained for three or more types of target base sequences.

[0021] In the state shown in Figure 2A, when an electric field is injected for a certain time T with the sample injection end 203 of the capillary on the negative electrode side and the sample elution end on the positive electrode side, the state shown in Figure 2B is obtained. Ignoring positive ions, the current I flowing between the negative electrode and the sample injection end 203 of the capillary, i.e., the current I flowing through the sample (measurement sample) 204, is carried by the negative ions injected into the capillary. Here, when the effective electric field strength in the sample near the sample injection end of the capillary is E and the cross-sectional area of ​​the capillary inner diameter is A, the number J of molecules (single-base extension reaction products) injected into the capillary is ｉ The average of the mobility and concentration is u ｉ and C ｉ and is represented by the following (Equation 1):

[0022] Here, single-base extension reaction product 1 corresponding to a certain target base sequence 1 corresponds to i=1, and single-base extension reaction product 2 corresponding to another target base sequence 2 corresponds to i=2. Mobility indicates the migration speed of each negative ion per unit electric field strength. The average charge of each molecule is expressed as q ｉ Then, the amount of charge injected into the capillary Q ｉ is expressed by the following (Equation 2):

[0023] In this case, I・T = Σ ｉ Q ｉ From the relationship above, when the mobility of the negative ions 205 in the sample other than the single-base extension reaction product is represented by i=0, the following (Equation 3) is obtained:

[0024] Assuming that the average charge amount q and average mobility μ of each single-base extension reaction product are equal and the total concentration is C, the total number of molecules injected into the capillary J and the total charge amount Q can be expressed as the following (Equation 4) and (Equation 5) from (Equation 1), (Equation 2), and (Equation 3), respectively:

[0025] In addition, the fluorescent signal intensity S of the peak of each single-base extension reaction product on the electropherogram obtained by fragment analysis of capillary electrophoresis is ｉ is the sensitivity coefficient m ｉ Using this, the following (Equation 6) is obtained:

[0026] In addition, the sensitivity coefficient m ｉ includes the average number of fluorescent dye labels per single-base extension reaction product, the excitation efficiency of the fluorophore, the quantum yield of the fluorophore, the light-collection efficiency of the emitted fluorescence, the sensitivity of the image sensor, etc. Using (Equation 6), the sum of the peak fluorescent signal intensities is given by the following (Equation 7) when the sensitivity coefficient is the same and m is used:

[0027] This (Equation 7) is the reason why, when applying the internal standard method for quantitative detection in actual capillary electrophoresis, the fluorescent signal intensity increases linearly with concentration in low-concentration samples, but the fluorescent signal intensity (corresponding to S) saturates in samples with concentrations above a certain level (when C becomes excessively large). Considering the relationship for each single-base extension reaction product, the following (Equation 8) can be expressed from (Equation 6) and (Equation 7):

[0028] That is, the fluorescent signal intensity S of each single-base extension reaction product relative to the total fluorescent signal intensity S of each single-base extension reaction product injected into the capillary by electric field injection is ｉ is the concentration C of each single-base extension reaction product to the total concentration C of each single-base extension reaction product in the sample. ｉ At the same time, from this (Equation 8), for example, for a single-base extension reaction product 1 for a certain target base sequence 1 and a single-base extension reaction product 2 for another target base sequence 2, the following (Equation 9) is derived:

[0029] By using this relationship, when the ratio of the fluorescent signal intensity of the peak of each single-base extension reaction product to the fluorescent signal intensity of the peak of the internal standard obtained in each capillary is compared, it becomes possible to quantify over a wide dynamic range without saturating the fluorescent signal intensity even for samples with concentrations above a certain level.

[0030] The present invention is a genetic analysis method that combines a single-base extension reaction and electrophoresis, in which an internal standard is added and electrophoresis is performed separately for each type of detection base species, thereby quantitatively detecting the target gene mutation with high sensitivity and accuracy. Therefore, in one aspect, the present invention provides a method for quantitatively analyzing genetic mutations, comprising the steps of: (a) adding a constant concentration of an internal standard labeled with a first fluorescent dye to a test sample; (b) dividing or preparing the test sample into portions equal to the number of base types to be detected; (c) adding a single-base extension primer for detecting a target base sequence to the test sample; (d) performing a single-base extension reaction using the test sample divided or prepared for each base type, the single-base extension primer for detecting the target base sequence, and a terminator for each base type labeled with a second fluorescent dye; (e) subjecting the products of the single-base extension reaction for each base type to electrophoresis; and (f) measuring the electrophoretic mobility and the fluorescence intensity of the fluorescent dye, and detecting a wild type and a mutant type of the target base sequence based on the fluorescence intensity. The present invention relates to a method for quantitatively analyzing genetic mutations, characterized by calculating a ratio of the fluorescence intensity of a second fluorescent dye at the peak of the product of the single-base extension reaction to the fluorescence intensity of a first fluorescent dye at the peak of the internal standard obtained for each base type, and quantifying the quantitative ratio of the single-base extension reaction products of each base type from the ratio.

[0031] In the present invention, the term "target base sequence" refers to a base sequence for which detection and quantification are desired. For example, the target base sequence is a base sequence containing a genetic mutation, and the target base sequence is detected (and quantified) by distinguishing between wild-type and mutant forms. Examples of such genetic mutations include, but are not limited to, single nucleotide polymorphisms (SNPs), insertion mutations (IN), deletion mutations (DEL), microsatellite repeat sequences, copy number variations (CNVs), and epigenetic mutations. Furthermore, the mutant form of the target base sequence may include one type of mutation or multiple types of mutations. For example, there are genetic mutations in which the wild-type and two to three types of mutations exist, and such multiple types of mutations can also be distinguished, detected, and quantified according to the present invention.

[0032] The test sample used in this method is not particularly limited as long as it is a nucleic acid sample for which a target base sequence is to be detected, and includes deoxyribonucleic acid (DNA), such as genomic DNA and cDNA, and ribonucleic acid (RNA), such as messenger RNA (mRNA), and fragments thereof. In the present invention, it is preferable to use, for example, cell-free DNA (cfDNA, DNA free in the blood) or circulating tumor DNA (ctDNA) as the test sample. Nucleic acid preparation from the sample can be performed using methods known in the art. Nucleic acid preparation kits are commercially available from many manufacturers, allowing for convenient purification of the target nucleic acid.

[0033] Hereinafter, each embodiment of the method for quantitative analysis of gene mutation according to the present invention will be described.

[0034] FIG. 3 is an explanatory diagram of an example of a method for quantitatively analyzing rare genetic mutations over a wide dynamic range using a single-base extension reaction by capillary electrophoresis. In one embodiment of the present invention, a constant concentration of internal standard is first added to a sample (test sample) containing a mixture of wild-type and mutant forms of a target base sequence. The internal standard may be one or more fluorescently labeled DNA fragments of known or fixed concentration, or a size standard bound to a fluorescent dye label for correcting electrophoretic mobility and base length. Next, the sample is divided into the same number of base species to be detected. While FIG. 3 shows the sample being divided into four base species, if two or three base species are to be detected, the sample is divided into two or three. When an internal standard such as a size standard that is not affected by the single-base extension reaction (i.e., changes little before and after the reaction) is used, adding the internal standard first reduces dispensing errors that occur when adding the internal standard after division, thereby maintaining a constant final concentration of the internal standard and improving quantification accuracy. The divided samples are subjected to a single-base extension reaction for each base type using a terminator labeled with one type of fluorescent dye.

[0035] When an internal standard that is affected by a single-base extension reaction is used, for example, it can be a single-base extension reaction product at a SNP site (gene mutation site) of the same base type as the SNP site to be detected (gene mutation site) and whose abundance ratio is known in advance (e.g., 100%). In this case, the minimum number of capillaries required is one, and, for example, by one capillary electrophoresis, the abundance ratio of A at SNP site 2 can be quantified from the ratio of the peak fluorescence intensity of the single-base extension reaction product at SNP site 2, where the proportion of A is unknown, to the peak fluorescence intensity of the single-base extension reaction product at SNP site 1, where the proportion of base A is 100%.

[0036] When four capillaries are used, internal standards are required for at least the four base types A, C, G, and T. When performing single-base extension reactions for each base type, errors such as nonspecific adsorption in reagent reactions can be suppressed by mixing not only fluorescent dye-labeled terminators for the target bases but also fluorescently unlabeled terminators for the remaining three non-target bases.

[0037] The efficiency of incorporating a terminator varies depending on the mutation site (SNP site), the base type, and the fluorescent dye label bound to the terminator. The excitation efficiency and quantum efficiency vary depending on the type of fluorophore in the fluorescent dye label. If the overall efficiency of these is taken as the sensitivity coefficient K(m, n) (m: mutation (SNP) number, n: base type), K(m, n) can be obtained from the slope of a calibration curve obtained in advance for each mutation (SNP) number and base type. Therefore, for example, for four types of base types A, C, G, and T, the fluorescence intensity peak of the internal standard obtained with the capillary for the A base is expressed as S ａ The fluorescence intensity peak of the single-base extension reaction product obtained in the capillary of base A is represented by T ａ The fluorescence intensity peak of the internal standard obtained with the C-base capillary is S ｃ The fluorescence intensity peak of the single-base extension reaction product obtained in the capillary with C base is represented by T ｃ The fluorescence intensity peak of the internal standard obtained with the capillary of G base is S ｇ The fluorescence intensity peak of the single-base extension reaction product obtained in the capillary with G base is T ｇ The fluorescence intensity peak of the internal standard obtained in the capillary of the T base is S ｔ The fluorescence intensity peak of the single-base extension reaction product obtained in the capillary of T base is T ｔ , then the abundance ratio of A, C, G and T is T ａ / S ａ , T ｃ / S ｃ , T ｇ / S ｇ and T ｔ / S ｔ This allows quantification while preventing saturation of the fluorescent signal intensity. ａ / S ａ , Tｃ / S ｃ , T ｇ / S ｇ and T ｔ / S ｔ Not only is quantification performed as ａ / S ａ , K(m,c)*T ｃ / S ｃ , K(m, g)*T ｇ / S ｇ , K(m,t)*T ｔ / S ｔ It is desirable to quantify the signal by the method described above. Alternatively, the primer for single-base extension reaction can be designed so that the fluorescent signal peak of the internal standard and the peak of the single-base extension reaction product of the target nucleotide sequence do not overlap on the electropherogram. In this case, the same fluorescent dye label may be attached to the single-base extension reaction product and the internal standard.

[0038] Therefore, in one embodiment, the steps are carried out in the order of the above-mentioned steps (a), (b), (c), (d), (e), and (f). Specifically, the method for quantitative analysis of genetic mutations of the present invention includes: (a) adding a constant concentration of an internal standard labeled with a first fluorescent dye to a test sample; (b) dividing or preparing the test sample into the number of types of base species to be detected; (c) adding a single-base extension primer for detecting a target base sequence to the test sample; (d) performing a single-base extension reaction using the test sample divided or prepared for each base species, the single-base extension primer for detecting the target base sequence, and a terminator for each base species labeled with a second fluorescent dye; (e) subjecting the product of the single-base extension reaction for each base species to electrophoresis; and (f) measuring the electrophoretic mobility and the fluorescence intensity of the fluorescent dye, and detecting the wild type and mutant type of the target base sequence based on the fluorescence intensity. The present invention relates to a method for quantitatively analyzing genetic mutations, characterized by calculating a ratio of the fluorescence intensity of a second fluorescent dye at the peak of the product of the single-base extension reaction to the fluorescence intensity of a first fluorescent dye at the peak of the internal standard obtained for each base type, and quantifying the quantitative ratio of the single-base extension reaction products of each base type from the ratio.

[0039] In another embodiment, the steps are carried out in the order of the above-mentioned steps (c), (a), (b), (d), (e), and (f). Specifically, the method for quantitative analysis of genetic mutations of the present invention includes: (c) adding a single-base extension reaction primer for detecting a target base sequence to a test sample; (a) adding a constant concentration of an internal standard labeled with a first fluorescent dye to the test sample; (b) dividing or preparing the test sample into the same number of base types to be detected; (d) performing a single-base extension reaction using the test sample divided or prepared for each base type, a single-base extension reaction primer for detecting the target base sequence, and a terminator for each base type labeled with a second fluorescent dye; (e) subjecting the products of the single-base extension reaction for each base type to electrophoresis; and (f) measuring the electrophoretic mobility and the fluorescence intensity of the fluorescent dye, and detecting the wild type and mutant type of the target base sequence based on the fluorescence intensity. The present invention relates to a method for quantitatively analyzing genetic mutations, characterized by calculating a ratio of the fluorescence intensity of a second fluorescent dye at the peak of the product of the single-base extension reaction to the fluorescence intensity of a first fluorescent dye at the peak of the internal standard obtained for each base type, and quantifying the quantitative ratio of the single-base extension reaction products of each base type from the ratio.

[0040] In step (a), a fixed concentration of an internal standard labeled with a first fluorescent dye is added to the test sample. The internal standard may be a DNA fragment of known or fixed concentration, or a size standard for correcting electrophoretic mobility and base length. Alternatively, the internal standard may be a single-base extension reaction product of known concentration, which is extended with a base of the same base type as the detection base type. The first fluorescent dye is useful for detecting the amount or abundance ratio of the internal standard in subsequent analysis, and any fluorescent dye known in the art may be used. Examples of fluorescent dyes include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), sulforhodamine (TR), tetramethylrhodamine (TRITC), carboxy-X-rhodamine (ROX), carboxytetramethylrhodamine (TAMRA), NED, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5'-hexachlorofluorescein CE-phosphoramidite (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), 5'-tetrachlorofluorescein CE-phosphoramidite (TET), rhodamine 110 (R110), rhodamine 6G (R6G), VIC (registered trademark), ATTO system, Alexa Fluor (registered trademark), Texas Fluor (registered trademark), Examples of fluorescent dyes that do not cause a shift in migration size include dR110 (carboxy-dichloro rhodamine 110), dR6G (dihydro rhodamine 6G), dTAMRA (tetramethyl rhodamine), dROX (carboxy-X-rhodamine), etc. There are no particular limitations on the type or introduction method of such fluorescent dyes, and various conventionally known means can be used.

[0041] The addition of the internal standard may be carried out at the beginning of the method, before the single base extension reaction, or before electrophoresis.

[0042] In step (b), the test sample is divided or prepared according to the number of types of detection base species. The detection base species corresponds to the type of genetic mutation to be detected (wild type and mutant type). For example, when a single target base sequence in which only one wild type and one mutant type exist is to be detected, there are two types of detection base species, and the test sample is divided into two or prepared in two (for example, two test samples are prepared by amplification). When two or more mutant types exist or when multiple types of target base sequences are to be detected, there are three or four types of detection base species. In one embodiment, the test sample is divided into two, three, or four. It is preferable to divide or prepare the test sample in equal volumes, but it does not have to be divided equally when dividing after adding an internal standard.

[0043] Steps (c) to (e) can be performed by a genetic analysis method that combines a single-base extension reaction and electrophoresis, and such a genetic analysis method is well known in the art, for example, as described in Non-Patent Document 1.

[0044] The single-base extension reaction is carried out using a single-base extension primer for detecting the target base sequence in the presence of a fluorescent dye-labeled terminator (dideoxynucleotide triphosphate: ddNTP) as a substrate. In step (c), the single-base extension primer for detecting the target base sequence is added.

[0045] The primer for the single-base extension reaction may be either DNA or RNA, and is determined depending on the types of the test sample and target base sequence, and the type of polymerase used in the single-base extension reaction. Preferably, the primer is DNA, and the single-base extension reaction is carried out using DNA or mRNA as the test sample template.

[0046] Each primer is designed so that at least a portion (the 3' end) has a sequence specific to the target base sequence, i.e., a sequence complementary to the target base sequence. Furthermore, the base length of the primer affects the base length (size) of the product of the single-base extension reaction, and therefore the electrophoretic mobility is determined by the base length of the primer. Therefore, in a genetic analysis method that combines single-base extension reaction and electrophoresis, it is necessary to appropriately design the base sequence and base length of the primer. For example, when attempting to detect multiple (e.g., two or more) target base sequences, primers are designed that have specific sequence portions for each target base sequence and have different base lengths so that differences in mobility occur in electrophoresis. The portion that causes the difference in base length does not have to be specific to the target base sequence (e.g., the 5' portion of the primer shown in Figure 1). Since a primer is required for each target base sequence, a number of primers are designed according to the type of target base sequence to be detected. In order to detect, for example, 5 or more types (e.g., 5 to 100 types), preferably 10 or more types (e.g., 10 to 100 types) of target base sequences, different primers can be used for each target base sequence.

[0047] Primer design techniques are well known in the art, and primers usable in the present invention are designed to satisfy conditions enabling specific annealing, for example, to have a length and base composition (melting temperature) that enable specific annealing. For example, the length that functions as a primer is preferably 10 bases or more, more preferably 15 to 50 bases, and even more preferably 15 to 30 bases, for example, approximately 20 bases. Furthermore, during design, it is preferable to confirm the GC content and melting temperature (Tm) of the primer. Known primer design software can be used to confirm the Tm. The designed primers can be chemically synthesized using known oligonucleotide synthesis techniques, but are typically synthesized using a commercially available chemical synthesizer.

[0048] In order to adjust electrophoretic mobility, primers may have different mobilities by including, in addition to sequence portions of different base lengths as described above, tags (molecular weights), such as interstrand-crosslinked double-stranded DNA tags. The inventors previously developed a fragment analysis method using capillary electrophoresis, and developed an analytical method that can simultaneously detect tens to hundreds of gene mutations. Specifically, by linking an interstrand-crosslinked double-stranded DNA tag to a primer and using it, the base length of the primer can be extended by changing the length of the double-stranded DNA tag, thereby stably extending the electrophoretic distance to 120 bp or more, thereby increasing the number of gene mutations that can be simultaneously detected. The double-stranded DNA tag has a length that can be distinguished by mobility and has at least one interstrand crosslink. In the present invention, "interstrand crosslink" means that one strand of a double-stranded DNA is crosslinked to the other strand at at least one location. The method for intramolecularly crosslinking two such strands is not particularly limited as long as it is a method known in the art. Preferably, interstrand crosslinking is achieved by photocrosslinking. A double-stranded DNA tag having an interstrand crosslink determines the migration distance (mobility) in electrophoresis. That is, by linking double-stranded DNA tags of different lengths to a primer, the migration distance in electrophoresis can be changed. Since capillary electrophoresis can detect nucleic acids up to approximately 600 bases long, the length of the double-stranded DNA tag can range from 1 to approximately 590 bases, excluding the base length (10 to 30 bases) of the primer portion that binds to the target base sequence. The base sequence of the double-stranded DNA tag is not particularly limited as long as it is a nucleic acid having an interstrand crosslink. Furthermore, double-stranded DNA tags can be chemically synthesized using known oligonucleotide synthesis techniques, but are typically synthesized using a commercially available chemical synthesizer.

[0049] In this case, if the multiple target base sequences include a target base sequence for which more quantitative genetic analysis is desired, it is advisable to arrange the target base sequence items for which more quantitative analysis is required in a base length range (generally set to around 50 to 100 bp, but this varies depending on the specifications of the capillary electrophoresis device) where the resolution and accuracy are high. Due to the nature of fragment analysis, the resolution of electrophoresis tends to decrease as the base length increases, so detection accuracy can be improved by using short-base primers for target base sequences for which more accurate quantification is required.

[0050] In step (d), a single-base extension reaction is carried out using a test sample divided or prepared for each base type, a single-base extension primer for detecting the target base sequence, and a terminator for each base type labeled with a second fluorescent dye. When carrying out the single-base extension reaction, it is preferable that the test sample is divided or prepared for each detection base type, and the single-base extension reaction is carried out for each divided or prepared test sample.

[0051] Single-base extension reactions are known in the art and are typically performed using a polymerase. The polymerase used is selected depending on the type of template (test sample) and the type of primer used. For example, a DNA-dependent or RNA-dependent DNA polymerase is used for a single-base extension reaction using a DNA primer with a DNA or RNA template, respectively.

[0052] The single-base extension reaction is widely known in the art, and for example, Non-Patent Document 1 describes a method for efficiently extending one base by a cycle reaction.

[0053] When a target base sequence is present, the primer hybridizes to the target base sequence, and a nucleotide is incorporated as a substrate from the 3' end of the primer by the synthesis reaction of the polymerase. At this time, by using, for example, a dideoxynucleotide (ddNTP) as the nucleotide (substrate) to be incorporated, the synthesis reaction is completed with only one base extension. For this reason, this ddNTP is also called a terminator.

[0054] In step (d), a terminator labeled with a second fluorescent dye is used as a substrate for the single-base extension reaction. Here, the terminator includes two or more terminators labeled with different types of second fluorescent dyes so that the types of bases corresponding to the detection base species can be distinguished. The second fluorescent dye is useful for easily detecting whether a substrate has been incorporated or for determining the type of incorporated base, and any fluorescent dye known in the art can be used. The second fluorescent dye can be any of those exemplified as the first fluorescent dye, and the first and second fluorescent dyes may be the same or different. Preferably, the first and second fluorescent dyes are different types of fluorescent dyes. For example, when attempting to determine the type of base, a combination of five fluorescent dyes that are excited and detected at different wavelengths can be used to distinguish between four types of bases and five types for reference (to detect and correct base length from a reference ladder DNA).

[0055] In one embodiment, in step (d), the single-base extension reaction may be performed in the presence of a terminator of a base type that is not labeled with a fluorescent dye and is different from the second fluorescent dye, in addition to the terminator of each base type labeled with the second fluorescent dye, thereby suppressing errors such as nonspecific adsorption of the reagent reaction.

[0056] After the single-base extension reaction, in step (e), the products of the single-base extension reaction are subjected to electrophoresis for each base type. The electrophoresis is not particularly limited as long as it is a measurement method capable of fragment analysis by electrophoresis, and for example, capillary electrophoresis (CE) or electrophoresis in a microchannel such as MEMS (Micro-Electro-Mechanical Systems) can be used. In a preferred embodiment, the electrophoresis is capillary electrophoresis (CE).

[0057] Electrophoresis, such as CE, is a technique for separating introduced components based on differences in mobility due to charge, size, shape, etc. Based on mobility, the type of target base sequence (based on the type of primer) can be identified. Furthermore, based on the signal from the fluorescent dye, the presence or absence of the target base sequence or the type of specific base in the target base sequence (based on the type of substrate incorporated by the single-base extension reaction), for example, wild type or mutant type, can be distinguished.

[0058] In step (f), the electrophoretic mobility and the fluorescence intensity of the fluorescent dye are measured, and the wild-type and mutant forms of the target base sequence are detected based on the fluorescence intensity. The ratio of the fluorescence intensity of the second fluorescent dye at the peak of the single-base extension reaction product to the fluorescence intensity of the first fluorescent dye at the peak of the internal standard obtained for each base type is calculated, and the quantitative ratio of the single-base extension reaction product for each base type can be quantified from this ratio. In other words, the wild-type and mutant forms of the target base sequence can be quantitatively detected.

[0059] As described above, the fluorescent signal intensity S of the internal standard peak obtained in each capillary, i.e., for each base type, is ｉ The fluorescent signal intensity T of the peak of the single base extension reaction product of each base type ｉ Ratio T ｉ / S ｉ The relative fluorescence intensity is calculated and the quantitative ratio of the single-base extension reaction product of each base type is quantified. Conventional internal standard methods are applicable only when the signal intensity is proportional to the concentration of the analyte. However, the internal standard method used in the present invention is applicable not only when the signal intensity is proportional to the concentration of the analyte, but also when it is not proportional, approaches saturation, or reaches saturation. By plotting the relative fluorescence intensity in this way, it is possible to quantify single-base extension reaction products (gene mutations) with high accuracy using the internal standard method for targets over a wider concentration range than conventional methods.

[0060] In one embodiment, in step (a), when the internal standard of a fixed concentration is a single-base extension reaction product of a known concentration that has been extended using a base of the same base type as the detection base species, the ratio of the fluorescence intensity of the second fluorescent dye at the peak of the single-base extension reaction product to the fluorescence intensity of the first fluorescent dye at the peak of the single-base extension reaction product of a known concentration is calculated, and the quantitative ratio of the single-base extension reaction product of each base species can be quantified from this ratio.

[0061] By quantitatively detecting the wild-type and mutant forms of a target base sequence, it is possible to quantify, for example, the abundance ratio of mutant sequences to wild-type sequences, which is necessary for cancer diagnosis, or the frequency of genetic mutations. In one embodiment, the target base sequences to be analyzed include wild-type sequences and mutant sequences, and the ratio of the mutant sequences to the wild-type sequences is in the range of 0.01% to 10%, for example, 0.01% to 1%, or even 0.01% to 0.1%, and the target base sequences can be analyzed in this manner. In this way, quantitative genetic analysis of the target base sequences can be performed.

[0062] In one embodiment, the method of the present invention further comprises, after performing steps (c) and (d), (g) capturing the test sample that has served as a template for the single-base extension reaction using a solid phase for capturing the test sample, and recovering a reaction solution containing the single-base extension reaction product, wherein steps (c), (d), and (g) are repeatedly performed using the same test sample a number of times equal to the number of types of detection base species, thereby obtaining single-base extension reaction products prepared for each detection base species corresponding to the test sample in step (b), and then steps (a), (e), and (f) are performed. In this embodiment, single-base extension reaction products corresponding to the test sample are prepared for each detection base species by repeatedly performing the single-base extension reaction using the test sample as a template a number of times equal to the number of types of detection base species.

[0063] In step (g), the test sample is captured using a solid phase. The solid phase is not particularly limited as long as it can capture the test sample. A solid phase with an immobilized probe, such as beads (preferably magnetic beads), a solid surface, or a fiber, commonly used in the field of genetic analysis, can be used. The immobilized probe varies depending on the type of test sample, and those skilled in the art can design and synthesize an appropriate probe and immobilize it on the solid phase. After capturing the test sample, the reaction solution containing the remaining single-base extension reaction product is recovered and subjected to the subsequent step. The captured test sample is reused as a template for a single-base extension reaction for another detection base type. By repeatedly performing the single-base extension reaction and capturing the test sample in steps (c), (d), and (g), a single-base extension reaction product for each detection base corresponding to the test sample is prepared (corresponding to step (b)).

[0064] FIG. 4 is an explanatory diagram of an example of an embodiment in which a decrease in sensitivity is prevented by capturing and recovering template DNA with magnetic beads when the template DNA (test sample) used in the single-base extension reaction is small. When the target base sequence in the test sample is used as template DNA, even if DNA is amplified by PCR or the like before the single-base extension reaction, there may be cases in which the amount of template DNA, particularly mutant template DNA, is small. Although there is a method of increasing the concentration by eluting in a small amount of solution in a column purification process after amplification, there is a possibility that the sensitivity may decrease when the single-base extension reaction product is divided due to division. Therefore, a single-base extension reaction of base type X (X is one of base types A, C, G, and T) is performed on the template DNA using a selective primer that reacts with the target base sequence and a terminator (fluorescently labeled terminator) bound to one type of fluorescent dye label. Here, the template DNA is captured with magnetic beads via a probe, and only the reaction solution containing the single-base extension reaction product is recovered. Since template DNA remains in the unrecovered solution, it is captured with magnetic beads to return it to a state where only template DNA remains. As before, a single-base extension reaction of the next base type is carried out using a selective primer that reacts with the target base sequence and a terminator (fluorescently labeled terminator) bound to one type of fluorescent dye label. Repeating this procedure the number of times required for the number of base types to be detected, e.g., the number of base types A, C, G, and T, can prevent a decrease in sensitivity when a small amount of template DNA is used in the single-base extension reaction. Even when the amount of template DNA is not small, this procedure can remove contaminants from the single-base extension reaction product (proteins, DNA, etc. remaining in the solution that may cause noise in the fluorescent signal), making it useful for achieving more stable detection sensitivity.

[0065] The above-described method for quantitative analysis of gene mutations according to the present invention can be carried out simply and quickly using an apparatus having the necessary configuration or a kit containing the necessary components.

[0066] Therefore, in another aspect, the present invention provides an apparatus for quantitatively analyzing genetic mutations (a genetic mutation quantitative analysis apparatus), the apparatus comprising: a measurement unit that performs single-base extension reactions, electrophoresis, and measurement of fluorescent signals; a data analysis unit that includes a measurement data storage unit that stores measurement data obtained by the measurement unit and a data processing device; and a control unit, wherein the measurement unit is configured to electrophorese the single-base extension reaction products for each type of detected base species and measure the fluorescent signals obtained from each electrophoresis, and the control unit is configured to control the data analysis unit to calculate the ratio of the fluorescent intensity of the fluorescent dye at the peak of the single-base extension reaction product to the fluorescent intensity of the fluorescent dye at the peak of the internal standard obtained for each base species.

[0067] The control unit may further include a reference database that stores previous measurement data, in which case the control unit is configured to compare the measurement data stored in the measurement data storage unit with the previous measurement data stored in the reference database and perform data processing.

[0068] The device according to the present invention may further comprise an output display.

[0069] In yet another aspect, the present invention provides a kit for quantitative analysis of gene mutations, the kit comprising: an internal standard at a constant concentration labeled with a first fluorescent dye; a primer for a single-base extension reaction for detecting a target base sequence; and a terminator for a single-base extension reaction labeled with a second fluorescent dye, wherein the terminator is contained in a different container for each base type to be used in the single-base extension reaction for each base type.

[0070] The internal standard, the primer for the single-base extension reaction, and the terminator are as described above. In the present invention, since the single-base extension reaction is performed for each detection base type, the terminators for each base type are stored in different containers so that they can be used separately.

[0071] In addition to the above components, the kit of the present invention may also include a buffer constituting a reaction solution, enzymes (polymerase, reverse transcriptase, etc.), a standard sample for calibration, etc. By providing a kit containing a primer, a terminator (substrate), and an internal standard to be used in the single-base extension reaction, quantitative analysis of gene mutations can be performed more quickly and easily.

[0072] The device and kit for carrying out the method for quantitatively analyzing gene mutations will be specifically described below.

[0073] FIG. 5 is a flowchart showing an example of a processing procedure for a quantitative analysis device and quantitative analysis kit for low-frequency gene mutations for carrying out the present invention. The present invention allows for the construction of a system that uses an internal standard to calculate the mutation percentage (the proportion of mutants relative to the wild type) of a target base sequence over a wide dynamic range. First, in step S501, the target base sequence and the target mutation percentage are determined. Next, in step S502, reaction reagents to be used are determined based on the amount of template DNA to be used. Specifically, the base lengths of usable primers are assigned according to the detection base length range specified by the analyzer, and the protocol (operating instructions) instructs the user on the necessity of a template DNA recovery step based on the amount of template DNA. Next, in step S503, a single-base extension reaction is performed. In step S504, the product of the single-base extension reaction is electrophoresed to obtain a fluorescent signal. At this time, by incorporating an internal standard that provides a fluorescent signal at a predetermined detection position (base length), the relationship between the detection timing of the electrophoretic fluorescent signal and base length can be more accurately determined. Step S505 describes the process of calculating the correlation between detection timing and base length from the fluorescent signal of the internal standard (size standard). Next, in step S506, the fluorescent signal peak at a predetermined detection timing (base length) is divided by the fluorescent signal peak of the internal standard, enabling quantification of the mutation percentage without saturating the fluorescent signal intensity even for samples above a certain concentration. At this time, as shown in step S507, the fluorescent signal peak calculation region is determined from the primer assignment of a predetermined base length based on information about the reaction reagent used, and an estimated sensitivity coefficient K(m, n) is calculated. Then, in step S508, the fluorescent signal of the target is compared with reference data stored in a reference database or reference data that outputs a baseline value based on the reaction reagent settings to determine whether the target's fluorescent signal is high. If the target's fluorescent signal is not higher than the reference data, the target is determined to have "no mutation" (step S509). If the target's fluorescent signal is higher than the reference data, the mutation percentage is calculated in step S510.

[0074] FIG. 6 is a block diagram showing an example of the functions of the quantitative analysis device for gene mutations of the present invention. The main components of the device are a measurement unit 601, a data analysis unit 602, a control unit 603, and an output display unit 604. In the measurement unit 601, a sample subjected to a single-base extension reaction is placed in a sample placement unit, and the fluorescence signal of the sample flowing through the electrophoresis unit is measured over time using a fluorescence measurement unit using capillary electrophoresis. The data analysis unit 602 includes a measurement data storage unit for storing the measurement data obtained by the measurement unit 601, and a program for executing this data processing can be implemented by software. As shown in the flowchart of FIG. 5, the data processing includes acquiring fluorescence signals at predetermined detection timings (base lengths), integrating the fluorescence signals at predetermined base lengths, correcting for signal drift caused by longer electrophoresis times, calculating the ratio of the fluorescence signal intensity of the peak of the single-base extension reaction product to the fluorescence signal intensity of the peak of the internal standard obtained in each capillary, and quantitatively calculating the abundance ratio of gene mutations. At this time, reference data (reference database) for electrophoresis data previously stored in the data analysis unit 602 can be used. Furthermore, the reference data (reference database) can be updated by transmitting and receiving information to and from an external network. All functional control of the measurement unit 601, data analysis unit 602, etc. can be realized by software, with a processor interpreting and executing programs stored in the memory of the control unit 603. Furthermore, some or all of the components, functional units, processing units, processing means, etc. can also be realized by hardware, for example, by designing them as integrated circuits. Information such as programs, files, and databases for each function can be stored in memory, a recording device such as a hard disk or solid-state drive (SSD), or a recording medium such as an IC card, SD card, or DVD. After data processing, a calculation is performed to determine whether the fluorescence signal correction using the correction value falls within the detectable range of the analyzer, and the results are output to the output display unit 604.

[0075] The block diagram shown here is an example of a system that is an integrated genetic analysis device, and the genetic analysis method of the present invention can be applied as long as it has the functions of a measurement unit 601, a data analysis unit 602, a control unit 603, and an output display unit 604.

[0076] FIG. 7 shows an example of a user interface screen output by the quantitative analysis device for genetic mutations of the present invention. As described above with reference to FIGS. 5 and 6 , the target gene (target base sequence), the required mutation percentage (mutation detection percentage), and the corresponding reaction reagents to be used are input into the device. Electrophoresis conditions, such as injection voltage, injection time, electrophoresis voltage, electrophoresis time, and temperature, are input, similar to the specifications of a typical capillary electrophoresis device, and the measurement results display these electrophoresis conditions as reference data. The electrophoresis measurement results are displayed on the screen, allowing the user to see signals derived from mutations in the target gene (target base sequence) that appear within a specific range of electrophoretic mobility (bp). For example, even if data is obtained by electrophoresis using four capillaries with four different base types, the electrophoresis data can be merged and displayed on the measurement results screen to show the abundance ratio of the genetic mutation selected by the user. This allows the detection of mutations (SNP detection) in specific base types in the target gene (target base sequence) to be displayed, and the mutation percentage can be calculated and displayed.

[0077] 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 invention, and the present invention is 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.

[0078] 101: Target base sequence (template DNA) 102: Primers with different base lengths for each target 103: Fluorescent dye 104: Single-base extension reaction product 201: Polymer solution 202: Capillary 203: Sample injection end 204: Sample (measurement sample) 205: Negative ions other than single-base extension reaction product 601: Measurement unit 602: Data analysis unit 603: Control unit 604: Output display unit

Claims

1. A method for quantitatively analyzing gene mutations, comprising: (a) adding a constant concentration of an internal standard labeled with a first fluorescent dye to a test sample; (b) dividing or preparing the test sample into portions corresponding to the number of base types to be detected; (c) adding a single-base extension primer for detecting a target base sequence to the test sample; (d) performing a single-base extension reaction using the test sample divided or prepared for each base type, the single-base extension primer for detecting the target base sequence, and a terminator for each base type labeled with a second fluorescent dye; (e) subjecting the product of the single-base extension reaction for each base type to electrophoresis; and (f) measuring the electrophoretic mobility and the fluorescence intensity of the fluorescent dye, and detecting the wild type and mutant type of the target base sequence based on the fluorescence intensity. A method for quantitatively analyzing gene mutations, characterized in that a ratio of the fluorescence intensity of a second fluorescent dye of the peak of the product of the single-base extension reaction to the fluorescence intensity of a first fluorescent dye of the peak of the internal standard obtained for each base type is calculated, and the quantitative ratio of the single-base extension reaction products of each base type is quantified from the ratio.

2. The method according to claim 1, wherein the steps are carried out in the following order: step (a), step (b), step (c), step (d), step (e) and step (f).

3. The method according to claim 1, wherein the steps are carried out in the following order: step (c), step (a), step (b), step (d), step (e) and step (f).

4. The method according to claim 1, wherein in step (d), a single-base extension reaction is carried out in the presence of a terminator of a base type different from the base type that is not labeled with a fluorescent dye, in addition to a terminator for each base type labeled with a second fluorescent dye.

5. The method according to claim 1, wherein in step (a), the constant concentration of the internal standard is a single-base extension reaction product of known concentration extended by a base of the same base type as the detected base type, and a ratio of the fluorescence intensity of a second fluorescent dye of the peak of the single-base extension reaction product to the fluorescence intensity of a first fluorescent dye of the peak of the single-base extension reaction product of known concentration is calculated, and the quantitative ratio of the single-base extension reaction products of each base type is quantified from the ratio.

6. The method according to claim 1, further comprising the step of: (g) capturing the test sample that has served as a template for the single-base extension reaction using a solid phase for capturing the test sample, and recovering a reaction solution containing the single-base extension reaction product, after performing steps (c) and (d), by repeatedly performing steps (c), (d), and (g) using the same test sample a number of times equal to the number of types of detection base types, thereby obtaining single-base extension reaction products prepared for each detection base type that correspond to the test sample in step (b), and then performing steps (a), (e), and (f).

7. The method of claim 6, wherein the solid phase is a magnetic bead.

8. The method of claim 1, wherein in step (b), the test sample is divided into two, three or four portions.

9. The method of claim 1, wherein the genetic variation comprises a single nucleotide polymorphism (SNP).

10. The method of claim 1, wherein the electrophoresis is capillary electrophoresis.

11. A quantitative analysis device for gene mutations for carrying out the method according to claim 1, comprising: a measurement unit that performs single-base extension reaction, electrophoresis, and measurement of fluorescent signals; a data analysis unit including a measurement data storage unit that stores measurement data obtained by the measurement unit and a data processing device; and a control unit, wherein the measurement unit is configured to electrophorese the single-base extension reaction product for each type of detected base type and measure the fluorescent signal obtained from each electrophoresis, and the control unit is configured to control the data analysis unit to calculate a ratio of the fluorescent intensity of the fluorescent dye at the peak of the single-base extension reaction product to the fluorescent intensity of the fluorescent dye at the peak of the internal standard obtained for each base type.

12. A kit for quantitative analysis of gene mutations for carrying out the method according to claim 1, comprising: an internal standard at a constant concentration labeled with a first fluorescent dye; a primer for a single-base extension reaction for detecting a target base sequence; and a terminator for a single-base extension reaction labeled with a second fluorescent dye, wherein the terminators are contained in different containers for each base type for use in the single-base extension reaction for each base type.

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