Quantitative analysis method of genetic mutation, quantitative analyzer, and quantitative analysis kit

Abstract

To provide a method and means for detecting genetic mutation more quantitatively in a sample of a wider concentration range (dynamic range), in a gene analysis method in which single base elongation reaction and electrophoresis are combined.SOLUTION: A quantitative analysis method of genetic mutation in which a fluorescent label marker of a known base length being an internal standard is mixed into a sample before single base extension reaction, the sample is divided only by the number of base types being subjects to be detected and electrophoresis is executed severally at different capillaries, and on the basis of a comparison of a ratio of fluorescence signal intensity of a peak of a single base extension reaction product to fluorescence signal intensity of a peak of the internal standard obtained in each capillary, an abundance ratio of genetic mutation is quantitatively determined.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a method for quantitatively analyzing gene mutations, particularly gene mutations that exist at low frequencies, as well as an apparatus and kit for quantitatively analyzing gene mutations based on said method. [Background technology]

[0002] With the advancement of genetic analysis technology, the relationship between various diseases and gene mutations is being elucidated. Somatic mutations, which are acquired gene mutations resulting from diseases such as cancer, are characterized in that the location of the mutation on the genome cannot be predicted, and the proportion of mutations present within an individual or tissue cannot be predicted. For example, tissue samples from tumor sites removed from cancer patients contain cancer cells and normal cells, and there is diversity in gene mutations even among cancer cells. Therefore, the proportion of cells present in a sample that have a gene mutation at a specific position of a specific gene may be very low. Therefore, a highly sensitive detection method is required to detect acquired gene mutations resulting from diseases. In addition, when selecting a treatment method or a therapeutic drug, not only the presence or absence of a gene mutation at a specific position of a target gene may be used as an index, but also the proportion of the mutation. Therefore, in addition to highly sensitive detection of gene mutations, quantification of the proportion of the mutations is also required.

[0003] One type of gene 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, the gene sequence derived from the target tumor can be selectively enriched by multiplex polymerase chain reaction (PCR) in formalin-fixed and paraffin-embedded tissue, and 120 known gene mutations in 13 cancer genes can be detected. In the measurement of KRAS G12D mutation in the human lung cancer A427 cell line and EGFR T790M mutation in the lung adenocarcinoma cell line NCI-H1975, it has been shown that the detection sensitivity of the mutant type relative to the wild type is about 3%. The allele frequency that can be detected by the single-base extension reaction of SNaPshot is typically about 5%. As described in Non-Patent Document 1, 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), varies widely, and the detection sensitivity is thought to be limited to a few percent due to unnecessary peaks other than the products obtained in the single-base extension reaction.

[0004] From the viewpoint of improving the detection sensitivity of gene mutations, Patent Document 1 reports a method for eliminating the influence of fluorescent signal crosstalk (changes in fluorescent signal intensity caused by interference between multiple types of fluorescent signals), which is a factor in reducing sensitivity when detecting somatic gene polymorphisms present in trace amounts. A method has been proposed in which a nucleic acid sample is divided, each reacted with each base type, electrophoresed in a separate flow path for each base type, and gene mutations are detected for each nucleic acid sample electrophoresed in each of the multiple flow paths based on waveform data obtained from the labeling signal for each base type (however, the specific degree of improvement in detection sensitivity is not described). Also, from the viewpoint of improving the quantitative accuracy, Patent Document 2 proposes a method for increasing the normalization accuracy to obtain information on trace gene mutations present in a target gene region with high sensitivity and high accuracy. A nucleic acid sample to be analyzed, which is labeled for each base type, is electrophoresed, the labeling signal for each base type is detected to generate waveform data of the detection intensity, other peak positions are selected for each peak position of the waveform data for each base type, the signal intensity of each peak position is calculated relative to the signal intensity of the other selected peak position, 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 gene mutations, for example when the abundance ratio of mutant to wild type is less than 1%, an approach of simply increasing the sample concentration is also conceivable. In this case, the internal standard method is used to quantify the mutant gene. In the internal standard method, the signal strength of each of the analyte and internal standard must be proportional to their concentrations, that is, the increase in the signal strength of each of the analyte and internal standard must be linear with respect to their concentrations. Assuming that the linearity of the signal strength is maintained when the sample concentration is increased, this characteristic enables quantitative detection of gene mutations. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 5723993 (U.S. Patent Application Publication No. 2014 / 0336949) [Patent Document 2] Patent No. 5789720 (U.S. Patent No. 10,274,459) [Non-patent literature]

[0007] [Non-Patent Document 1] Dias-Santagata, D. et al., EMBO Molecular Medicine Vol. 2, pp. 146-158 (2010) Summary of the Invention [Problem to be solved by the invention]

[0008] One of the gene mutation detection techniques is the detection of gene mutations (e.g., SNPs) by single-base extension reaction products using fragment analysis of capillary electrophoresis. However, the detection sensitivity of mutants relative to wild type is generally limited to a few percent. In particular, when single-base extension reaction products in which different fluorescent dye labels are bound to four types of bases are electrophoresed in each capillary, in addition to the effects of fluorescent signal crosstalk, errors in signal intensity occur in color conversion (unmixing of fluorescent signals) for base type identification, impairing quantitation. In order to accurately and quantitatively detect low-frequency gene mutations of 1% or less, it is necessary to devise a method for acquiring fluorescent signals and a method for data processing. In addition, when detecting gene mutations with a sensitivity of 1% or less, it is necessary to detect fluorescent signals originating from wild type and mutant types in a sample over a wider concentration range (dynamic range) than before. However, in actual capillary electrophoresis, when the internal standard method is applied for quantitative detection, there is a problem that, although the increase in fluorescent signal intensity with respect to concentration increases linearly in low-concentration samples, the fluorescent signal intensity saturates in samples with a certain concentration or higher. [Means for solving the problem]

[0009] As a result of intensive research to solve the above-mentioned problems, the inventors 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 means for solving the above-mentioned problems that can be realized by applying the internal standard method to samples with a high dynamic range, in which quantification can be performed without saturating the fluorescent signal intensity even for samples with a certain concentration or higher, by comparing the ratio of the fluorescent signal intensity of the peak of the internal standard obtained in each capillary to the fluorescent signal intensity of the peak of the single-base extension reaction product.

[0010] Thus, in one aspect, the present invention provides a method for quantitative analysis of 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 a test sample in an amount equal to 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 a test sample divided or prepared for each base type, a single-base extension primer for detecting a 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 to electrophoresis for each of the base types; (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. Including, The present invention provides a method for quantitatively analyzing gene mutations, characterized in that a ratio of the fluorescence intensity of a second fluorescent dye of a peak of the product of the single-base extension reaction to the fluorescence intensity of a first fluorescent dye of the internal standard peak 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.

[0011] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: A measurement unit that performs a single base extension reaction, electrophoresis, and measurement of a fluorescent signal; a data analysis unit including a measurement data storage unit that stores the measurement data obtained by the measurement unit and a data processing device; Control unit and Equipped with the measurement unit is configured to electrophorese the single-base extension reaction product for each type of detection base species and measure a fluorescent signal obtained from each electrophoresis; The control unit is configured to control the data analysis unit to calculate a ratio of the fluorescence intensity of the fluorescent dye at a peak of the single-base extension reaction product to the fluorescence intensity of the fluorescent dye at an internal standard peak obtained for each base type.

[0012] In yet another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a fixed concentration of an internal standard labeled with a first fluorescent dye; A single base extension reaction primer for detecting a target base sequence, and A terminator for single-base extension reactions labeled with a second fluorescent dye Including, The present invention provides a kit for quantitative analysis of gene mutations for carrying out the method for quantitative analysis of gene mutations, characterized in that the terminators are contained in different containers for each base type so as to be used in a single-base extension reaction for each base type. Effect of the Invention

[0013] According to the present invention, in detecting gene mutations (SNPs) in single-base extension reaction products using fragment analysis of capillary electrophoresis, gene mutations present at a low frequency of 1% or less can be quantitatively measured, thereby improving the accuracy of disease diagnosis related to genetic abnormalities such as cancer. Without special processing of electrophoretic data or changes to hardware, only by ingenuity in the analysis method mainly related to reagents, quantitative analysis of gene mutations with a mutant type abundance ratio of 1% or less is possible. Problems, configurations, and effects other than those described above will be made clear in the description of the embodiments below. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is an explanatory diagram of capillary electrophoresis fragment analysis using a single-base extension reaction to quantitatively measure the abundance ratio of mutant to wild-type of a target base sequence. [Diagram 2] FIG. 2 is a schematic diagram showing electric field injection of a sample (measurement sample) in a capillary electrophoresis device. [Diagram 3] FIG. 1 is an explanatory diagram of a method for quantitatively analyzing low-frequency gene mutations in a high dynamic range by capillary electrophoresis using a single-base extension reaction. [Figure 4] FIG. 1 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 amount of template DNA (test sample) used in a single-base extension reaction is small. [Diagram 5] 1 is a flow chart showing an example of a processing procedure for carrying out the present invention using a quantitative analysis device and a quantitative analysis kit for low-frequency gene mutations. [Figure 6] FIG. 2 is a block diagram showing an example of functions of the genetic mutation quantitative analysis device of the present invention. [Figure 7] FIG. 2 is a diagram showing an example of a user interface screen output by the genetic mutation quantitative analysis device of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, an example of an embodiment of the present invention will be described with reference to the drawings.

[0016] FIG. 1 is an explanatory diagram of capillary electrophoresis fragment analysis that quantitatively measures the abundance ratio of a mutant type to a wild type of a target base sequence using a single-base extension reaction. In general, a target base sequence 101 is used as a template DNA, and a primer 102 having a different base length (molecular weight) is used for each target to perform a single-base extension reaction, and the primer is designed so that the electrophoretic mobility of the resulting product is changed. 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 position of the primer 102 corresponding to the gene mutation in a single-base extension reaction. The gene mutation is identified by detecting the fluorescence of the fluorescent dye label 103 at the 3'-end of this single-base extension reaction product 104, which is obtained by converting double-stranded DNA into a single strand by formamide treatment and heat denaturation. At this time, the electrophoretic mobility of each single-base extension reaction product can be changed by using a primer having a different base length for each target.

[0017] By using primers with different base lengths for each target, academic research to date has shown that more than 20 known types of gene mutations can be detected with a single electrophoresis run. Furthermore, this technology can be used not only to detect single nucleotide polymorphisms (SNPs), in which only one base is mutated, but also to detect gene sequences of specific lengths by designing the primers. In addition, the same principle can be used to detect insertions (IN) and deletions (DEL), which are types of gene mutations.

[0018] The cause of the decrease in detection sensitivity and quantitativeness during fluorescence detection is the effect of fluorescent signal crosstalk from adjacent products when electrophoresed in a capillary, because single-base extension reaction products, in which different fluorescent dyes are bound to the four types of bases, are electrophoresed in each capillary (i.e., one capillary). Furthermore, even in color conversion (unmixing of fluorescent signals) for identifying base types, errors in fluorescent signal intensity occur, impairing quantitativeness. For this reason, software for analyzing electrophoretic data has been prepared and efforts have been made to reduce fluorescent signal crosstalk and color conversion errors. The cause of the color conversion error is the overlap of the fluorescence spectra of fluorescent dye labels, such as x-rhodamine (ROX), rhodamine 110 (R110), rhodamine 6G (R6G), tetramethylrhodamine (TAMRA), fluorescein or its derivatives, fluorescein isothiocyanate (FITC), Alexa 488, Alexa 532, cy3, cy5, Texas red, etc., and when the excitation wavelength of the laser light mounted on the capillary electrophoresis device is limited, it is technically difficult to completely separate the fluorescence spectra of the fluorescent dye labels used for the four types of bases (eliminate the overlap). In addition, in addition to the fluorescent dye labels used for the four types of bases, a size standard bound to a different fluorescent dye label is used as an internal standard to correct the electrophoretic mobility and base length. This allows mixing of an internal standard at a fixed concentration, correction of electrophoretic mobility and base length, and quantification of the single-base extension reaction product. However, in actual capillary electrophoresis, when attempting to apply this internal standard method for quantitative detection, it has been confirmed that, although the fluorescence signal intensity increases linearly with concentration in low-concentration samples, the fluorescence signal intensity saturates in samples with concentrations above a certain level. The reason for this will be explained in the next section.

[0019] Fig. 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, with Fig. 2A showing the state before electric field injection and Fig. 2B showing 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 made for single-base extension reaction products obtained for three or more types of target base sequences.

[0020] In the state shown in A of Fig. 2, when an electric field is injected for a certain period of time T with the sample injection end 203 of the capillary as the negative electrode and the sample elution end as the positive electrode, the state shown in B of Fig. 2 is obtained. If positive ions are ignored, 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 specimen) 204, is carried by the negative ions injected into the capillary. Here, if 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, then the number of molecules (single-base extension reaction products) injected into the capillary, J, i is the average of its mobility and concentration, u i and C i and is represented by the following (Equation 1):

number

[0021] Here, the single-base extension reaction product 1 corresponding to a certain target base sequence 1 corresponds to i=1, and the 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 q i Then, the amount of charge injected into the capillary, Q i is expressed by the following (Equation 2):

number

[0022] In this case, I·T=Σ iQ i From the above relationship, when the mobility of the negative ions 205 in the sample other than the single-base extension reaction product is expressed as i=0, the following (Equation 3) is obtained:

number

[0023] Assuming that the average charge amount q and the 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:

number

[0024] 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 i is the sensitivity coefficient m i Using this, we obtain the following (Equation 6):

number

[0025] 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 taken as m:

number

[0026] This (Equation 7) is the very reason why, when attempting to apply the internal standard method for quantitative detection in actual capillary electrophoresis, the fluorescence signal intensity increases linearly with concentration in low-concentration samples, but the fluorescence 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):

number

[0027] That is, the fluorescence signal intensity S of each single-base extension reaction product relative to the total fluorescence signal intensity S of each single-base extension reaction product injected into the capillary by electric field injection is i is the ratio of the concentration of each single-base extension product in the sample, C, to the total concentration of each single-base extension product, C. 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:

number

[0028] By using this relationship, when comparing the ratio of the fluorescent signal intensity of the peak of each single-base extension reaction product to the fluorescent signal intensity of the internal standard peak obtained in each capillary, it is possible to quantify over a wide dynamic range without saturating the fluorescent signal intensity even for samples with a certain concentration or above.

[0029] The present invention relates to a method for quantitatively detecting a target gene mutation with high sensitivity and accuracy in a genetic analysis method that combines a single-base extension reaction and electrophoresis, by adding an internal standard and performing electrophoresis separately for each type of detection base. Thus, in one aspect, the present invention provides a method for quantitatively analyzing a genetic mutation, comprising: (a) adding a constant concentration of an internal standard labeled with a first fluorescent dye to a test sample; (b) dividing or preparing a test sample in an amount equal to 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 a test sample divided or prepared for each base type, a single-base extension primer for detecting a 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 to electrophoresis for each of the base types; (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. Including, The present invention relates to 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.

[0030] In the present invention, the target base sequence refers to a base sequence that is desired to be detected and quantified. For example, the target base sequence is a base sequence that includes a genetic mutation, and the target base sequence is detected (and quantified) by distinguishing between a wild type and a mutant type. Examples of such genetic mutations include, but are not limited to, single nucleotide polymorphism (SNP), insertion mutation (IN: Insertion), deletion mutation (DEL: Deletion), microsatellite repeat sequences, copy number polymorphism (CNV), and epigenetic mutation. In addition, the mutant type of the target base sequence may include one type of mutant type or multiple types of mutant types. For example, there is a genetic mutation that has a wild type and two to three types of mutant types, and such multiple types of mutant types can also be distinguished and detected and quantified according to the present invention.

[0031] The test sample used in this method is not particularly limited as long as it is a nucleic acid sample to be detected for a target base sequence, and includes deoxyribonucleic acid (DNA), such as genomic DNA, 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 blood) and circulating tumor DNA (ctDNA) as the test sample. Preparation of nucleic acid from a sample can be performed by a method known in the art. Kits for preparing nucleic acid are available from many manufacturers, and it is possible to easily purify the target nucleic acid.

[0032] Hereinafter, each embodiment of the method for quantitatively analyzing gene mutation according to the present invention will be described.

[0033] FIG. 3 is an explanatory diagram of an example of a method for quantitatively analyzing low-frequency gene mutations in a high dynamic range by capillary electrophoresis using a single-base extension reaction. In one embodiment of the present invention, a constant concentration of an internal standard is first added to a sample (test sample) in which a wild type and a mutant type of a target base sequence are mixed. As the internal standard, one or more types of fluorescently labeled DNA fragments with known or fixed concentrations, or a size standard bound with a fluorescent dye label for correcting electrophoretic mobility and base length, etc. are used. Next, the sample is divided by the number of types of base species to be detected. FIG. 3 shows a state in which the sample is divided by the number of four types of bases, but when two or three types of bases are to be detected, the sample is divided into two or three. When an internal standard that is not affected by a single-base extension reaction (changes little before and after the reaction), such as a size standard, is used as the internal standard, the addition of the internal standard first can reduce dispensing errors that occur when the internal standard is added after division, so that the final concentration of the internal standard can be constant and the quantitative accuracy can be improved. For each divided sample, a single-base extension reaction is carried out for each base type using a terminator labeled with one type of fluorescent dye.

[0034] When an internal standard that is affected by a single-base extension reaction is used, it can be, for example, a single-base extension reaction product at an SNP site with a known abundance ratio (e.g., 100%) that has the same base type as the SNP site (gene mutation site) to be detected. In this case, the minimum number of capillaries required is one, and for example, by capillary electrophoresis using one capillary, 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 ratio of A is unknown, to the peak fluorescence intensity of the single-base extension reaction product at SNP site 1, where the ratio of base type A is 100%.

[0035] When using four capillaries, at least internal standards are required for the four base types A, C, G, and T. When performing single-base extension reactions for each base type, errors such as non-specific adsorption in reagent reactions can be suppressed by mixing not only the fluorescent dye-labeled terminators of the target base but also fluorescent unlabeled terminators of the remaining three non-target bases.

[0036] The efficiency of incorporating a terminator varies depending on the mutation site (SNP site), 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 calculated 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 A base is T a , S is the fluorescence intensity peak of the internal standard obtained with the C-base capillary. c The fluorescence intensity peak of the single-base extension reaction product obtained in the capillary of C base is T c , S is the fluorescence intensity peak of the internal standard obtained in the G-base capillary. g The fluorescence intensity peak of the single-base extension reaction product obtained in the capillary with G base is Tg , the fluorescence intensity peak of the internal standard obtained in the capillary of 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 without saturating the fluorescence signal intensity. a / S a , T c / S c , T g / S g and T t / S t In addition to quantifying as the above, multiply the sensitivity coefficient, K(m,a)*T 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 preferable to quantify the amount of the single-base extension reaction primer by the method described above. Alternatively, the single-base extension reaction primer may 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 base sequence do not overlap on the electropherogram. In this case, the same fluorescent dye label may be bound to the single-base extension reaction product and the internal standard.

[0037] Therefore, in one embodiment, the steps are carried out in the order of the above-mentioned steps (a), (b), (c), (d), (e) and (f). (a) adding a constant concentration of an internal standard labeled with a first fluorescent dye to a test sample; (b) dividing or preparing a test sample in an amount equal to 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 a test sample divided or prepared for each base type, a single-base extension primer for detecting a 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 to electrophoresis for each of the base types; (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. Including, The present invention relates to 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.

[0038] 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 quantitatively analyzing gene mutations of the present invention includes the steps of: (c) adding a single-base extension primer for detecting a target base sequence to the test sample; (a) adding a constant concentration of an internal standard labeled with a first fluorescent dye to a test sample; (b) dividing or preparing a test sample in an amount equal to the number of types of base species to be detected; (d) performing a single-base extension reaction using a test sample divided or prepared for each base type, a single-base extension primer for detecting a 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 to electrophoresis for each of the base types; (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. Including, The present invention relates to 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.

[0039] In step (a), a constant concentration of an internal standard labeled with a first fluorescent dye is added to the test sample. As the internal standard, a DNA fragment of known or fixed concentration, or a size standard for correcting electrophoretic mobility and base length, etc. can be used. Alternatively, as the internal standard, a single-base extension reaction product of known concentration extended by a base of the same base type as the detection base type can be used. The first fluorescent dye is useful for detecting the amount or ratio of the internal standard in the subsequent analysis, and a fluorescent dye known in the art can 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 series, Alexa Fluor (registered trademark) series, Texas Examples of fluorescent dyes that do not cause a shift in electrophoretic 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 and introduction method of such fluorescent dyes, and various conventionally known means can be used.

[0040] The addition of the internal standard may be performed at the beginning of the method, before the single base extension reaction, or before electrophoresis.

[0041] In step (b), the test sample is divided or prepared by the number of types of detection base types. The detection base types correspond to the types of gene mutations to be detected (wild type and mutant type). For example, when a single type of target base sequence in which only one type of wild type and mutant type exists is to be detected, the detection base types are two types, 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 types of mutant types exist or when multiple types of target base sequences are to be detected, the detection base types are three or four types. In one embodiment, the test sample is divided into two, three or four. The test sample is preferably divided (equally divided) or prepared in the same volume, but it does not have to be equally divided when dividing after adding an internal standard.

[0042] Steps (c) to (e) can be carried out by a genetic analysis method that combines a single-base extension reaction and electrophoresis. Such a genetic analysis method is well known in the art, for example, as described in Non-Patent Document 1.

[0043] The single-base extension reaction is carried out using a single-base extension primer for detecting a target base sequence in the presence of a terminator (dideoxynucleotide triphosphate: ddNTP) labeled with a fluorescent dye as a substrate. In step (c), the single-base extension primer for detecting a target base sequence is added.

[0044] The primer for single-base extension reaction may be either DNA or RNA, and is determined according to the type of 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 as a template.

[0045] Each primer is designed so that at least a portion (the 3' end side) has a sequence specific to the target base sequence, i.e., a sequence complementary to the target base sequence. The base length of the primer also affects the base length (size) of the product of the single-base extension reaction, and therefore the electrophoretic mobility is determined from the base length of the primer. Therefore, in a genetic analysis method that combines a single-base extension reaction and electrophoresis, it is necessary to appropriately design the base sequence and base length of the primer. For example, when detecting multiple (e.g., two or more) target base sequences, primers are designed that have a specific sequence portion for each target base sequence and have different base lengths so that a difference in mobility occurs in electrophoresis. The portion for causing the difference in base length does not have to be specific to the target base sequence (e.g., the 5' side portion of the primer shown in Figure 1). Since a primer is required for each target base sequence, the number of primers is designed according to the type of target base sequence to be detected. In order to detect, for example, 5 or more types (eg, 5 to 100 types), preferably 10 or more types (eg, 10 to 100 types), of target base sequences, different primers can be used for each target base sequence.

[0046] Primer design techniques are well known in the art, and primers that can be used in the present invention are designed to satisfy conditions that allow specific annealing, for example, to have a length and base composition (melting temperature) that allow 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, about 20 bases. In addition, when designing, it is preferable to confirm the GC content and melting temperature (Tm) of the primer. To confirm Tm, known software for primer design can be used. The designed primer can be chemically synthesized by known oligonucleotide synthesis techniques, but is usually synthesized using a commercially available chemical synthesis device.

[0047] In order to adjust the electrophoretic mobility, the primer may have different mobilities by being provided with a tag (molecular weight), for example, an interstranded double-stranded DNA tag, in addition to the sequence portion with different base lengths as described above. The present inventors previously developed a fragment analysis method using capillary electrophoresis, and developed an analysis method capable of expanding the number of gene mutations that can be detected simultaneously to several tens to several hundreds of types. Specifically, by linking a double-stranded DNA tag that is interstranded 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, and the electrophoretic distance can be stably extended to 120 bp or more, thereby making it possible to increase the number of gene mutations that can be detected simultaneously. 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 and the other strand in the double-stranded DNA are crosslinked at at least one point. The method of intramolecularly crosslinking such two strands is not particularly limited as long as it is a method known in the art. Preferably, the interstrand crosslinking is by photocrosslinking. A double-stranded DNA tag having an interstrand bridge 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 a nucleic acid having a base length of up to about 600 bases can be detected in capillary electrophoresis, the length of the double-stranded DNA tag can be in the range of 1 to about 590 bases, excluding the base length (10 to 30 bases) of the primer 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 bridge. In addition, the double-stranded DNA tag can be chemically synthesized by a known oligonucleotide synthesis method, but is usually synthesized using a commercially available chemical synthesis device.

[0048] 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 region (generally set to around 50 to 100 bp, but differing 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 that detection accuracy can be improved by using short base primers for target base sequences for which more accurate quantitative analysis is required.

[0049] 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 reaction primer for detecting a target base sequence, and a terminator for each base type labeled with a second fluorescent dye. When carrying out a single-base extension reaction, it is preferable that the test sample is divided or prepared for each detection base type, and a single-base extension reaction is carried out for each divided or prepared test sample.

[0050] Single-base extension reactions are known in the art, and are typically single-base extension reactions using polymerases. The polymerase used is selected depending on the type of template (test sample) and the type of primer used. For example, DNA-dependent or RNA-dependent DNA polymerases are used for single-base extension reactions using DNA primers with DNA or RNA as templates, respectively.

[0051] Single-base extension reactions are widely known in the art, and for example, Non-Patent Document 1 describes a method for efficiently extending one base by a cycle reaction.

[0052] 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 terminated with only one base extension. Therefore, this ddNTP is also called a terminator.

[0053] 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 types of terminators labeled with different types of second fluorescent dyes so that the types of bases corresponding to the detection base types can be distinguished. The second fluorescent dye is useful for easily detecting whether or not the substrate has been incorporated, or for determining the type of the incorporated base, and any fluorescent dye known in the art can be used. As the second fluorescent dye, those exemplified in the first fluorescent dye can be used, and the first fluorescent dye and the second fluorescent dye may be the same type or different types. Preferably, the first fluorescent dye and the second fluorescent dye are different types of fluorescent dye. For example, when trying to determine the type of base, five types of fluorescent dyes that are excited and detected at different wavelengths can be used in combination to distinguish four types of bases and five types for reference (for detecting and correcting the base length from a reference ladder DNA).

[0054] In one embodiment, in step (d), the single-base extension reaction may be performed in the presence of a terminator of a base type different from the second fluorescent dye-labeled base type, which is not labeled with a fluorescent dye, in addition to the terminator of each base type labeled with a second fluorescent dye. This can suppress errors such as nonspecific adsorption of the reagent reaction.

[0055] 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 performing 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).

[0056] Electrophoresis, for example CE, is a technique for separating introduced components based on differences in mobility based on charge, size, shape, etc. Based on the mobility, the type of target base sequence (based on the type of primer) can be identified. In addition, based on the signal from the fluorescent dye, it is possible to distinguish the presence or absence of the target base sequence or the type of a specific base in the target base sequence (based on the type of substrate incorporated by the single-base extension reaction), for example, between wild type and mutant type.

[0057] In step (f), the electrophoretic mobility and the fluorescence intensity of the fluorescent dye are measured, and the wild type and mutant type of the target base sequence are detected based on the fluorescence intensity. In this case, the ratio of the fluorescence intensity of the second fluorescent dye of the peak of the single-base extension reaction product to the fluorescence intensity of the first fluorescent dye of the internal standard peak obtained for each base type is calculated, and the amount ratio of the single-base extension reaction product of each base type can be quantified from the ratio, that is, the wild type and mutant type of the target base sequence can be quantitatively detected.

[0058] As described above, the fluorescence signal intensity S of the internal standard peak obtained in each capillary, i.e., for each base type, is i The fluorescence signal intensity T of the peak of the single-base extension reaction product of each base type against i The ratio of T i / S iis obtained, 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, but 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 the single-base extension reaction product (gene mutation) with high accuracy using the internal standard method for a target with a wider concentration range than before.

[0059] In one embodiment, in step (a), when the constant concentration of the internal standard is a known concentration of a single-base extension reaction product extended by a base of the same base type as the detection base type, 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 known concentration is calculated, and the quantitative ratio of the single-base extension reaction products of each base type can be quantified from this ratio.

[0060] By quantitatively detecting the wild type and mutant type of the target base sequence, it is possible to quantitatively determine 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 sequence can be analyzed when the multiple target base sequences to be analyzed include wild type sequences and mutant sequences, and the content ratio of mutant sequences to wild type sequences is in the range of 0.01% to 10%, for example, in the range of 0.01% to 1%, or even in the range of 0.01% to 0.1%. In this way, it is possible to quantitatively perform genetic analysis on the target base sequence.

[0061] Also in one embodiment, the method of the present invention further comprises: After carrying out 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. Further comprising: Step (c), step (d) and step (g) are repeatedly performed using the same test sample for the number of types of detection base species, thereby obtaining, in step (b), single-base extension reaction products prepared for each detection base species corresponding to the test sample; Then, steps (a), (e) and (f) are carried out. In this embodiment, a single-base extension reaction using the test sample as a template is repeated the same number of times as the number of types of detection base species, thereby preparing single-base extension reaction products corresponding to the test sample for each detection base species.

[0062] 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, and a solid phase with a probe immobilized thereon, such as beads (preferably magnetic beads), a solid surface, or a fiber, which is commonly used in the field of genetic analysis, can be used. The probe to be immobilized varies depending on the type of the test sample, and a person skilled in the art can design and synthesize an appropriate probe and immobilize it on the solid phase. After the test sample is captured, the reaction solution containing the remaining single-base extension reaction product is collected 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, and the single-base extension reaction and capture of the test sample in steps (c), (d), and (g) are repeatedly performed to prepare a single-base extension reaction product for each detection base corresponding to the test sample (corresponding to step (b)).

[0063] FIG. 4 is an explanatory diagram of an example of an embodiment in which a template DNA (test sample) used in a single-base extension reaction is captured and collected by magnetic beads to prevent a decrease in sensitivity when the amount of 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 the template DNA, even if DNA is amplified by PCR or the like before the single-base extension reaction, there may be a case where 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 a decrease in sensitivity due to division occurs when dividing the single-base extension reaction product. Therefore, a single-base extension reaction of base type X (X is any 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 (fluorescence-labeled terminator) bound to one type of fluorescent dye label. Here, the template DNA is captured by magnetic beads via a probe, and only the reaction solution containing the single-base extension reaction product is collected. Since template DNA remains in the unrecovered solution, it is captured by magnetic beads to return it to a state of only template DNA, and as before, a selective primer that reacts with the target base sequence and a terminator (fluorescently labeled terminator) bound to one type of fluorescent dye label are used to carry out a single-base extension reaction of the next base type. By repeating this operation for the number of base types to be detected, for example, the number of base types A, C, G, and T, it is possible to prevent a decrease in sensitivity when the template DNA used in the single-base extension reaction is small. Even if the template DNA is not small, this operation 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 obtaining more stable detection sensitivity.

[0064] The above-mentioned method for quantitatively analyzing 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.

[0065] Therefore, in another aspect, the present invention provides an apparatus for quantitatively analyzing genetic mutations (a quantitative analysis apparatus for genetic mutations), the apparatus comprising: A measurement unit that performs a single base extension reaction, electrophoresis, and measurement of a fluorescent signal; a data analysis unit including a measurement data storage unit that stores the measurement data obtained by the measurement unit and a data processing device; Control unit and Equipped with the measurement unit is configured to electrophorese the single-base extension reaction product for each type of detection base species and measure a fluorescent signal obtained from each electrophoresis; The control unit is configured to control the data analysis unit to calculate a ratio of the fluorescent intensity of the fluorescent dye of the peak of the single-base extension reaction product to the fluorescent intensity of the fluorescent dye of the internal standard peak obtained for each base type.

[0066] The control unit may further comprise a reference database for storing previous measurement data; In that case, the control unit is configured to compare the measurement data stored in the measurement data storage unit with previous measurement data stored in the reference database and perform data processing.

[0067] The device according to the present invention may further comprise an output display.

[0068] In yet another aspect, the present invention provides a kit for quantitative analysis of genetic mutations, the kit comprising: a fixed concentration of an internal standard labeled with a first fluorescent dye; A single base extension reaction primer for detecting a target base sequence, and A terminator for single-base extension reactions labeled with a second fluorescent dye Including, The terminators are contained in different containers for each base type so as to be used in a single-base extension reaction for each base type.

[0069] The internal standard, the primer for single-base extension reaction, and the terminator are as described above. In the present invention, since a single-base extension reaction is performed for each detection base type, the terminators for each base type are stored in different containers and are in a state where they can be used separately.

[0070] In addition to the above-mentioned components, the kit according to the present invention may contain a buffer constituting a reaction solution, enzymes (polymerase, reverse transcriptase, etc.), a standard sample for calibration, etc. By providing a primer, a terminator (substrate), and an internal standard used in a single-base extension reaction as a kit, it becomes possible to perform quantitative analysis of gene mutations more quickly and easily.

[0071] The following provides a specific explanation of an apparatus and a kit for carrying out the method for quantitatively analyzing gene mutations.

[0072] FIG. 5 is a flow chart showing an example of a processing procedure in a quantitative analysis device and a quantitative analysis kit for a gene mutation that exists at a low frequency for carrying out the present invention. In the present invention, a system can be constructed that uses an internal standard to calculate the mutation % (the ratio of the mutation type to the wild type) of a target base sequence in a high dynamic range. First, in step S501, a target base sequence and a target mutation % are determined. Next, in step S502, a reaction reagent to be used is determined from the amount of template DNA to be used. Specifically, the base length of a primer that can be used is assigned according to the detection base length range that is the specification of the analysis device, and the necessity of a template DNA recovery step is instructed to the user in a protocol (operation manual) from 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 mixing an internal standard that can obtain a fluorescent signal at a predetermined detection position (base length), it is possible to more accurately determine the relationship between the detection timing of the fluorescent signal of electrophoresis and the base length. Step S505 describes a process of calculating the correlation between the detection timing and the base length from the fluorescent signal of the internal standard (size standard). Next, in step S506, the fluorescent signal peak at the predetermined detection timing (base length) is divided by the fluorescent signal peak of the internal standard, so that the fluorescent signal intensity does not saturate even in samples with a certain concentration or more, and the mutation % can be quantified. At this time, as shown in step S507, the peak calculation region of the fluorescent signal is determined from the primer assignment of the predetermined base length based on the information of the reaction reagent used, and the expected sensitivity coefficient K (m, n) is calculated. Then, in step S508, the fluorescent signal is compared with the reference data stored in the reference database, or the reference data that outputs the baseline value with the setting of the reaction reagent, and it is determined whether the fluorescent signal of the target is high. If the fluorescent signal of the target is not higher than the reference data, the target is determined to be "no mutation" (step S509). If the fluorescent signal of the target is higher than the reference data, the mutation % is calculated in step S510.

[0073] 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 that has undergone a single-base extension reaction is placed in the sample placement unit, and the fluorescent signal of the sample flowing through the electrophoresis unit over time is measured by the fluorescent measurement unit using capillary electrophoresis. The data analysis unit 602 is provided with a measurement data storage unit for storing the measurement data obtained in the measurement unit 601, and a program for executing the data processing can be realized by software. The contents of the data processing include, as shown in the flowchart of FIG. 5, acquisition of a fluorescent signal at a predetermined detection timing (base length), integration of a fluorescent signal at a predetermined base length, correction calculation of a signal drift caused by an increase in electrophoresis time, calculation of 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, and quantitative calculation of the abundance ratio of gene mutations. At this time, it is also possible to use reference data (reference database) of electrophoretic data stored in advance in the data analysis unit 602. Furthermore, it is also possible to update the reference data (reference database) by transmitting and receiving information to and from an external network. All of the functional controls of the measurement unit 601, the data analysis unit 602, etc. can be realized by software by having a processor interpret and execute a program stored in the memory of the control unit 603. In addition, it is also possible to realize a part or all of each configuration, functional unit, processing unit, processing means, etc. by hardware, for example, by designing them as integrated circuits. Information such as programs, files, databases, etc. of each function can be stored in a memory, a recording device such as a hard disk or SSD (Solid State Drive), or a recording medium such as an IC card, SD card, or DVD. After data processing, it is determined by calculation whether the correction of the fluorescent signal by the correction value falls within the detectable range of the analysis device, and the results are output on the output display unit 604.

[0074] Note that 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 the measurement unit 601, data analysis unit 602, control unit 603, and output display unit 604.

[0075] 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. As described above with reference to FIG. 5 and FIG. 6, the target gene (target base sequence), the required mutation percentage (mutation detection percentage), and the reaction reagents used according to them are input to the device. As with the specifications of a normal capillary electrophoresis device, the electrophoresis conditions such as injection voltage, injection time, electrophoresis voltage, electrophoresis time, and temperature are input, and the measurement results display these electrophoresis conditions as reference data. The electrophoresis measurement results are displayed on the screen, making it possible to show the user a signal derived from a mutation in the target gene (target base sequence) shown in a specific range of electrophoretic mobility (bp). For example, even if the data is electrophoresis data for four base types each using four capillaries, the measurement result screen can display the mutation detection (SNP detection) shown in a specific base type of the target gene (target base sequence) type, and calculate and display the mutation percentage.

[0076] The present invention is not limited to the above-described embodiment, and includes various modified examples. For example, the above-described embodiment has been described in detail to clearly explain the present invention, and is not necessarily limited to those including all of the configurations described. In addition, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. In addition, it is possible to add, delete, or replace a part of the configuration of each embodiment with another configuration. [Explanation of symbols]

[0077] 101: Target 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 port 204: Sample (measurement sample) 205: Negative ions other than single base extension products 601: Measurement section 602: Data Analysis Department 603: Control unit 604: Output display section

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 a test sample in an amount equal to 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 a test sample divided or prepared for each base type, a single-base extension primer for detecting a 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 to electrophoresis for each of the base types; (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. Including, 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. In step (a), the constant concentration of the internal standard is a single-base extension reaction product having a known concentration, which is extended by a base having the same base type as the detection base type; 2. The method according to claim 1, further comprising calculating a ratio of a fluorescence intensity of a second fluorescent dye at a peak of the single base extension reaction product to a fluorescence intensity of a first fluorescent dye at a peak of the single base extension reaction product of the known concentration, and quantifying the quantitative ratio of the single base extension reaction products of each base type from the ratio.

6. After carrying out 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. Further comprising: Step (c), step (d) and step (g) are repeatedly performed using the same test sample for the number of types of detection base types, thereby obtaining, in step (b), single-base extension reaction products prepared for each detection base type corresponding to the test sample; The method of claim 1 , further comprising carrying out 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. 2. 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 measurement unit that performs a single base extension reaction, electrophoresis, and measurement of a fluorescent signal; a data analysis unit including a measurement data storage unit that stores the measurement data obtained by the measurement unit and a data processing device; Control unit and Equipped with the measurement unit is configured to electrophorese the single-base extension reaction product for each type of detection base species and measure a fluorescent signal obtained from each electrophoresis; 2. A quantitative analysis device for gene mutations for carrying out the method according to claim 1, characterized in that the control unit is configured to control the data analysis unit to calculate a ratio of the fluorescent intensity of the fluorescent dye of the peak of the single-base extension reaction product to the fluorescent intensity of the fluorescent dye of the internal standard peak obtained for each base type.

12. a fixed concentration of an internal standard labeled with a first fluorescent dye; A single base extension reaction primer for detecting a target base sequence, and A terminator for single-base extension reaction labeled with a second fluorescent dye Including, 2. A kit for quantitative analysis of genetic mutations for carrying out the method of claim 1, characterized in that the terminators are contained in different containers for each base type so as to be used in a single-base extension reaction for each base type.

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