Methods and kits for genetic analysis

Abstract

To provide methods and kits for genetic analysis allowing for accurate determination on whether plural genetic mutations present in close proximity are in a cis or trans positional relationship and quantification of the genetic mutations.SOLUTION: A method for genetic analysis comprises the steps of: providing probes corresponding to respective plural genetic mutations; conducting an amplification reaction using a solution comprising a primer for amplifying a region comprising the plural genetic mutations, the probes, a test biological sample and an enzyme; then measuring the binding of the probes to the amplification product by melting curve analysis varying a temperature of the solution; and determining whether the plural gene mutations are on the same allele (cis form) or on different alleles (trans form) in the DNA contained in the biological sample, on the basis of the result of the melting curve analysis.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a genetic analysis method and a genetic analysis kit for analyzing multiple gene mutations, more specifically, to a genetic analysis method and a genetic analysis kit for analyzing multiple gene mutations using a melting curve analysis probe. The present invention also relates to a method and a kit for evaluating a trait (e.g., drug responsiveness) associated with multiple gene mutations. [Background technology]

[0002] Non-small cell lung cancer accounts for 80-90% of all lung cancers. Driver gene mutations, including EGFR (Epidermal Growth Factor Receptor), are frequently observed, and molecular targeted drugs have been developed for these mutations. EGFR tyrosine kinase inhibitors (EGFR-TKIs), which are molecular targeted drugs for EGFR gene mutations, have been developed as follows: gefitinib and erlotinib as first generation drugs, and afatinib and dacomitinib as second generation drugs. However, even if these EGFR-TKIs are effective in patients with EGFR gene mutations, a resistance mutation called T790M occurs about one year after the start of treatment. Therefore, osimertinib was developed as a third generation EGFR-TKI that is effective in patients with T790M mutations. However, a new problem has arisen in that C797S occurs as a resistance mutation to osimertinib, making osimertinib ineffective.

[0003] In recent years, it has become clear that the therapeutic effect changes depending on the positional relationship of the gene mutations T790M and C797S (Non-Patent Document 1). When T790M and C797S exist in trans form (exist on different alleles), osimertinib is effective for T790M, and first and second generation EGFR-TKIs are effective for C797S, so treatment is successful with a combination of existing EGFR-TKIs. On the other hand, when T790M and C797S exist in cis form (exist on the same allele), it has been found that all EGFR-TKIs, including existing EGFR-TKI combinations, are ineffective. By determining the positional relationship of the mutations related to sensitivity and resistance to such treatments, it is possible to develop effective therapeutic drugs and determine whether or not to administer the therapeutic drug, so a method for distinguishing between cis and trans gene mutations is important.

[0004] The following methods have been reported to distinguish between cis and trans mutations of T790M and C797S. First, hydrolysis probes labeled with different fluorescent dyes corresponding to T790M and C797S are prepared. DNA polymerase, primers, probes and enzymes required for PCR are added to the sample, and digital PCR is performed. In digital PCR, the reaction solution is divided into microcompartments such as wells or droplets, and amplification is performed in the microcompartments. In a trans-type sample, T790M and C797S exist in different genes, so one microcompartment contains only one gene mutation, and only one fluorescence is detected. In a cis-type sample, T790M and C797S exist in the same gene, so one microcompartment contains both genes, and two types of fluorescence are detected. Therefore, by counting the number of microcompartments where only one type of fluorescence is detected and the number of microcompartments where two types of fluorescence are detected, it is possible to determine whether the gene mutation present in the sample is cis or trans. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2018-108063 A (US2019 / 0352699A1) [Non-patent literature]

[0006] [Non-Patent Document 1] Uchibori,et al.,Nat.Commun.,8,14768,2017 [Non-Patent Document 2] Nakagawa,et al.,Anal.Chem.,92,11705-11713,2020 Summary of the Invention [Problem to be solved by the invention]

[0007] However, in digital PCR using hydrolysis probes labeled with two types of fluorescent dyes, if the copy number is high, the probability that T790M and C797S trans DNAs will simultaneously enter one microcompartment during fractionation increases, resulting in false detection as cis DNA. In addition, since T790M and C797S trans DNAs will stochastically enter one microcompartment at the same time, it is difficult to accurately quantify the ratio when cis and trans DNAs are mixed.

[0008] Therefore, an object of the present invention is to provide a DNA detection method and a DNA detection kit that can accurately determine whether multiple types of closely spaced gene mutations are in a cis or trans positional relationship and quantify them. [Means for solving the problem]

[0009] The present inventors have developed a technology that combines digital PCR with melting curve analysis by using a non-degradable probe such as a molecular beacon instead of a hydrolysis probe, and identifies the genotype of a target gene with high sensitivity and high multiplexing by melting curve analysis after amplification (Patent Document 1, Non-Patent Document 2). When a molecular beacon designed to correspond to multiple types of gene mutations in close proximity is prepared and a melting curve analysis is performed, it was found that the shape of the melting curve changes between the cis type and the trans type due to steric hindrance between the molecular beacons, and the melting temperature (Tm) calculated from the melting curve is also different. Based on the difference in the melting curve and melting temperature, it was found that the cis type and the trans type of multiple gene mutations can be determined and quantified, and the present invention was completed.

[0010] The present invention, in one aspect, comprises: preparing probes corresponding to each of a plurality of gene mutations; carrying out an amplification reaction using a solution containing primers for amplifying a region containing the plurality of gene mutations, the probe, a test biological sample, and an enzyme; After the step of carrying out the amplification reaction, a step of measuring the binding between the amplification product and the probe by melting curve analysis in which the temperature of the solution is changed; determining whether the multiple gene mutations in the DNA contained in the test biological sample are in cis form on the same allele or in trans form on different alleles based on the results of the melting curve analysis; The present invention provides a method for genetic analysis, comprising:

[0011] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: 1. A method of performing a genetic analysis on a subject's biological sample to assess a trait associated with a plurality of genetic variations, comprising: carrying out the above-mentioned method using a test biological sample from a subject, and determining whether multiple gene mutations in the test biological sample from the subject are in cis form on the same allele or in trans form on different alleles; assessing whether the subject has a trait associated with the plurality of genetic variations. The present invention provides a method comprising:

[0012] In a further aspect, the present invention provides a method for producing a method for the treatment of a cancer A primer pair including a forward primer and a reverse primer for amplifying a region including multiple gene mutations; a plurality of melting curve analysis probes each corresponding to a plurality of gene mutations, the probes binding to a product amplified using the forward primer and the reverse primer; Including, The probe has different melting curve characteristics or melting temperatures when the multiple gene mutations are present on the same allele and when they are present on different alleles. Effect of the Invention

[0013] The present invention provides a method and kit for gene analysis that can determine or quantify with high accuracy and high sensitivity whether multiple types of adjacent gene mutations are cis or trans in terms of their positional relationship, and is therefore useful in fields such as basic research, testing, and drug discovery that involve gene detection and genotype discrimination. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram showing an example of a method for measuring the melting temperature of DNA using a fluorescently labeled probe. [Diagram 2] FIG. 1 is a schematic diagram showing an example of a method for measuring the melting temperature of trans-type (A) and cis-type (B) gene mutations using a fluorescently labeled probe according to an embodiment of the present invention. [Diagram 3] FIG. 1 shows examples of melting curves when trans-type (A and B) and cis-type (C to F) mutations are measured using fluorescently labeled probes in one embodiment of the present invention. [Figure 4] FIG. 2 is a diagram showing an example of a first database storing temperatures for gene mutations in cis and trans in one embodiment of the present invention. [Diagram 5] 1 is a flow chart showing one embodiment of a genetic analysis method for determining whether a gene mutation in a biological sample is in cis or trans using melting curve analysis. [Figure 6] 1 is a flow chart showing one embodiment of a genetic analysis method for quantifying gene mutation copy number and cis / trans ratio of gene mutations by combining melting curve analysis and digital PCR. [Figure 7] FIG. 1 shows the measurement results when only T790M mutation, only C797S mutation, cis-type and trans-type genes were amplified in real-time PCR and melting curve analysis was performed in an example of the present invention. [Figure 8] FIG. 13 shows the measurement results when only T790M mutation, only C797S mutation, cis-type and trans-type genes were amplified in digital PCR and melting curve analysis was performed in an example of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The objectives, features, advantages, and ideas related thereto of the present invention will be apparent to those skilled in the art from the description of this specification. From the description of this specification, those skilled in the art can easily reproduce the present invention. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustrative or explanatory purposes, and do not limit the present invention thereto. It is apparent to those skilled in the art that various modifications and alterations can be made based on the description of this specification without departing from the intent and scope of the present invention disclosed in this specification.

[0016] (1) Genetic analysis method In one aspect, the present invention provides a method of genetic analysis, the method comprising: preparing probes corresponding to each of a plurality of gene mutations; carrying out an amplification reaction (e.g., PCR) using a solution containing primers for amplifying a region containing the multiple gene mutations, the probe, a test biological sample, and an enzyme; After the step of carrying out the amplification reaction, a step of measuring the binding between the amplification product and the probe by melting curve analysis in which the temperature of the solution is changed; determining whether the multiple gene mutations are on the same allele (cis type) or on different alleles (trans type) in the DNA contained in the test biological sample based on the results of the melting curve analysis; Includes.

[0017] As used herein, genetic analysis refers to determining whether multiple genetic mutations to be analyzed are present in a test biological sample, determining whether multiple genetic mutations are on the same allele (cis type) or on different alleles (trans type), measuring the abundance ratio or abundance of the cis and trans forms of multiple genetic mutations to be analyzed, monitoring the presence of the cis and trans forms of multiple genetic mutations, and the like.

[0018] In the present invention, the test biological sample is not particularly limited as long as it is a biological sample containing DNA that contains or may contain a gene mutation to be analyzed, and may be, for example, a body fluid sample such as blood or urine, tissue, or cultured cells, or may be DNA (genomic DNA, circulating DNA, etc.) extracted and purified from such samples, or may be DNA (cDNA, etc.) derived from mRNA extracted and purified from a sample. Alternatively, synthesized DNA may be used as the test biological sample for the design of suitable probes and primers. When the test biological sample is a body fluid sample, tissue, cultured cells, etc., it is preferable to pre-treat the DNA, etc., to make it available for the amplification reaction performed by the method of the present invention. Methods for preparing DNA from such samples are known in the art, and kits for easily purifying DNA and kits for extracting mRNA and synthesizing cDNA are also commercially available.

[0019] In the present invention, the genetic mutations to be analyzed are multiple genetic mutations that are desired to be determined as being on the same allele (cis type) or on different alleles (trans type). "On the same allele (cis type)" means that multiple genetic mutations exist on the same allele of two alleles in a chromosome, and "on different alleles (trans type)" means that multiple genetic mutations exist on different alleles in two alleles in a chromosome. Since it is desirable to perform melting curve analysis with each probe, not with the same probe, for multiple genetic mutations, it is desirable that the distance between the mutations is 5 bases or more. In addition, it is desirable that the distance between the mutations is closer than 50 bases so that a change in the melting curve occurs due to steric hindrance between the probes. Therefore, the multiple genetic mutations are, for example, 5 to 60 bases, preferably 5 to 40 bases apart. However, the genetic mutations may exist farther apart as long as a change in the melting curve occurs due to steric hindrance between the probes.

[0020] Specific examples of gene mutations to be analyzed include, but are not limited to, T790M and C797S in the epidermal growth factor receptor (EGFR) gene, T790M and L858R, exon 19 deletion mutation (activating mutation) in the EGFR gene, any combination of R88, R108, E453, E542, E545, E726, and H1047R mutations in the PIK3CA gene, and resistance mutations C1156Y, I1171N, I1171S, I1171T, F1174C, V1180L, L1196M, G1202R, G1202 deletion, D1203N, E1210K, G1269A, etc. in the ALK gene.

[0021] In the method of the present invention, a probe corresponding to each of a plurality of gene mutations is prepared. The probe is designed so that at least a part of the probe has a sequence specific to the base sequence of the gene mutation to be analyzed, that is, a sequence complementary to the base sequence of the gene mutation. Since a probe is required for each of a plurality of gene mutations, a number of probes corresponding to the type of gene mutation to be analyzed are prepared. Probe design methods are well known in the art, and probes usable in the present invention are designed to have a length and base composition (melting temperature) that allow specific binding (hybridization). For example, as a length that functions as a probe, the base length of the sequence portion specific to the gene mutation 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 of the probe and the melting temperature (Tm) of the probe. To confirm Tm, known probe design software can be used.

[0022] In the present invention, it is desirable that the ends of the probes are closer than 20 bases to each other so that the melting curve changes due to steric hindrance between the probes for each of the multiple gene mutations. If the ends of the probes overlap, the amount of binding decreases, but if the affinity of the probe is high, the fluorescence intensity of the fluorescent dye of the probe is high, or the fluorescence detection sensitivity of the detector is high, it is possible to detect the probes, so there may be an overlapping area between the ends of the probes. It is desirable that the ends of the probes are separated by 2 bases or more, or even 4 bases or more. If the ends of the probes are separated by 10 bases or more, it is desirable to adjust the amount of change in the melting curve due to steric hindrance by inserting a linker between the base sequence of the probe and the fluorescent dye or by changing the size of the fluorescent dye.

[0023] The probe contains a fluorescent dye, or a fluorescent dye and a quenching dye. When the probe contains a fluorescent dye, the fluorescent dye is used to measure the binding between the amplification product and the probe. In one embodiment, the 3'-end sequence and the 5'-end sequence of the probe have complementary sequence portions, and in a free state (when not bound to the amplification product), the 3'-end sequence and the 5'-end sequence of the probe bind to form a stem-loop structure. In other words, the probe is preferably a molecular beacon.

[0024] The designed probes can be chemically synthesized by known oligonucleotide synthesis techniques, but are usually synthesized using a commercially available chemical synthesis apparatus.

[0025] Next, an amplification reaction is carried out using a solution containing primers for amplifying a region containing multiple gene mutations, the above-mentioned probe, a test biological sample, and an enzyme.

[0026] Primer design techniques are also 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 primer design software 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.

[0027] The enzyme used in the amplification reaction is a polymerase that is capable of carrying out an extension reaction using DNA as a template, and any known polymerase can be used.

[0028] The amplification step can be carried out by any amplification reaction known in the art. Preferably, the amplification step is carried out by an asymmetric nucleic acid amplification reaction. In this case, the amount of either the forward primer or the reverse primer added is increased to carry out the amplification reaction. For asymmetric nucleic acid amplification reaction, see, for example, Anal.Chem., 92, 11705-11713, 2020 (Non-Patent Document 2).

[0029] In one embodiment, the binding between the amplification product and the probe is measured by melting curve analysis in which the temperature of the solution is changed. Since the binding changes depending on the melting temperature, the melting temperature of the double strand of the amplification product and the probe can be calculated from the change in binding accompanying the change in temperature of the solution. Alternatively, the binding between the amplification product and the probe is measured for each cycle of the temperature change of the solution, and the target gene mutation can be determined from the relationship between the number of cycles and the change in binding.

[0030] By performing the amplification reaction and melting curve analysis in the manner described above, it is possible to determine whether multiple gene mutations are present in cis form or in trans form in the DNA in the test biological sample based on the difference between the melting temperature or melting curve characteristics when multiple gene mutations are present in cis form and the melting temperature or melting curve characteristics when multiple gene mutations are present in trans form.

[0031] The gene analysis method of the present invention will be specifically described with reference to the schematic diagrams of FIGS.

[0032] Fig. 1 is a schematic diagram showing an example of a method for measuring the melting temperature of DNA using a fluorescently labeled probe. Examples of the fluorescently labeled probe include a molecular beacon and a Pleiades probe, but Fig. 1 will be explained here using a molecular beacon as an example.

[0033] The molecular beacon, a fluorescently labeled probe 102, is configured as an oligonucleotide and has a sequence complementary to the sequence (amplified product) between the primer pair used in the amplification reaction to amplify the target gene. The molecular beacon also has complementary sequence portions at both ends, with a fluorescent dye 103 at one end and a quenching dye (quencher) 104 at the other end. In the initial state (free state) of the amplification reaction, the molecular beacon 102 exists alone and free, as shown in B of FIG. 1. At that time, the molecular beacon 102 forms a stem loop at the complementary sequence portion, and the fluorescent dye 103 and the quencher 104 are close to each other, so no fluorescence is emitted. In the first denaturation step, when the sample solution is heated, it takes on a highly flexible structure as shown in C of FIG. 1, but the fluorescent dye and the quenching dye are not always separated, so the fluorescence remains quenched. In the annealing step, when the temperature is lowered to about room temperature, the loop portion of the molecular beacon 102 anneals to the DNA 101 amplified in the sample solution as shown in A of FIG. 1. This causes the fluorescent dye 103 and the quencher 104 to constantly separate, so the fluorescently labeled probe 102 emits strong fluorescence. In the next extension step, the molecular beacon 102 is released, and the state becomes again as shown in B of FIG. 1, and the fluorescence is quenched. In the next denaturation step, the state becomes again as shown in C of FIG. 1, and the fluorescence remains quenched. Since this step is repeated in the amplification reaction, the fluorescence intensity may be measured at some stage during heating or cooling. The same method can be used to measure the fluorescence intensity after the amplification reaction is completed. After the amplification reaction is completed, the fluorescence intensity may be measured by heating or cooling just to measure the fluorescence intensity. A melting curve is created by plotting the change in fluorescence intensity with the temperature change at this time, and the melting temperature can be calculated from the melting curve.

[0034] In the molecular beacon 102 used here, the combination of the fluorescent dye 103 and the quencher 104 is not particularly limited as long as it is a combination that is generally used in real-time PCR. For example, examples of the fluorescent dye 103 include FAM, VIC, ROX, Cy3, Cy5, etc., and examples of the quencher 104 include TAMRA, BHQ1, BHQ2, BHQ3, etc. All of them are commonly used and are commercially available.

[0035] When two types of gene mutations having different sequences are to be analyzed, the sequences of molecular beacons 102 that specifically bind to each of the gene mutations of interest can be prepared, and by binding different fluorescent dyes, it is possible to distinguish and detect the two gene mutations of interest in one reaction system. Alternatively, by designing the sequences of the molecular beacons so that the melting temperatures of the two types of molecular beacons are different, it is possible to distinguish and detect the two gene mutations of interest in one reaction system, even if the same fluorescent dye is bound to them.

[0036] FIG. 2 is a schematic diagram showing an example of a method for measuring the melting temperatures of trans- and cis-type gene mutations using a fluorescently labeled probe according to an embodiment of the present invention. In FIG. 2, the T790M mutation and the C797S mutation of the EGFR gene are shown as examples of gene mutations. T790M and C797S are gene mutations that occur 19 bases apart, and a fluorescently labeled probe (e.g., a molecular beacon) is prepared for each mutation. When a molecular beacon is used as the fluorescently labeled probe, the molecular beacon is designed to have a specific sequence in the loop such that the T790M or C797S mutation is near the center, and the loop sequence is 10 to 30 bases long. As shown in FIG. 2A, in the trans-type gene, T790M and C797S exist on separate alleles, so that only the fluorescently labeled probe (molecular beacon) 204 corresponding to T790M binds to the trans-type DNA 201 containing the T790M mutation, and only the fluorescently labeled probe (molecular beacon) 205 corresponding to C797S binds to the trans-type DNA 202 containing the C797S mutation. As shown in FIG. 2B, in the cis-type DNA 203, T790M and C797S exist on the same allele, so that the T790M-compatible molecular beacon 204 and the C797S-compatible molecular beacon 205 bind simultaneously. As a result, in the cis-type DNA 203, steric hindrance occurs between the T790M-compatible molecular beacon 204 and the C797S-compatible molecular beacon 205, and the binding of the molecular beacon is reduced compared to the trans-type DNAs 201 and 202.

[0037] When the specificity of the fluorescently labeled probes (molecular beacons) corresponding to T790M and C797S is low and they bind weakly to the wild types (T790 and C797), the fluorescently labeled probe corresponding to T790M and the fluorescently labeled probe corresponding to C797S bind to the trans-type DNA as well, resulting in a binding pattern similar to that of the cis-type. In this case, a probe (blocker) corresponding to a wild-type gene that is not modified with a fluorescent dye or quencher is used to inhibit non-specific binding of the fluorescently labeled probe and to prevent steric hindrance from occurring in the trans-type DNA. Specifically, the fluorescently labeled probe and blocker corresponding to T790M can bind to DNA having T790M, and the fluorescently labeled probe and blocker corresponding to C797S can bind to DNA having C797S without steric hindrance. A probe (blocker) corresponding to the wild-type gene can be appropriately designed by a person skilled in the art based on the base sequence of the wild-type gene. Therefore, in one embodiment, in the step of performing the amplification reaction, the amplification reaction can be performed by further adding a probe corresponding to the wild-type gene to the solution.

[0038] FIG. 3 shows an example of melting curves when trans- and cis-type mutations are measured using a fluorescently labeled probe. The melting curve when a trans-type mutation is measured is shown in FIG. 3A, and the differential curve of the melting curve is shown in FIG. 3B. As shown in FIG. 3A, at low temperatures, the fluorescently labeled probe binds to the corresponding DNA and shows high fluorescence intensity, and as the temperature rises, the fluorescently labeled probe dissociates from the corresponding DNA, decreasing the fluorescence intensity. When the differential curve is calculated from FIG. 3A, a high peak is shown at 60°C as shown in FIG. 3B, and the temperature of the peak is the melting temperature (Tm) 301. On the other hand, an example of a melting curve when a cis-type mutation is measured is shown in FIG. 3C, and the differential curve is shown in FIG. 3D. In the case of the cis-type, steric hindrance occurs between the fluorescently labeled probes corresponding to each mutation, so the binding amount of the fluorescently labeled probe decreases and the fluorescence intensity at low temperatures decreases, and after the fluorescently labeled probe corresponding to one of the mutations dissociates, the steric hindrance is eliminated and the fluorescence intensity changes to the same as in the trans-type, as shown in FIG. 3C. When the differential curve is obtained, the peak height is lowered as shown in FIG. 3D, and the peak temperature (melting temperature) 301 is shifted to the higher temperature side. Another example of the melting curve when measuring a cis-type mutation is shown in FIG. 3E, and its differential curve is shown in FIG. 3F. In the example of FIG. 3E, the fluorescently labeled probe corresponding to one of the mutations dissociates around 55° C., which relieves steric hindrance, increasing the amount of binding of the fluorescently labeled probe corresponding to the other mutation, and the fluorescence intensity increases from 55° C. to 60° C. Therefore, in FIG. 3F, which is the differential curve of FIG. 3E, a large negative peak 302 is generated between 55° C. and 60° C. In addition, the peak melting temperature 301 is shifted to the higher temperature side.

[0039] The melting curve features that can be used to distinguish between trans- and cis-type mutations include the following: melting temperature, the left-right balance of the width of the peak shape in the differential melting curve, the magnitude of the minimum value on the low-temperature side of the maximum value in the differential melting curve, the left-right slope of the peak in the differential melting curve and its balance (which can be calculated by second derivative), the height of the maximum value and the height of the minimum value in the differential melting curve, and the ratio thereof. Using any one or any combination of these features, it is possible to determine whether multiple gene mutations are present and, if present, whether they are trans-type or cis-type.

[0040] FIG. 4 is a diagram showing an example of the first database storing the temperatures for each of the gene mutations in the cis and trans forms. As explained in FIG. 3, the melting temperature changes between the cis and trans forms. Therefore, the melting temperatures for T790M and C797S in the cis and trans forms are measured in advance using control DNA and stored in the database 400 as shown in FIG. 4. By measuring a biological sample with unknown gene mutation information and measuring the melting temperature, it is possible to determine whether the gene mutation in the biological sample is cis or trans by comparing it with the melting temperature in the database 400. If the melting temperature of the trans form of T790M is observed and neither the trans form nor the cis form of C797S is observed, it can also be determined that only T790M is present and C797S is not present. Therefore, in one embodiment, the first database that stores the melting temperature information of each of the cis and trans forms of multiple gene mutations can be used to compare with the melting temperature of the DNA contained in the test biological sample, and determine whether multiple gene mutations are cis or trans.In this case, for example, the first database is used to count the copy number of gene mutations, the copy number of cis gene mutations, and the copy number of trans gene mutations for the DNA contained in the test biological sample.

[0041] In FIG. 4, T790M and C797S are used as examples of gene mutations to be stored in the database 400, but the gene mutations are not limited to these and are also not limited to two types, but may be three or more types.

[0042] In addition, as an example of information to be stored in the database, the feature amount of the shape of the melting curve and its differential curve may be as shown in FIG. 3, for example, a depression added to a sigmoid curve such as the melting curve E in FIG. 3, or the magnitude of a negative peak such as the differential curve of the melting curve F in FIG. 3. Therefore, in one embodiment, a second database storing features of melting curves corresponding to each of the cis and trans forms of multiple gene mutations can be used to compare with the melting curve for DNA contained in the test biological sample to determine whether the multiple gene mutations are cis or trans. The features of the melting curve stored in such a second database include, but are not limited to, at least one selected from the group consisting of melting temperature, left-right balance of the width of the peak shape in the differential curve of the melting curve, the magnitude of the minimum value on the low-temperature side of the maximum value in the differential curve of the melting curve, the left-right slope of the peak in the differential curve of the melting curve and the balance thereof, the height of the maximum value and the height of the minimum value in the differential curve of the melting curve, and the ratio thereof. In this case, for example, the second database is used to count the copy numbers of gene mutations, cis-type gene mutations, and trans-type gene mutations for DNA contained in the test biological sample.

[0043] 5 is a flow chart showing an embodiment of a gene analysis method for determining whether a gene mutation in a biological sample is cis-type or trans-type using melting curve analysis. First, a probe corresponding to each of multiple types of adjacent mutations is designed (S501). The probe, biological sample, enzyme, and primers are mixed and an amplification reaction (PCR) is performed (S502). A melting curve between the probe corresponding to each mutation and the amplified DNA is obtained by melting curve analysis (S503). The melting temperature and the characteristics of the melting curve are compared with values ​​in a database to determine whether the adjacent mutations are present in the same allele or different alleles (S504).

[0044] The amplification step by PCR can be carried out by any amplification reaction known in the art. For example, it is preferable to use a quantifiable amplification reaction as the amplification reaction, for example, real-time PCR, digital PCR, etc. can be used. Preferably, the amplification step is carried out by an asymmetric nucleic acid amplification reaction. In this case, the amount of either the forward primer or the reverse primer added is increased to carry out the amplification reaction. For asymmetric nucleic acid amplification reaction, please refer to the description in, for example, Anal.Chem., 92, 11705-11713, 2020 (Non-Patent Document 2).

[0045] The binding between the amplification product and the probe is measured with a change in temperature. Since the binding changes depending on the melting temperature, the melting temperature of the double strand of the amplification product and the probe can be calculated from the change in binding that accompanies the temperature change.

[0046] The binding between the amplified product and the probe is measured for each temperature change cycle, and the target gene mutation can be detected from the relationship between the number of cycles and the change in binding. If the fluorescent dye of the probe is different for each mutation, the binding between the amplified product and the probe is measured for each temperature change cycle while changing the fluorescent filter, and the ratio of the initial concentrations of multiple target gene mutations in the DNA of the test biological sample can be calculated from the relationship between the number of cycles and the change in binding.

[0047] The binding between the amplification product and the probe is measured for each cycle of temperature change, and the relationship between the number of cycles and the change in binding is compared with the change in binding in a sample containing a known concentration of the target gene mutation, allowing the initial concentration of the target gene mutation, the concentration of which is unknown, in the DNA of the test biological sample to be calculated.

[0048] For example, the T790M probe is modified with the fluorescent dye FAM, and the C797S probe is modified with the fluorescent dye Cy5. Each probe is mixed with a biological sample containing the DNA of the target gene mutation, enzyme, and primers, and real-time PCR is performed. In real-time PCR, the fluorescence intensity due to the binding of the probe and the amplified product is measured while changing the fluorescent filter for each temperature change cycle. The relationship between the number of cycles and the fluorescence intensity is compared with the change in fluorescence intensity of a sample containing a known concentration of the target gene mutation, and the initial concentrations of T790M and C797S in the biological sample can be calculated. After real-time PCR, a melting curve analysis is performed to obtain the melting curves of FAM and Cy5, their differential curves, and the melting temperatures calculated from the differential curves. The melting temperatures obtained in advance using control DNA are compared with the values ​​in a database that stores the melting temperatures, and it is determined whether T790M and C797S are in the cis or trans configuration. When the melting temperature is intermediate between the cis and trans forms, or when the shape of the melting curve is intermediate between the cis and trans forms, it is determined that both cis and trans forms are present.

[0049] FIG. 6 is a flow chart showing an embodiment of a gene analysis method for quantifying the copy number of a gene mutation and the ratio of cis-type and trans-type of a gene mutation by combining melting curve analysis and digital PCR. First, a probe corresponding to each of multiple adjacent mutations is designed (S601). The probe, biological sample, enzyme, and primer are mixed, divided into wells, and PCR is performed (S602). A melting curve of the probe corresponding to each mutation and the amplified product is obtained by melting curve analysis (S603). For each well, the melting temperature and melting curve characteristics are compared with the values ​​in the database to determine whether the adjacent mutations are present in the same allele, different alleles, only one mutation, or neither mutation (S604). Finally, wells containing DNA of a mutation present in the same allele, wells containing DNA of a mutation present in different alleles, wells containing DNA of only one mutation, and wells containing neither mutation are counted, and the initial concentration of each is calculated by Poisson correction (S605).

[0050] For example, the T790M probe is modified with the fluorescent dye FAM, and the C797S probe is modified with the fluorescent dye Cy5. Each probe is mixed with a biological sample containing the nucleic acid of the target gene, enzyme, and primers, and then divided into wells. After PCR, melting curve analysis is performed to obtain the melting curves of FAM and Cy5, their differential curves, and the melting temperatures calculated from the differential curves. For each well, the melting temperatures obtained in advance using a control nucleic acid are compared with the values ​​in a database to determine whether T790M and C797S are in the cis or trans position. Even if one each of trans-type T790M and C797S nucleic acids are placed in the same well, the corresponding melting temperatures are obtained, so there is no risk of misjudging them as cis. Wells containing cis-type nucleic acids, wells containing one each of trans-type T790M and C797S nucleic acids, wells containing T790M nucleic acids, wells containing C797S nucleic acids, and wells containing neither mutation are counted, and the initial concentration of each is calculated using Poisson correction.

[0051] (2) Methods for evaluating traits associated with multiple gene mutations As described above, the method of the present invention can determine whether multiple gene mutations are present and, if present, whether they are cis-type or trans-type, and can therefore be used to evaluate traits associated with the gene mutations, such as drug responsiveness.

[0052] Thus, in one aspect, the present invention provides a method of performing a genetic analysis on a subject's test biological sample to assess a trait associated with a plurality of genetic variations, the method comprising: performing the genetic analysis method using a biological sample from a subject, and determining whether multiple genetic mutations in the biological sample from the subject are in cis form on the same allele or in trans form on different alleles; Assessing whether a subject has a trait associated with multiple genetic variations Includes.

[0053] Traits associated with multiple gene mutations include, for example, susceptibility to disease or disorders, or drug responsiveness. For example, the drug responsiveness of lung cancer patients differs between the cis and trans forms of the EGFR gene mutations T790M and C797S, and all EGFR-TKIs are effective for patients without gene mutations, and when the mutations T790M and C797S exist in the trans form, the EGFR-TKIs gefitinib, erlotinib, afatinib, and dacomitinib are effective for proteins with the mutation C797S, and the EGFR-TKI osimertinib is effective for proteins with the mutation T790M, while when the mutations T790M and C797S exist in the cis form, the above drugs are not effective.

[0054] Furthermore, for example, when the above-mentioned trait is drug responsiveness, after determining multiple gene mutations in the subject, an appropriate drug can be selected and administered to the subject. Therefore, in another aspect, the present invention provides a method for treating or preventing a disease in a subject, comprising the steps of determining multiple gene mutations associated with drug responsiveness using a biological sample from the subject, evaluating the drug responsiveness of the subject, and administering an appropriate drug to the subject. EGFR gene mutations T790M and C797S are specifically described as an example. A biological sample from a lung cancer patient, such as blood, is prepared. As described above, the DNA in the biological sample is examined for the presence or absence of EGFR gene mutations T790M and C797S, and if a gene mutation is present, whether it is cis or trans. If the target lung cancer patient does not have a gene mutation, at least one drug selected from all EGFR-TKIs is administered. When the mutation T790M and the mutation C797S are present in the trans form, the subject is administered at least one drug selected from EGFR-TKI gefitinib, erlotinib, afatinib, and dacomitinib, and osimertinib. On the other hand, when the subject lung cancer patient has the mutation T790M and C797S in the cis form, the efficacy of the EGFR-TKI drug is low, and it is necessary to select a treatment method or a therapeutic drug other than the above-mentioned EGFR-TKI drug (for example, brigatinib, an ALK tyrosine kinase inhibitor). In this way, the subject can be administered and treated with a drug having the optimal effect, and unnecessary drug administration can be avoided.

[0055] (3) Genetic analysis kit The above-mentioned method of the present invention can be carried out more easily and simply by using a kit containing a plurality of fluorescently labeled probes for melting curve analysis corresponding to at least a plurality of gene mutations.

[0056] That is, in one aspect, the present invention provides a genetic analysis kit, the kit comprising: A primer pair including a forward primer and a reverse primer for amplifying a region including multiple gene mutations; a plurality of melting curve analysis probes each corresponding to a plurality of gene mutations, the probes binding to products amplified using a forward primer and a reverse primer; Including, The probes have different melting curve characteristics or melting temperatures when the multiple genetic variations are present on the same allele versus when they are present on different alleles.

[0057] The forward primer and reverse primer are as described in the previous section. In one embodiment, the concentration of the forward primer and the concentration of the reverse primer included in the kit of the present invention are different, and one of the primers is higher (due to asymmetric nucleic acid amplification reaction). It is preferable that the concentration of the primer that synthesizes the complementary strand of the melting curve analysis probe is higher.

[0058] The kit of the present invention may further include other components necessary for carrying out an amplification reaction, such as a DNA polymerase, a substrate, etc. In addition, the kit may include instructions describing procedures and protocols for determining multiple gene mutations.

[0059] When there are multiple sets of each of the multiple gene mutations to be analyzed, multiple primers and multiple probes may be prepared according to the gene mutations to be detected, and added simultaneously to perform an amplification reaction (e.g., asymmetric nucleic acid amplification reaction) on a biological sample containing DNA of the multiple gene mutations to be analyzed. By designing the multiple probes to have different melting temperatures with the DNA of each gene mutation to be analyzed or different types of fluorescent dyes, it is possible to distinguish the type of gene mutation to be analyzed contained in the solution from the fluorescent color and melting temperature of the solution after the amplification reaction.

[0060] The gene analysis kit of the present invention is useful for determining the presence or absence of multiple gene mutations and for determining the positional relationship (cis type or trans type) of these gene mutations on the alleles.

[0061] Furthermore, the genetic analysis kit of the present invention can be used to evaluate traits associated with multiple gene mutations (eg, susceptibility to a disease or disorder, drug responsiveness, etc.). EXAMPLES

[0062] [Example 1] In this example, an example is shown in which only the T790M mutation in the EGFR gene, only the C797S mutation, and the cis and trans forms of both mutations are amplified in real-time PCR and discriminated by melting curve analysis.

[0063] Genomic DNA (final concentration 133 molecules / μL) of the EGFR gene with only the T790M mutation, only the C797S mutation, and both cis and trans mutations was prepared, and the forward primer (final concentration 0.25 μM), reverse primer (final concentration 2.0 μM), fluorescently labeled probe corresponding to the T790M mutation (final concentration 0.5 μM), fluorescently labeled probe corresponding to the C797S mutation (final concentration 0.5 μM), blocker for the wild type corresponding to T790M (final concentration 0.5 μM), blocker for the wild type corresponding to C797S (final concentration 0.5 μM), and 1x master mix (containing DNA polymerase and dNTP) were added to prepare a PCR reaction solution. At this time, the concentrations of the primer pairs were added asymmetrically so that the complementary DNA strand of the fluorescently labeled probe was excessively amplified (the concentration of the reverse primer added was set high). The sequences of the primers, probes, and blockers are as follows.

[0064] Forward primer: 5'-CCTCACCTCCACCGTGCA-3' (SEQ ID NO: 1) Reverse primer: 5'-TCTTTGTGTTCCCGGACATAGTC-3' (SEQ ID NO: 2) Fluorescently labeled probe corresponding to the T790M mutation: 5'-TCATCATGCAGCTCAT-3' (SEQ ID NO: 3) Fluorescently labeled probe corresponding to the C797S mutation: 5'-CTTCGGCAGCCTCCTG-3' (SEQ ID NO: 4) Blocker for wild type corresponding to T790M: 5'-GCT C ATC ACG CA GC T-3' (SEQ ID NO: 5) (the underlined part represents the special base LNA) Blocker for wild type corresponding to C797S: 5'-CTTCGG CTG CCTCCT-3' (SEQ ID NO: 6) (the underlined part represents the special base LNA)

[0065] The forward primer and reverse primer correspond to the sequences of 2348-2360 and 2398-2420 in the DNA sequence of the human EGFR gene. EasyBeacon (PentaBase) was used for both fluorescently labeled probes, which have hydrophobic special bases near both ends and are designed to form hydrophobic bonds within the molecule in the free fluorescently labeled probe. The fluorescently labeled probe corresponding to the T790M mutation has FAM as a fluorescent dye bound to the 5' end and BHQ-1 as a quencher bound to the 3' end, while the fluorescently labeled probe corresponding to the C797S mutation has Cy5 as a fluorescent dye bound to the 5' end and BHQ-2 as a quencher bound to the 3' end. Furthermore, the blocker for the wild type contains the special base LNA (Locked Nucleic Acid) known to have the effect of increasing the Tm value in the sequence, and a special base (terminator) that inhibits the extension reaction is bound to the 3' end.

[0066] The PCR reaction was carried out at 95°C for 20 seconds, followed by 60 cycles of (95°C, 1 second → 60°C, 20 seconds). After the reaction, the change in fluorescence intensity was observed using a real-time PCR device while heating from 50°C to 95°C, and the melting curve was measured and analyzed.

[0067] FIG. 7A shows a melting curve measured with a FAM filter for observing the fluorescent dye FAM bound to the fluorescently labeled probe corresponding to the T790M mutation. FIG. 7B shows a differential curve of the melting curve of FIG. 7A. In FIG. 7B, when comparing the melting temperatures, which are the peak tops of the differential curves of the melting curves of each genomic DNA, it can be seen that the melting temperature of the cis genomic DNA is 2 to 3° C. higher than that of the genomic DNA containing only the T790M mutation and the trans genomic DNA (shift in melting temperature indicated by arrow 701).

[0068] Figure 7C shows melting curves measured with a Cy5 filter to observe the fluorescent dye Cy5 bound to the fluorescently labeled probe corresponding to the C797S mutation. Comparing the shapes of the melting curves of each genomic DNA, the genomic DNA with only the C797S mutation and the trans-type genomic DNA are sigmoid curves, whereas the cis-type genomic DNA shows a dip where the fluorescence intensity decreases and increases from 50°C to 65°C. The dip shape of the melting curve shown by 702 is considered to be due to the reaction explained below. Figure 7B shows that the melting temperature at which the fluorescently labeled probe corresponding to the T790M mutation binds to the cis-type genomic DNA is 61°C, and Figure 7A shows that the amount of probe binding is reduced because the fluorescence intensity decreases in the high temperature range above 55°C. Therefore, at temperatures above 55°C, the binding of the fluorescently labeled probe corresponding to the T790M mutation to cis-type genomic DNA decreases, while the binding of the fluorescently labeled probe corresponding to the C797S mutation to cis-type genomic DNA becomes easier, resulting in an increase in fluorescence intensity at temperatures above 55°C.

[0069] FIG. 7D shows the differential curve of the melting curve measured with a Cy5 filter for observing the fluorescent dye Cy5 bound to the fluorescently labeled probe corresponding to the C797S mutation shown in FIG. 7C. As in the results for T790M in FIG. 7B, it can be seen that the melting temperature of the cis genomic DNA is 2° C. higher than that of the genomic DNA with only the C797S mutation and the trans genomic DNA. Furthermore, the cis genomic DNA has a large negative peak around 60° C. in FIG. 7D, which corresponds to the depression between 50° C. and 65° C. in FIG. 7C (the negative peak on the lower side than the melting temperature of the differential curve of the melting curve, indicated by the arrow 703). When a sample containing a mixture of cis genomic DNA and trans genomic DNA or genomic DNA with only the C797S mutation is measured, the rate of shift in melting temperature and the depth of the large negative peak around 60°C in the differential melting curve are smaller than when a sample containing only cis genomic DNA is measured. By evaluating the degree of shift compared to when a sample containing only cis genomic DNA is measured, the mixture ratio of cis genomic DNA and trans genomic DNA or genomic DNA with only the C797S mutation can be estimated.

[0070] In this way, by performing melting curve analysis using fluorescently labeled probes corresponding to adjacent gene mutations, it is possible to determine from the shape of the melting curve and the melting temperature value whether the gene mutations are on the same allele (cis type) or on different alleles (trans type), whether only one mutation is present, or whether no mutations are present at all.

[0071] [Example 2] In this example, an example is shown in which only the T790M mutation in the EGFR gene, only the C797S mutation, and the cis and trans forms of both mutations are amplified in digital PCR and discriminated by melting curve analysis.

[0072] Genomic DNA (final concentration 133 molecules / μL) of only the T790M mutation of the EGFR gene, only the C797S mutation, the cis type and the trans type of both mutations were prepared, and the forward primer (final concentration 0.25 μM) required for PCR, the reverse primer (final concentration 2.0 μM), the fluorescently labeled probe corresponding to the T790M mutation (final concentration 0.5 μM), the fluorescently labeled probe corresponding to the C797S mutation (final concentration 0.5 μM), the blocker for the wild type corresponding to T790M (final concentration 0.5 μM), the blocker for the wild type corresponding to C797S (final concentration 0.5 μM), and 1x master mix (containing DNA polymerase and dNTP) were added to prepare a PCR reaction solution. At this time, the primer pair was added so that the concentration was asymmetric so that the complementary DNA strand of the fluorescently labeled probe was excessively amplified (the concentration of the reverse primer added was set high). The primers, probes, and blockers were the same as those used in Example 1.

[0073] Afterwards, 15μL of PCR reaction solution was divided into 20,000 wells and DNA was amplified by PCR. The PCR reaction was carried out at 96℃ for 10 minutes, followed by 59 cycles of (60℃, 2 minutes → 98℃, 30 seconds), and finally at 60℃ for 2 minutes. After the reaction, the chip with the wells was heated on a temperature-controlled stage from 50℃ to 85℃, while the change in fluorescence intensity of each well was observed using a digital PCR device compatible with melting curve analysis, and the melting curve was measured and analyzed.

[0074] FIG. 8A shows a melting curve obtained by performing digital PCR on trans-type genomic DNA and measuring it with a FAM filter for observing the fluorescent dye FAM bound to the fluorescently labeled probe corresponding to the T790M mutation. FIG. 8B shows a melting curve obtained by performing digital PCR on cis-type genomic DNA and measuring it with a FAM filter. FIG. 8C shows a graph showing the relationship between the melting temperature and the fluorescence intensity calculated from the melting curve in FIG. 8A. FIG. 8D shows a graph showing the relationship between the melting temperature and the fluorescence intensity calculated from the melting curve in FIG. 8B. Comparing FIG. 8C and FIG. 8D, it can be seen that the melting temperature of the well containing the cis-type genomic DNA is higher than the melting temperature of the well containing the T790M mutation in the trans-type genomic DNA.

[0075] FIG. 8E shows a melting curve obtained by performing digital PCR on trans-type genomic DNA and measuring it with a filter for Cy5 to observe the fluorescent dye Cy5 bound to the fluorescently labeled probe corresponding to the C797S mutation. FIG. 8F shows a melting curve obtained by performing digital PCR on cis-type genomic DNA and measuring it with a filter for FAM. As shown in FIG. 8F, a depression 801 in the melting curve is observed in the well containing the cis-type genomic DNA. FIG. 8G shows a graph showing the relationship between the melting temperature and the fluorescence intensity calculated from the melting curve in FIG. 8E. FIG. 8H shows the relationship between the melting temperature and the fluorescence intensity calculated from the melting curve in FIG. 8F. By comparing FIG. 8G and FIG. 8H, it can be seen that the melting temperature of the well containing the cis-type genomic DNA is higher than the melting temperature of the well containing the C797S mutation in the trans-type genomic DNA.

[0076] In this way, by performing digital PCR using fluorescently labeled probes corresponding to each mutation in adjacent gene mutations and performing melting curve analysis, it is possible to determine for each well whether the gene mutations are on the same allele (cis type), on different alleles (trans type), whether only one mutation is present, or whether no mutations are present, based on the shape of the melting curve and the melting temperature value. As a result, it is possible to count and quantify genes on the same allele (cis type), genes on different alleles (trans type), and genes with only one mutation, based on the determination results for each well. [Explanation of symbols]

[0077] 101...DNA 102...Fluorescently labeled probe 103...Fluorescent dye 104…Quencher 201…Trans-type DNA containing the T790M mutation 202…C797S mutation in trans DNA 203…Cis-type DNA containing the T790M and C797S mutations Fluorescently labeled probe for the 204…T790M mutation Fluorescently labeled probe for the 205…C797S mutation 301...Melting temperature 302: Negative peak below the melting temperature 400...Database 701...Melting temperature shift 702... Depression in the melting curve 703...Negative peak on the lower side of the melting temperature of the differential curve of the melting curve 801... Melting curve depression

Claims

1. preparing probes corresponding to each of a plurality of gene mutations; carrying out an amplification reaction using a solution containing primers for amplifying a region containing the plurality of gene mutations, the probe, a test biological sample, and an enzyme; After the step of carrying out the amplification reaction, a step of measuring the binding between the amplification product and the probe by melting curve analysis in which the temperature of the solution is changed; determining whether the multiple gene mutations in the DNA contained in the test biological sample are in cis form on the same allele or in trans form on different alleles based on the results of the melting curve analysis; A genetic analysis method comprising:

2. the probe comprises a fluorescent dye, or comprises a fluorescent dye and a quencher dye; The method of claim 1 , wherein the fluorescent dye is used to measure binding between the amplification product and the probe.

3. The method according to claim 1, wherein the 3'-end sequence and the 5'-end sequence of the probe have complementary sequence portions, and the probe forms a stem-loop structure in a free state.

4. The method of claim 1 , wherein the step of performing an amplification reaction is performed by an asymmetric nucleic acid amplification reaction.

5. The method of claim 1, further comprising: comparing a melting temperature for DNA contained in the test biological sample with a first database storing melting temperature information corresponding to each of the cis and trans forms of the multiple gene mutations to determine whether the multiple gene mutations are in the cis form or the trans form.

6. The method according to claim 5, further comprising using a first database to count the copy number of the gene mutation, the copy number of the cis-type gene mutation, and the copy number of the trans-type gene mutation for DNA contained in the test biological sample.

7. The method of claim 1, further comprising: comparing a melting curve for DNA contained in the test biological sample with a second database storing melting curve characteristics corresponding to the cis and trans forms of the plurality of gene mutations to determine whether the plurality of gene mutations are in the cis or trans form.

8. 8. The method according to claim 7, wherein the features of the melting curve stored in the second database include at least one selected from the group consisting of melting temperature, left-right balance of the width of the peak shape in the differential curve of the melting curve, magnitude of the minimum value on the low-temperature side of the maximum value in the differential curve of the melting curve, left-right slopes of the peak in the differential curve of the melting curve and their balance, height of the maximum value and height of the minimum value in the differential curve of the melting curve, and a ratio thereof.

9. The method according to claim 7, further comprising using a second database to count the copy number of the gene mutation, the copy number of the cis-type gene mutation, and the copy number of the trans-type gene mutation for DNA contained in the test biological sample.

10. The method according to claim 1 , wherein in the step of carrying out the amplification reaction, a probe corresponding to a wild-type gene is further added to the solution to carry out the amplification reaction.

11. 1. A method of performing a genetic analysis on a subject's biological sample to assess a trait associated with a plurality of genetic variations, comprising: A step of carrying out the method according to claim 1 using a subject's biological sample to determine whether multiple gene mutations in the subject's biological sample are in cis form on the same allele or in trans form on different alleles; assessing whether the subject has a trait associated with the plurality of genetic variations. The method includes:

12. The method of claim 11 , wherein the trait comprises disease or disorder susceptibility or drug responsiveness.

13. A primer pair including a forward primer and a reverse primer for amplifying a region including multiple gene mutations; a plurality of melting curve analysis probes each corresponding to a plurality of gene mutations, the probes binding to a product amplified using the forward primer and the reverse primer; Including, A gene analysis kit, wherein the probe has different melting curve characteristics or melting temperatures when the multiple gene mutations are present on the same allele and when they are present on different alleles.

14. A genetic analysis kit according to claim 13 for carrying out the method according to claim 1.

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