Method for producing genomic DNA as reference substance for calibration used in analysis of mutation, and method for analyzing genomic DNA, cell, and target sequence mutation

Abstract

This method for producing genomic DNA as a reference substance for calibration used in analysis of mutation of a target sequence involves a step for introducing a mutated target sequence and an internal reference sequence into genomic DNA.

Description

Method for producing genomic DNA as a calibration standard for use in mutation analysis, and method for analyzing mutations in genomic DNA, cells, and target sequences

[0001] The present invention relates broadly to methods for producing genomic DNA as a calibration standard for use in mutation analysis, and to methods for analyzing mutations in genomic DNA, cells, and target sequences.

[0002] Chemotherapeutic agents that target abnormal enzymes with amino acid mutations are called molecularly targeted drugs. In order to administer molecularly targeted drugs, it is necessary to first check for the presence of the amino acid mutation using a diagnostic drug, which is called a companion diagnostic drug. Among companion diagnostic drugs, diagnostic drugs that indirectly check for amino acid mutations by detecting gene mutations are called gene mutation diagnostic drugs.

[0003] The sensitivity of gene mutation diagnostic agents, particularly the sensitivity for detecting mutant genes, is defined by the variant allele frequency (VAF), which is an index of the frequency of mutant genes, and the detection sensitivity is evaluated using a calibration reference material with a defined VAF. For diagnostic standardization and quality control, it is necessary to use a common standard such as a calibration reference material, and known examples of such materials include plasmids, yeast, and genomic DNA (Non-Patent Documents 1 and 2).

[0004] A known method for determining the VAF of a calibration reference material is to use the copy number of a mutant gene in the calibration reference material measured by droplet digital PCR (ddPCR), for example. When using the copy number of a mutant gene, a validated droplet digital PCR reagent is required for each mutant gene. However, if a validated droplet digital PCR reagent for the target mutant gene is not commercially available, it is necessary to design and validate a probe to be used in droplet digital PCR. Therefore, the more types of mutant genes of interest there are, the more effort is required.

[0005] H White et al. A certified plasmid reference material for the standardization of BCR-ABL1 mRNA quantification by real-time quantitative PCR. Leukemia. 2015, 29, 369-376. Xin He et al. Development of a new genetic reference material system based on Saccharomyces cerevisiae cells. Molecular Therapy: Methods & Clinical Development. 2021, 20, 473-482.

[0006] To determine VAF, the copy number of the mutant gene must be accurately measured, and ddPCR is the most reliable validated method available at present.

[0007] The problem to be solved by the present invention is to provide a novel method for producing a calibration reference material that enables accurate calculation of VAF using ddPCR regardless of the copy number of the mutant gene. Another problem to be solved by the present invention is to provide a novel method for producing a calibration reference material that enables easy calculation of VAF for multiple calibration reference materials having various mutant genes without preparing validated droplet digital PCR reagents for each mutant gene.

[0008] The present inventors have conducted extensive research into the step of introducing a mutant gene into genomic DNA in a method for producing genomic DNA as a calibration reference material, and as a result have discovered a novel method for producing genomic DNA as a calibration reference material that enables VAF to be calculated without using the copy number of the mutant gene.

[0009] That is, this application encompasses the following inventions: [1] A method for producing genomic DNA as a calibration standard material used in analyzing mutations in a target sequence, the method comprising the step of introducing a mutated target sequence and an internal standard sequence into genomic DNA. [2] The method according to [1], wherein the introduced target sequence and internal standard sequence are derived from a recombinant vector. [3] The method according to [2], wherein the recombinant vector is a transposon-based vector. [4] The method according to [2] or [3], wherein the recombinant vector is produced by a method comprising: 1) a step of cleaving a recombinant vector having a base sequence in the following order: a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence; the two first restriction enzyme recognition sequences are arranged so that the two cleavage sites for the first restriction enzyme are located on opposite sides of the second restriction enzyme recognition sequence; the first restriction enzyme recognition sequence is a type IIS restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence; and 2) a step of ligating a double-stranded nucleic acid comprising a region consisting of a polymorphic base in a target sequence and its 5'- and 3'-flanking sequences and their complementary sequences. [5] The method according to any one of [1] to [4], wherein the introduced sequence further comprises a selectable marker. [6] The method according to any one of [1] to [5], wherein the mutation is a polymorphism. [7] The method according to any one of [1] to [6], wherein the analysis is a companion diagnostic. [8] The method according to any one of [1] to [7], wherein the calibration standard material is used in validation of a companion diagnostic. [9] Genomic DNA produced by the method according to any one of [1] to [8].

[10] A cell having one or more copies of genomic DNA into which a mutated target sequence and an internal standard sequence have been introduced.

[11] The cell according to

[10] , wherein the number of chromosomes is diploid.

[12] A method for analyzing mutations in a target sequence, comprising: 1) subjecting genomic DNA into which a mutated target sequence and an internal standard sequence have been introduced to an amplification reaction; and 2) calculating the copy number of the introduced internal standard sequence and a different internal standard sequence present in the genomic DNA.

[13] The method of

[12] , further comprising a step of calculating a variant allele frequency (VAF) based on the copy number.

[14] The method of

[13] , wherein the VAF is a percentage of a value calculated from the following formula: (copy number of the introduced internal standard sequence - copy number of a different internal standard sequence present in the genomic DNA) / copy number of the introduced internal standard sequence.

[15] The method of any one of

[12] to

[14] , wherein the method is used to evaluate the sensitivity of a reagent for detecting a mutation.

[16] The method of

[15] , wherein the evaluation of the sensitivity is used as an index for validation of the reagent for detecting the mutation.

[17] The method of any one of

[12] to

[16] , wherein the amplification reaction is performed by PCR.

[18] The method of

[17] , wherein the PCR is droplet digital PCR.

[0010] According to the present invention, it is possible to produce genomic DNA as a calibration reference material, which allows VAF to be calculated by using the copy number of an internal standard gene derived from genomic DNA, without using the copy number of a mutant gene.

[0011] FIG. 5 shows a schematic diagram of the arrangement of a first restriction enzyme recognition sequence and a second restriction enzyme recognition sequence in a recombinant vector when the first restriction enzyme recognition sequence cleaves a site 1 to 5 bases away from the recognition sequence. FIG. 5 shows a schematic diagram of the case where a double-stranded nucleic acid is inserted and ligated after treatment with a first restriction enzyme recognition sequence that cleaves a site 1 to 5 bases away from the recognition sequence. FIG. 5 shows a structural diagram of Tol2 pDNA (SEQ ID NO: 1). To confirm the arbitrariness and robustness of the second internal control gene, the copy numbers of the AGO1 gene, ERBB2 gene, and TMEM11 gene were analyzed in HEL299 cells without transfection and in HEL299 cells with the gene stably introduced. The left side of FIG. 5 shows the relationship between the copy number of the BRAF WT gene and the copy number of the TMEM11 gene. The center of FIG. 5 shows the relationship between the copy number of the BRAF V600E1 gene and the value obtained by subtracting the copy number of the TMEM11 gene from the copy number of the PTEN gene. The right side of Figure 5 shows the relationship between VAF calculated from the copy number of the BRAF WT gene and the copy number of the BRAF V600E1 gene, and VAF calculated from the copy number of the PTEN gene and the copy number of the TMEM11 gene. The left side of Figure 6 shows the relationship between the copy number of the KRAS WT gene and the copy number of the AGO1 gene. The center of Figure 6 shows the relationship between the copy number of the KRAS G12A gene and the value obtained by subtracting the copy number of the AGO1 gene from the copy number of the PTEN gene. The right side of Figure 6 shows the relationship between VAF calculated from the copy number of the KRAS WT gene and the copy number of the KRAS G12A gene, and VAF calculated from the copy number of the PTEN gene and the copy number of the AGO1 gene.

[0012] Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described, but the scope of the present invention should not be interpreted as being limited to the following embodiment.

[0013] (Method for producing genomic DNA as a calibration reference material for use in analyzing mutations in a target sequence) In a first embodiment, a method for producing genomic DNA as a calibration reference material for use in analyzing mutations in a target sequence is provided, the method comprising the step of introducing a mutated target sequence and an internal standard sequence into the genomic DNA.

[0014] As used herein, "mutation" refers to one or more bases in which a base substitution, deletion, or insertion has occurred, or the state in which the base substitution or the like has occurred. "Mutation" can be used interchangeably with "polymorphism." The position of the base where the base substitution, deletion, or insertion has occurred, the number of bases substituted or deleted, and the number of bases inserted can be set appropriately depending on the purpose. The type of mutation is not particularly limited, but examples include missense mutation, nonsense mutation, frameshift mutation, and silent mutation. In this embodiment, the mutation may be a polymorphism.

[0015] As used herein, the term "calibration reference material" refers to a material that serves as a reference when evaluating analytical methods or diagnostic agents, or when calibrating an instrument. Calibration reference materials may also be referred to as calibration standards. As used herein, "genomic DNA as a calibration reference material used in analyzing mutations in a target sequence" broadly refers to genomic DNA containing mutated genes.

[0016] As used herein, "genomic DNA" refers to the DNA that constitutes all of the genetic information (genome) contained in a cell.

[0017] As used herein, the term "internal standard sequence" refers to any gene sequence different from the target sequence, derived from the genomic DNA into which the mutated target sequence and internal standard sequence are introduced. Preferably, the internal standard sequence is one for which a validated droplet digital PCR reagent is commercially available. Furthermore, the gene sequence is preferably a region near the centromere, where transposon transfer and viral gene insertion are unlikely to occur. The target sequence and the internal standard sequence may be sequences derived from different gene loci or different chromosomes. It is not necessary to select a sequence such as a housekeeping gene that is constitutively expressed in cells and exhibits little expression fluctuation as the internal standard sequence. Examples of internal standard sequences include the PTEN gene, AGO1 gene, TMEM11 gene, and ERBB2 gene.

[0018] As used herein, "droplet digital PCR" refers to a digital PCR technology that measures the absolute amount of a target sequence by counting nucleic acid molecules encapsulated in droplets (droplets) of a volume-homogenized water-in-oil emulsion. Unlike real-time PCR, which quantifies the relative concentration of target DNA / RNA using a calibration curve, droplet digital PCR allows for absolute quantification of target DNA / RNA concentration without the use of a calibration curve. Droplet digital PCR is commonly used as a validated method for determining VAF because it can quantify target DNA / RNA concentration with high accuracy and sensitivity.

[0019] Step of Introducing Mutated Target Sequence and Internal Standard Sequence into Genomic DNA The method of this embodiment includes the step of introducing the mutated target sequence and internal standard sequence into genomic DNA.

[0020] The target sequence is derived from genomic DNA.

[0021] The mutated target sequence and the internal standard sequence may be introduced into the genomic DNA separately, but are preferably introduced into the genomic DNA together. That is, the sequence introduced into the genomic DNA preferably contains both the mutated target sequence and the internal standard sequence. Furthermore, the introduced sequence may contain a selection marker in addition to the mutated target sequence and the internal standard sequence. By including a selection marker, genomic DNA into which the mutated target sequence and the internal standard sequence have been introduced can be efficiently selected and recovered. Examples of selection markers include antibiotic resistance genes and genes that express fluorescent proteins.

[0022] The locations where the mutated target sequence and internal standard sequence are introduced may be arbitrary, but it is preferable that they are different from the same region of the internal standard sequence as the target sequence in the genomic DNA and the internal standard sequence to be introduced.

[0023] The method for introducing the mutated target sequence and the internal standard sequence into genomic DNA may be appropriately determined by those skilled in the art, but it is preferable to introduce them using a recombinant vector, i.e., the mutated target sequence and the internal standard sequence are preferably derived from a recombinant vector.

[0024] As used herein, the term "recombinant vector" refers to a nucleic acid used to recombine a gene of interest, which carries the gene of interest up to the gene sequence to be recombined. There are no particular limitations on the recombinant vector, as long as it can be incorporated into genomic DNA and achieve stable expression. Examples of recombinant vectors include non-viral vectors such as transposon vectors and plasmid vectors, and viral vectors, with transposon vectors being preferred. Furthermore, a preferred example of a transposon vector is the Tol2 transposon vector.

[0025] When the recombinant vector is a transposon vector, the mutated target sequence and internal standard sequence are introduced into the genomic DNA by using a transposon system, when the recombinant vector is a viral vector, by infecting the host with the viral vector, or when the recombinant vector is a non-viral vector, by introducing the recombinant vector into the host by transformation. When a transposon system is used, a transposase vector or mRNA that expresses a transferase (transposase) may be used together with a transposon vector having the mutated target sequence and internal standard sequence.

[0026] A recombinant vector incorporating a mutated target sequence and an internal standard sequence may be produced by a method based on a nucleic acid amplification reaction such as PCR, but is preferably produced by a method that does not use a nucleic acid amplification reaction and includes a restriction enzyme treatment step and a ligation step. For example, a recombinant vector having a mutated target sequence can be produced without using a nucleic acid amplification reaction by treating the recombinant vector with a restriction enzyme, inserting a double-stranded nucleic acid containing the mutated target sequence into the cleavage site, and then ligating the resulting product. More specifically, for example, the target nucleic acid can be produced by a method comprising the steps of: 1) cleaving a recombinant vector with a first restriction enzyme, the recombinant vector having a base sequence arranged in the following order: a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and another first restriction enzyme recognition sequence, the two first restriction enzyme recognition sequences being arranged so that the cleavage sites for the two first restriction enzymes are on opposite sides of the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence being a type IIS restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence being a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence; and 2) ligating a double-stranded nucleic acid comprising a region consisting of the polymorphic base in the target sequence, its 5'- and 3'-flanking sequences, and their complementary sequences. This method is described in detail below.

[0027] The method for producing the recombinant vector includes a step of cleaving, with the first restriction enzyme, a recombinant vector having a base sequence in which a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and another first restriction enzyme recognition sequence are arranged in that order.

[0028] As used herein, the first restriction enzyme (which may be one or two) is a type IIS restriction enzyme. Type IIS restriction enzymes generally refer to restriction enzymes characterized in that the restriction enzyme recognition sequence and cleavage site are separated. Among type IIS restriction enzymes, type IIS restriction enzymes that have all cleavage sites on one side, away from the restriction enzyme recognition sequence, are preferred. Furthermore, the recognition sequence of a type IIS restriction enzyme does not have to be a palindromic sequence. Examples of type IIS restriction enzymes include AlwI, AlwXI, Alw26I, BbsI, BbvI, BbvII, BcefI, BccI, BcgI, BciVI, BinI, BmrI, BpmI, BsaI, BseRI, BsgI, BsmAI, BsmBI, BspMI, BsrDI, BstF5I, EarI, Eco31I, Eco57I, Esp3I, and Esp3I. Examples of suitable ribonucleotides include I, FauI, FokI, GsuI, HgaI, HinGUII, HphI, Ksp632I, MboII, MmeI, Mn1I, NgoVIII, PaqCI, PleI, PsrI, RleAI, SapI, SfaNI, TaqII, Tth111II, AcuI, BsmI, BsrI, BsmFI, BseMII, BspQI, and BtgZI. Of these, BsmI is preferred.

[0029] Furthermore, as used herein, in a base sequence arranged in the order of a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence, the two first restriction enzyme recognition sequences may be distinguished as follows: the former as first restriction enzyme recognition sequence A, and the latter as first restriction enzyme recognition sequence B. Furthermore, when viewed from either single-stranded sequence of the double-stranded sequence, the second restriction enzyme recognition sequence on the 5' side may be distinguished as the 5'-side first restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence on the 3' side may be distinguished as the 3'-side first restriction enzyme recognition sequence.

[0030] The two first restriction enzyme recognition sequences may be restriction enzyme recognition sequences recognized by the same first restriction enzyme, or may be restriction enzyme recognition sequences recognized by different first restriction enzymes. When the two first restriction enzyme recognition sequences are restriction enzyme recognition sequences recognized by the same first restriction enzyme, the two first restriction enzyme recognition sequences may be the same sequence. When the two first restriction enzyme recognition sequences are restriction enzyme recognition sequences recognized by the same first restriction enzyme, and the first restriction enzyme is a restriction enzyme that recognizes multiple base sequences, the two first restriction enzyme recognition sequences may be different sequences. Furthermore, when the two first restriction enzyme recognition sequences are restriction enzyme recognition sequences recognized by different first restriction enzymes, the two first restriction enzyme recognition sequences may be different sequences. When the two first restriction enzyme recognition sequences are restriction enzyme recognition sequences recognized by different first restriction enzymes, and the different first restriction enzymes recognize the same sequence, the two first restriction enzyme recognition sequences may be the same sequence. The two first restriction enzyme recognition sequences may be different from each other as described above, but are preferably the same sequence, and more preferably are the same sequence recognized by the same first restriction enzyme.

[0031] As used herein, the term "second restriction enzyme" refers to any restriction enzyme different from the first restriction enzyme, but preferably has a cleavage site within a base sequence arranged in the order of the first restriction enzyme recognition sequence, the second restriction enzyme recognition sequence, and the first restriction enzyme recognition sequence, and has only one cleavage site in the recombinant vector. Furthermore, it is preferable that the double-stranded nucleic acid inserted in step 2) contains a region consisting of the polymorphic base in the target sequence, its 5'- and 3'-adjacent sequences, and their complementary sequences, does not have a cleavage site. For example, although not particularly limited, NotI is preferred.

[0032] In a base sequence in which a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and another first restriction enzyme recognition sequence are arranged in this order, the first restriction enzyme recognition sequences are preferably spaced at least 3 bases apart, and more preferably spaced at least 6 bases apart. In one embodiment, the number of bases between the first restriction enzyme recognition sequences is selected from the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 bases.

[0033] The base sequence arranged in the order of a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence may have a linker sequence having any number of bases. The base sequences may be arranged in the order of a first restriction enzyme recognition sequence, a linker, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence, or in the order of a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, a linker, and a first restriction enzyme recognition sequence. The base sequences may also be arranged in the order of a linker, a first restriction enzyme recognition sequence, a linker, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence, or in the order of a linker, a first restriction enzyme recognition sequence, a linker, a second restriction enzyme recognition sequence, a linker, and a first restriction enzyme recognition sequence. Alternatively, the bases may be arranged in the order of a first restriction enzyme recognition sequence, a linker, a second restriction enzyme recognition sequence, a first restriction enzyme recognition sequence, a linker; alternatively, the bases may be arranged in the order of a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, a linker, a first restriction enzyme recognition sequence, a linker; alternatively, the bases may be arranged in the order of a first restriction enzyme recognition sequence, a linker, a second restriction enzyme recognition sequence, a linker, a first restriction enzyme recognition sequence, a linker; alternatively, the bases may be arranged in the order of a linker, a first restriction enzyme recognition sequence, a linker, a second restriction enzyme recognition sequence, a linker, a first restriction enzyme recognition sequence, a linker; alternatively, the bases may be arranged in the order of a linker, a first restriction enzyme recognition sequence, a linker, a second restriction enzyme recognition sequence, a linker, a first restriction enzyme recognition sequence, a linker; alternatively, the bases may be arranged in the order of a linker, a first restriction enzyme recognition sequence, a linker, a second restriction enzyme recognition sequence, a linker, a first restriction enzyme recognition sequence, a linker. The number of bases constituting the linker sequence is not particularly limited as long as the desired effect is obtained.

[0034] In one embodiment, the number of bases constituting the linker sequence is selected from the group consisting of 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases and 10 bases.

[0035] In a base sequence in which the first restriction enzyme recognition sequence, the second restriction enzyme recognition sequence, and the first restriction enzyme recognition sequence are arranged in this order, the two first restriction enzyme cleavage sites are arranged on opposite sides of the second restriction enzyme recognition sequence. For example, when the first restriction enzyme recognition sequence cleaves a site 1 to 5 bases away from the recognition sequence, the first and second restriction enzyme recognition sequences are arranged so that the underlined cleavage site (the bond between the two underlined bases is cleaved) is 5'-NNNNNN-(first first restriction enzyme recognition sequence)-(second restriction enzyme recognition sequence)-(second first restriction enzyme recognition sequence)-NNNNNN-3', and the complementary strand is 3'-NNNNNN-(first first restriction enzyme recognition sequence)-(second restriction enzyme recognition sequence)-(second first restriction enzyme recognition sequence)-NNNNNN-5'. A schematic diagram of the arrangement of the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence in a recombinant vector when the first restriction enzyme recognition sequence cleaves a site 1 to 5 bases away from the recognition sequence is shown in Figure 1. In Figure 1, N represents any base, but is a part of the target gene sequence near the site where the mutation is to be inserted.

[0036] As described above, a recombinant vector containing a base sequence in which a first restriction enzyme recognition sequence and a second restriction enzyme recognition sequence are arranged preferably has only two cleavage sites for the first restriction enzyme, i.e., the number of first restriction enzyme recognition sequences in the recombinant vector is preferably only two.

[0037] The base sequence containing the first and second restriction enzyme recognition sequences is cleaved with the first restriction enzyme. The reaction conditions, such as the reaction time and treatment temperature with the first restriction enzyme, can be appropriately determined by those skilled in the art based on the type of the first restriction enzyme.

[0038] When a recombinant vector having a base sequence arranged in the order of a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence is cleaved with the first restriction enzyme, the site in the recombinant vector having a base sequence arranged in the order of the first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence is excised.

[0039] The method for producing the recombinant vector further comprises the step of ligating a double-stranded nucleic acid comprising a region consisting of the polymorphic base in the target sequence and its 5'- and 3'-adjacent sequences, and their complementary sequences.

[0040] As used herein, ligation refers to a reaction in which nucleic acids are linked together via a phosphodiester bond using a ligase. The ligase used in the ligation step, as well as the reaction conditions such as the time and temperature for the reaction with the ligase, can be appropriately determined by those skilled in the art. Commercially available ligation reagents may also be used.

[0041] The polymorphic base in the target sequence, its 5'- and 3'-flanking sequences, and its complementary sequence preferably have 10 to 200 base pairs. In one embodiment, the polymorphic base in the target sequence, its 5'- and 3'-flanking sequences, and its complementary sequence have 10 to 150 base pairs, preferably 15 to 100 base pairs.

[0042] The ligation step is preferably performed after the step of cleaving with the first restriction enzyme. A double-stranded nucleic acid containing a region consisting of a polymorphic base in the target sequence, its 5'- and 3'-flanking sequences, and their complementary sequences is inserted and ligated into the site excised by the first restriction enzyme treatment, which contains a base sequence arranged in the following order: first restriction enzyme recognition sequence, second restriction enzyme recognition sequence, first restriction enzyme recognition sequence. Figure 2 shows a schematic diagram of the recombinant vector shown as an example in Figure 1 being treated with a first restriction enzyme recognition sequence that cleaves a site 1 to 5 bases away from the recognition sequence, followed by insertion of a double-stranded nucleic acid and ligation. In Figure 2, N represents any base, but is a portion of the target gene sequence near the site where the mutation is to be inserted. Furthermore, if the cleavage sequence with the first restriction enzyme has a 5'- or 3'-overhanging end, N in the region that forms a double strand with the 5'- or 3'-flanking sequence and a portion of their complementary sequence is a portion of the 5'- or 3'-flanking sequence and a portion of their complementary sequence.

[0043] When the cleavage sequence obtained by the first restriction enzyme treatment, which is a site having a base sequence arranged in the order of the first restriction enzyme recognition sequence, the second restriction enzyme recognition sequence, and the first restriction enzyme recognition sequence, has a 5'- or 3'-protruding end, the double-stranded nucleic acid to be ligated preferably has a 3'- or 5'-protruding end that forms a strand complementary to the 5'- or 3'-protruding end. When the cleavage sequence produced in the recombinant vector by the first restriction enzyme treatment has only blunt ends, the double-stranded nucleic acid to be ligated may be a nucleic acid having blunt ends.

[0044] The method for producing a recombinant vector may further include a step of annealing the polymorphic base and its 5'- and 3'-flanking sequences in the target sequence with their complementary sequences. The annealing step is preferably performed before the ligation step.

[0045] As used herein, annealing refers to a reaction in which a single-stranded nucleic acid and its complementary single-stranded nucleic acid form a double strand. Generally, double-stranded nucleic acids are formed by treatment at a specific annealing temperature. The annealing temperature is appropriately set by those skilled in the art and is determined, for example, based on the Tm value specific to the sequence. The annealing step is performed by treating the polymorphic base, its 5'- and 3'-adjacent sequences, and its complementary sequence at an annealing temperature calculated based on these sequences, etc. After forming single-stranded nucleic acids by thermal denaturation, they may be treated at the annealing temperature. The thermal denaturation treatment is performed, for example, at about 95°C. Alternatively, annealing may be performed, for example, by stepwise cooling from the temperature of the thermal denaturation treatment.

[0046] By the above-described method for producing a recombinant vector, a portion having a base sequence arranged in the order of a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence is excised from the recombinant vector, and a double-stranded nucleic acid containing a polymorphic base is ligated, thereby producing a recombinant vector containing a polymorphic base.

[0047] The recombinant vector into which a double-stranded nucleic acid has been inserted includes a recombinant vector into which, after digestion with a first restriction enzyme, a double-stranded nucleic acid having a nucleotide sequence in the order of the excised first restriction enzyme recognition sequence, the second restriction enzyme recognition sequence, and the first restriction enzyme recognition sequence has been inserted, rather than a double-stranded nucleic acid containing a polymorphic base, and which has then undergone self-ligation.Furthermore, the recombinant vector also includes a vector that has not been digested at all by the first restriction enzyme, or a vector that has been digested at only one site by the first restriction enzyme.

[0048] Therefore, by treating the recombinant vector with a second restriction enzyme, it is possible to confirm whether or not a double-stranded nucleic acid containing a polymorphic base has been inserted into the recombinant vector.

[0049] When the mutated target sequence and the internal standard sequence are introduced into genomic DNA using a recombinant vector, for example, the mutated target sequence and the internal standard sequence can be incorporated into the genomic DNA by transforming a cell containing the genomic DNA with the recombinant vector.

[0050] Transformation may be performed by any method known to those skilled in the art, such as electroporation or heat shock. Transformation may also be performed using a commercially available kit.

[0051] After transformation, selection, growth, and recovery of cells containing genomic DNA into which a mutated target sequence and an internal standard sequence have been introduced may be carried out by any method known to those skilled in the art. When a selection marker is introduced into the genomic DNA together with the mutated target sequence and the internal standard sequence, for example, when an antibiotic resistance gene or a gene that expresses a fluorescent protein is introduced, cells containing genomic DNA into which a mutated target sequence and an internal standard sequence have been introduced can be selected by adding an antibiotic or observing under a fluorescence microscope.

[0052] It is preferable that the introduction of the mutated target sequence and internal standard sequence into the genomic DNA be finally confirmed by amplifying a region containing the mutated target sequence and internal standard sequence from the genomic DNA and performing sequence analysis of the amplified product.

[0053] Cells containing genomic DNA in which the introduction of the mutated target sequence and internal standard sequence has been confirmed are collected, and genomic DNA is extracted from the cells, thereby obtaining genomic DNA as the desired calibration standard material.

[0054] Calculation of VAF of Genomic DNA as Calibration Standard The genomic DNA used as calibration standard produced by the method of this embodiment contains a mutated target sequence, and therefore can be used to analyze a wide range of target sequence mutations. For example, the copy number of the mutated target sequence in the genomic DNA used as calibration standard can be used to analyze target sequence mutations.

[0055] Herein, the internal standard sequence introduced into genomic DNA together with the mutated target sequence is referred to as the first internal standard sequence. Because the target sequence and the first internal standard sequence are derived from genomic DNA, the genomic DNA into which the mutated target sequence and the first internal standard sequence are introduced is genomic DNA having a non-mutated wild-type target sequence and a first internal standard sequence. Therefore, when one copy of the mutated target sequence and the first internal standard sequence are introduced into genomic DNA having one copy each of the wild-type target sequence and the first internal standard sequence, the resulting genomic DNA will have one copy of the wild-type target sequence, one copy of the mutated target sequence, and two copies of the first internal standard sequence.

[0056] Therefore, the copy number of the mutated target sequence in the genomic DNA used as the calibration standard material produced by the method of this embodiment is equal to the copy number of the first internal standard sequence increased by being introduced together with the mutated target sequence. The increase in the copy number of the first internal standard sequence can be calculated by subtracting the copy number of the first internal standard sequence in the genomic DNA before gene introduction from the copy number of the first internal standard sequence in the genomic DNA after gene introduction (Equation 1).

[0057] [Formula 1] [Number of copies of mutated target sequence] = [Number of copies of first internal standard sequence after gene transfer] - [Number of copies of first internal standard sequence before gene transfer]

[0058] Furthermore, herein, any internal standard sequence that is present in genomic DNA and that is different from the first internal standard sequence is referred to as the second internal standard sequence. The first internal standard sequence and the second internal standard sequence may be sequences derived from different loci or different chromosomes. When the genomic DNA is derived from a cell with a diploid chromosome number, the copy number of the first internal standard sequence and the copy number of the second internal standard sequence in the genomic DNA before the mutated target sequence and internal standard sequence are introduced are equal. Even after the mutated target sequence and internal standard sequence are introduced, the copy number of the second internal standard sequence does not change (Equation 2).

[0059] [Formula 2] [Number of copies of first internal standard sequence before gene introduction] = [Number of copies of second internal standard sequence]

[0060] Therefore, from [Equation 1] and [Equation 2], the copy number of the mutated target sequence in the genomic DNA used as the calibration reference material produced by the method of this embodiment can be calculated by subtracting the copy number of the second internal standard sequence from the copy number of the first internal standard sequence in the genomic DNA after the mutated target sequence and the first internal standard sequence have been introduced (Equation 3).

[0061] [Formula 3] [Number of copies of mutated target sequence] = [Number of copies of first internal standard sequence after gene transfer] - [Number of copies of second internal standard sequence]

[0062] Furthermore, when the genomic DNA is derived from cells with a diploid chromosome number, the copy number of the wild-type target sequence and the copy number of the second internal standard sequence are equal, and this relationship does not change even after the mutated target sequence and internal standard sequence are introduced (Equation 4).

[0063] [Formula 4] [Copy number of wild-type target sequence] = [Copy number of second internal standard sequence]

[0064] Furthermore, VAF is generally calculated using the copy number of the mutated target sequence according to the formula [Equation 5].

[0065] [Formula 5] VAF (%) = {[number of copies of mutated target sequence] / ([number of copies of mutated target sequence] + [number of copies of wild-type target sequence])} * 100

[0066] Therefore, the VAF of genomic DNA as a calibration standard can be calculated from [Equation 3] to [Equation 5] using the copy numbers of the first internal standard sequence and the second internal standard sequence (Equation 6). [Equation 6] VAF (%) = {([Copy number of first internal standard sequence after gene introduction] - [Copy number of second internal standard sequence]) / ([Copy number of first internal standard sequence after gene introduction] - [Copy number of second internal standard sequence] + [Copy number of second internal standard sequence])} * 100 = {([Copy number of first internal standard sequence after gene introduction] - [Copy number of second internal standard sequence]) / [Copy number of first internal standard sequence after gene introduction]} * 100

[0067] The genomic DNA is preferably derived from cells with a diploid number of chromosomes. However, if the number of chromosomes is not diploid, the VAF can be calculated in the same manner as above by selecting the first internal standard sequence and the second internal standard sequence so that the first internal standard sequence, the second internal standard sequence, and the target sequence are each carried on the same number of chromosomes.

[0068] Therefore, by producing genomic DNA as a calibration reference material using the method of this embodiment, which includes a step of introducing a mutated target sequence and an internal standard sequence into genomic DNA, it is possible to calculate the VAF of the calibration reference material by using the copy numbers of two types of internal standard genes without using the copy number of the mutant gene. In other words, the method of this embodiment makes it possible to accurately calculate the VAF of the calibration reference material without preparing validated droplet digital PCR reagents for each mutant gene. Because validated droplet digital PCR reagents for two types of internal standard genes can be used in common for calibration reference materials having each mutant gene, it is possible to simply calculate the VAF of multiple calibration reference materials.

[0069] The method of this embodiment may be a method for producing genomic DNA as a calibration reference material, which allows for easy calculation of VAF.

[0070] Analysis of mutations in target sequences using genomic DNA as a calibration standard The calibration standard produced by the method of this embodiment and having a specified VAF or mutant gene copy number can be used to evaluate the sensitivity of a companion diagnostic.

[0071] As used herein, a companion diagnostic refers to a diagnostic agent for detecting the presence of an amino acid mutation in preparation for administration of a molecularly targeted drug, which is a chemotherapeutic agent that targets an abnormal enzyme having the amino acid mutation. Among companion diagnostic agents, for example, the sensitivity of a gene mutation diagnostic agent, particularly the sensitivity for detecting a mutant gene, is defined by the VAF or the copy number of the mutant gene, and is evaluated using a calibration reference material for which the VAF or the copy number of the mutant gene is specified.

[0072] As used herein, the detection sensitivity of a gene mutation diagnostic agent refers to the minimum frequency of mutant genes that the diagnostic agent can detect in a sample tested by the diagnostic agent, and is defined using the VAF, which is an index of mutant gene frequency. The detection sensitivity of a gene mutation diagnostic agent may also refer to the minimum amount of mutant genes that the diagnostic agent can detect in a sample tested by the diagnostic agent, in which case it is defined using the copy number of the mutant gene. It may also be defined by the copy number calculated from the VAF, or by the VAF calculated from the copy number. The detection sensitivity of mutant genes is synonymous with minimum detection sensitivity, minimum detectable genetic abnormality frequency, detection limit, etc.

[0073] The detection sensitivity can be evaluated, for example, by detecting mutations using a mutation gene diagnostic agent in genomic DNA as a calibration reference material with a specified VAF. It can also be evaluated, for example, by detecting mutations using a mutation gene diagnostic agent in a sample containing a calibration reference material with a specified copy number of the mutant gene. A specific example of a method for evaluating detection sensitivity is described below. For example, when evaluating using a calibration reference material with a specified VAF of 1%, if the calibration reference material with the specified VAF is tested with the gene mutation diagnostic agent whose detection sensitivity is to be evaluated and the mutation can be detected, the detection sensitivity (VAF) of this gene mutation diagnostic agent can be evaluated as 1% or higher. Furthermore, for example, when an evaluation is performed using a calibration reference material in which the mutant gene is specified to have 30 copies, if a sample containing the calibration reference material in which the copy number is specified is tested with a gene mutation diagnostic agent whose detection sensitivity is to be evaluated, and the mutation can be detected, the detection sensitivity of this gene mutation diagnostic agent can be evaluated as 30 copies or more, and if the tested sample contains a total of 3,000 copies of the gene, the detection sensitivity (VAF) can be evaluated as 1% or more.

[0074] The evaluation of detection sensitivity can be used as an index for validation of companion diagnostic agents such as gene mutation diagnostic agents.

[0075] Therefore, the method of this embodiment may be a method for producing genomic DNA as a calibration reference material to be used in companion diagnostics, a method for producing genomic DNA as a calibration reference material to be used in evaluating the sensitivity of a companion diagnostic, a method for producing genomic DNA as a calibration reference material to be used in evaluating the sensitivity of a gene mutation diagnostic, or a method for producing genomic DNA as a calibration reference material to be used for validation of a companion diagnostic.

[0076] The present embodiment also provides genomic DNA produced by a method comprising the step of introducing a mutated target sequence and an internal standard sequence into genomic DNA.

[0077] The aspects of the method for producing genomic DNA as a calibration standard material in the first embodiment also apply to the second and third embodiments described below.

[0078] (Cells containing genomic DNA into which a mutated target sequence and an internal standard sequence have been introduced) In a second embodiment, cells are provided that contain one or more copies of genomic DNA into which a mutated target sequence and an internal standard sequence have been introduced.

[0079] The copy number of the genomic DNA into which the mutated target sequence and internal standard sequence have been introduced, which is contained in the cell of this embodiment, can be appropriately determined by those skilled in the art depending on the purpose.

[0080] Furthermore, the cells provided in this embodiment can be used as a calibration reference material, and tissues containing cells as a calibration reference material can also be used as a calibration reference material. Thus, this embodiment also provides cells as a calibration reference material and tissues as a calibration reference material. Examples of tissues as a calibration reference material include formalin-fixed, paraffin-embedded (FFPE) tissue samples.

[0081] The cells of this embodiment may be used to produce genomic DNA as a calibration standard.

[0082] When the cells of this embodiment are used as a calibration reference material, the number of chromosomes in the cells is preferably diploid.

[0083] In this embodiment, the VAF of a calibration reference material derived from a cell and having each mutation can be accurately calculated using the copy number of the internal standard sequence, without using the copy number of each mutant sequence. Furthermore, since a validated droplet digital PCR reagent for the internal standard gene can be commonly used for calibration reference materials having each mutant gene, without preparing a validated droplet digital PCR reagent for each mutant gene, the VAF of multiple calibration reference materials can be easily calculated.

[0084] The cells of this embodiment may be cells used in companion diagnostics, cells used in evaluating the sensitivity of a companion diagnostic agent, cells used in evaluating the sensitivity of a gene mutation diagnostic agent, or cells used for validation of a companion diagnostic agent.

[0085] (Method for analyzing mutations in a target sequence) In a third embodiment, a method for analyzing mutations in a target sequence is provided, comprising: 1) subjecting genomic DNA into which a mutated target sequence and an internal standard sequence have been introduced to an amplification reaction; and 2) calculating the copy numbers of the introduced internal standard sequence and different internal standard sequences present in the genomic DNA.

[0086] The method of this embodiment includes a step of subjecting genomic DNA into which a mutated target sequence and an internal standard sequence have been introduced to an amplification reaction. The amplification reaction may be carried out by any method known to those skilled in the art, but is preferably carried out by PCR, and more preferably by droplet digital PCR. The conditions for each stage of the amplification reaction, such as the temperature setting, reaction time, and reagents used, can be appropriately determined by those skilled in the art. When carrying out the amplification reaction by droplet digital PCR, it is preferable to use a validated droplet digital PCR reagent containing a probe validated for each gene to be amplified.

[0087] In the next step, it is preferable to amplify the region containing the first internal standard sequence and the region containing the second internal standard sequence so that it is possible to calculate the copy number of the first internal standard sequence introduced together with the mutated target sequence and the second internal standard sequence, which is an arbitrary internal standard sequence different from the first internal standard sequence and present in the genomic DNA. Furthermore, a region containing any sequence other than the first internal standard sequence and the second internal standard sequence may be amplified together, as long as it is possible to calculate the copy numbers of the first internal standard sequence and the second internal standard sequence. It is preferable to use different primers for amplifying the region containing the first internal standard sequence and the region containing the second internal standard sequence. Since the first internal standard sequence is derived from genomic DNA, it is preferable to amplify both the region containing the first internal standard sequence introduced together with the mutated target sequence and the region containing the first internal standard sequence originally present in the genomic DNA.

[0088] Step of Calculating Copy Number The method of this embodiment further includes a step of calculating the copy numbers of the introduced first internal standard sequence and a different second internal standard sequence present in the genomic DNA. The copy numbers can be calculated by quantitative analysis in the amplification reaction step. The copy number of the mutated target sequence in the genomic DNA can be calculated using the copy number of the first internal standard sequence and the copy number of the second internal standard sequence. Furthermore, the VAF of the genomic DNA can also be calculated using the copy number of the first internal standard sequence and the copy number of the second internal standard sequence. Therefore, the method of this embodiment may further include a step of calculating the VAF.

[0089] Calculating VAF For example, the method further includes calculating VAF using the following [Equation 7]: The percentage of the value calculated from [Equation 7] can be used as the VAF of the genomic DNA.

[0090] [Formula 7] ([number of copies of the introduced internal standard sequence] - [number of copies of a different internal standard sequence present in the genomic DNA]) / [number of copies of the introduced internal standard sequence]

[0091] The above [Equation 7] can also be expressed as the following [Equation 8].

[0092] [Formula 8] ([number of copies of first internal standard sequence] - [number of copies of second internal standard sequence]) / [number of copies of first internal standard sequence]

[0093] Furthermore, the values ​​calculated from the above [Equation 7] and [Equation 8] are the same as or equivalent to the value calculated from the following [Equation 9], which is generally used to calculate VAF.

[0094] [Formula 9] [Number of copies of mutated target sequence] / ([Number of copies of mutated target sequence]+[Number of copies of wild-type target sequence])

[0095] The method of this embodiment can calculate the copy number of a mutated target sequence in genomic DNA and the VAF, and therefore can widely analyze mutations in target sequences in genomic DNA.

[0096] When genomic DNA is used as a calibration reference material, the VAF calculated by the method of this embodiment can be used to evaluate the sensitivity of a reagent for detecting mutations, for example, a companion diagnostic agent such as a gene mutation diagnostic agent.

[0097] Therefore, the method of this embodiment may be a method for analyzing mutations in a target sequence used in a companion diagnostic, a method for analyzing mutations in a target sequence used in evaluating the sensitivity of a companion diagnostic, a method for analyzing mutations in a target sequence used in evaluating the sensitivity of a gene mutation diagnostic, or a method for analyzing mutations in a target sequence used for validating a companion diagnostic.

[0098] Example 1 <HEL299 Cell Culture> HEL299 cells, which are normal diploids (https: / / www.atcc.org / products / ccl-137) and do not have mutations in major oncogenes (https: / / www.cellosaurus.org / CVCL_2480), were obtained from ATCC and used. HEL299 cells stored in a -150°C freezer were quickly thawed in a 37°C water bath and suspended in complete medium (EMEM supplemented with 10% fetal bovine serum (Fujifilm Wako Pure Chemical Industries, Cat: 051-07615) pre-warmed to 37°C. After centrifugation (130 x g, 5 min, room temperature), the pellet was resuspended in fresh complete medium and seeded into a T-25 flask. The cells were incubated at 37°C and 5% CO ２ , CO under saturated water vapor conditions ２ Culture was initiated in an incubator. The culture supernatant was removed from the subconfluent cells, and the culture surface was washed with an appropriate amount of D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No. 045-29795) preheated to 37°C. Trypsin / EDTA solution (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No. 201-16945) was added, followed by incubation in CO ２ The cells were placed in an incubator to detach. Complete medium preheated to 37°C was added to the detached cells in an amount five times greater than that of Trypsin / EDTA, and the cell suspension was collected in a conical tube (hereinafter referred to as PET treatment). After centrifugation (130 x g, 5 min, room temperature), the pellet was resuspended in fresh complete medium, and the cell number and viability were evaluated by Trypan blue staining. The cells were then seeded into a new flask and incubated at 37°C, 5% CO ２ , CO under saturated water vapor conditions ２ Culture was initiated in an incubator.

[0099] <HEL299 Gene Transfection> Cells cultured for three passages after cell thawing were seeded at 600,000 cells / well on a 6-well multiplate and cultured for approximately 24 hours. After confirming that the cells were 80-90% confluent, transfection was performed using a Lipofectamine 3000 kit (Thermo Fisher Scientific L, Cat: 3000-008) according to the kit's package insert. The transfection conditions were: Lipofectamine volume 5.0 μL / well, transfected DNA volume 1.25 μg / well, and a Tol2 pDNA (SEQ ID NO: 1 or 2) / Tol2 Transposase pDNA (SEQ ID NO: 3) ratio of 2:1. An example of Tol2 pDNA (SEQ ID NO: 1) is shown in Figure 3. After removing the cell culture supernatant, 1000 μL / well of complete medium pre-warmed to 37°C was added. In a sterilized microtube, 125 μL of Lipofectamine 3000 was added to each well, vortexed for 3 seconds, and then spun down (Liquid 1). To a separate microtube, 125 μL of OptiMEM (Gibco, Cat: 31985-062), each dose of pDNA, and 2 μL of P3000 reagent per μg of pDNA were added and mixed by tapping. Liquid 1 was then added, and the mixture was further mixed by tapping. The mixture was then left to stand at room temperature for 15 minutes. The Lipofectamine:pDNA complex was then added dropwise to the cells using a Dropwise tube, mixed well, and then spun down in CO . ２ After 2 hours or more had passed, 1000 μL / well of complete medium preheated to 37° C. was added, and the cells were cultured overnight.

[0100] <Maintenance culture of transgene-stable recombinant HEL299 cells> Cells 20 hours or more after transfection were observed under a microscope and a fluorescence microscope (Tol2 pDNA contains EGFP, and transfected cells are GFP-positive) to confirm transfection. The culture supernatant was removed, and 2000 μL / well of complete medium pre-warmed to 37°C was added. After further overnight culture, the medium was replaced with complete medium supplemented with 0.4 μg / mL Puromycin and cultured for an additional 4 days. The complete medium containing Puromycin was removed, and fresh complete medium was added, and the cells were cultured until they again reached confluence. When the cells became subconfluent, the medium was replaced with complete medium supplemented with 0.4 μg / mL Puromycin and cultured for an additional 3 days. The complete medium containing Puromycin was removed, and fresh complete medium was added, and the cells were cultured until they again reached confluence. When the cells became subconfluent, they were harvested by PET treatment and scaled up to T-75 flasks. The next day, the medium was replaced with complete medium supplemented with 0.4 μg / mL Puromycin, and drug selection was performed for 3 days, confirming that the GFP positivity rate was 100%. After that, when the cells became subconfluent, they were subcultured up to P+4, and the remaining cells were collected and suspended at 1 million cells / mL using Cellbanker (TaKaRa, Cat: CB011), dispensed into cryotubes at 1 mL per tube, and cryopreserved.

[0101] <gDNA Extraction from HEL299 Cells> HEL299 cells stably transfected with the transgene or parental non-transfected HEL299 cells were harvested, washed twice with D-PBS, and cryopreserved as cell pellets. gDNA was purified from the cryopreserved cell pellets using a QIAamp DNA Mini QIAcube Kit (QIAGEN, Cat. C 51326) or a Blood & Cell Culture DNA Mini Kit (QIAGEN, Cat. 13323). The gDNA concentration was determined using a Quant-iT dsDNA HS Assay Kit (Thermo Fisher Scientific, Cat. Q32854).

[0102] <PCR amplification of integrated sequence> In order to confirm that the sequence between the "left end of Tol2" and the "right end of Tol2" of the Tol2 pDNA shown in Figure 3 was introduced into HEL299 cells, 10 ng of gDNA derived from HEL299 cells into which the introduced gene had been stably integrated was used as a template, and 300 nM Tol2 Seq PTEN primer (SEQ ID NO: 4), 300 nM Tol2 Seq CMV primer (SEQ ID NO: 5), and 0.5X KOD One PCR Master Mix (TOYOBO, Cat: KMM-101) were used as a reaction solution. 30 cycles of PCR were performed, each cycle consisting of 98°C for 10 seconds, 55°C for 5 seconds, and 68°C for 5 seconds. The reaction products were separated by electrophoresis on a 1.0% agarose / TAE gel and analyzed by SYBR The product was stained with Gold staining reagent for 10 minutes to detect nucleic acids. When a single band of interest was detected, the DNA sequence of the product was determined by direct sequencing.

[0103] <Direct sequencing of PCR product> The concentration of the PCR product of the integrated sequence was determined using a Qubit dsDNA BR Assay kit (Thermo Fisher Scientific, Cat: Q32853), and 10 fg of the product was treated with ExoSAP-IT Exp reagent (applied bisystems, Cat: 75001.200.UL) and used as a template for sequence analysis at Eurofins Genomics using the two primers (SEQ ID NOs: 4 and 5) used in the PCR of the integrated sequence.

[0104] <Copy number measurement by ddPCR> Using gDNA derived from HEL299 cells stably integrated with the transgene or parental non-transfected HEL299 cells, the PTEN gene (ddPCR Copy Number Assay: PTEN (FAM), Bio-Rad, Cat: 10031240), BRAF WT gene and BRAF V600E1 gene (ddPCR Mut assay, VAL BRAF V600E, Bio-Rad, Cat: 1080337), KRAS WT gene and KRAS G12A gene (ddPCR Mut assay, KRAS p.G12A, The copy numbers of the following genes were measured by ddPCR according to the protocol recommended by Bio-Rad: AGO1 gene (ddPCR Copy Number Assay: AGO1(HEX), Bio-Rad, Cat: 10031243), ERBB2 gene (ddPCR CNV assay, ERBB2, Bio-Rad, Cat: 10031240), and TMEM11 gene (ddPCR CNV assay, TMEM11, Bio-Rad, Cat: 10031243). Based on the copy numbers of these genes per genome amount, the copy number of the introduced gene per cell and the variant allele frequency (hereinafter referred to as VAF) were calculated according to the following formula.

[0105] Average number of copies of introduced gene per cell = {([PTEN gene copy number] - [reference gene copy number]) / [reference gene copy number]} * 2 or ([mutant gene copy number] / [wild-type gene copy number]) * 2

[0106] VAF (%) = {([PTEN gene copy number] - [reference gene copy number]) / [PTEN gene copy number]} * 100 or {[mutant gene copy number] / ([mutant gene copy number] + [wild-type gene copy number])} * 100

[0107] Example 2 <Result 1> The present invention determines the copy number of a sequence incorporated into genomic DNA by simultaneously measuring the copy numbers of an arbitrary first internal standard gene (in this example, a sequence of the PTEN gene that is the detection target for ddPCR) designed into a sequence to be incorporated into genomic DNA and an arbitrary second internal standard gene that is different from the first internal standard gene or the gene to be incorporated into genomic DNA, such as a BRAF mutant gene or a KRAS mutant gene. In this example, in order to confirm the arbitrariness and robustness of the second internal standard gene, the experiment was carried out to confirm that the copy numbers of the AGO1 gene, ERBB2 gene, and TMEM11 gene were the same between HEL299 cells that had not been transfected with a gene and HEL299 cells into which an introduced gene had been stably incorporated.

[0108] The copy number of each second internal standard gene was calculated using non-transfected HEL299 cells (pink circle) and three types of cell lines in which a transgene was stably integrated using the Tol2 system, and the results are shown in Figure 4. As is clear from the results, when each gene is plotted on the X axis / Y axis, the relationship is X ≒ Y, and it was considered that the copy number of the second internal standard gene per cell was constant regardless of which internal standard gene was measured.

[0109] Example 3 <Result 2> Using HEL299 cells stably incorporating a partial sequence of the human BRAF V600E1 gene (BRAF V600E1 / HEL299), the BRAF WT gene copy number, BRAF V600E1 gene copy number, PTEN gene copy number, and TMEM11 gene copy number were calculated. The specimens used in the test were four types of independently established BRAF V600E1 / HEL299 cells. The validated ddPCR reagents used were BRAF wild-type (WT), BRAF V600E1, PTEN, and TMEM11. As a result of determining the copy number by ddPCR, the relationship between the copy numbers of each gene in the plot was X≈Y, confirming that the BRAF WT gene copy number = the TMEM11 gene copy number ( Figure 5 , left), and the BRAF V600E1 gene copy number = [PTEN gene copy number - TMEM11 gene copy number] ( Figure 5 , center). The relationship between the VAF calculated from the BRAF WT gene copy number and the BRAF V600E1 gene copy number and the VAF calculated from the copy number of the first internal standard gene (PTEN gene) and the copy number of the second internal standard gene (TMEM11 gene) was also X≈Y, confirming the high accuracy of the VAF calculated by the present invention ( Figure 5 , right).

[0110] Example 4 <Result 3> Using HEL299 cells stably integrated with a partial sequence of the human KRAS G12A gene (KRAS G12A / HEL299), the KRAS WT gene copy number, KRAS G12A gene copy number, PTEN gene copy number, and AGO1 gene copy number were calculated. Five types of independently established KRAS G12A / HEL299 cells were used as specimens. Validated ddPCR reagents for KRAS WT, KRAS G12A, PTEN, and AGO1 were used. As a result of determining the copy numbers by ddPCR, the relationship between the copy numbers of each gene in the plot was X≈Y, confirming that the KRAS WT gene copy number = AGO1 gene copy number ( Figure 6 , left), and the KRAS G12A gene copy number = [PTEN gene copy number - AGO1 gene copy number] ( Figure 6 , center). The relationship between the VAF calculated from the KRAS WT gene copy number and the KRAS G12A gene copy number and the VAF calculated from the copy number of the first internal standard gene (PTEN gene) and the copy number of the second internal standard gene (AGO1 gene) was also X≈Y, confirming the high accuracy of the VAF calculated by the present invention ( Figure 6 , right).

[0111] The base sequences used in this example are shown in Table 1.

[0112]

Claims

1. A method for producing genomic DNA as a calibration reference substance used in the analysis of mutations in a target sequence, the method comprising the step of introducing a mutated target sequence and an internal standard sequence into the genomic DNA.

2. The method according to claim 1, wherein the target sequence and the internal standard sequence to be introduced are derived from a recombinant vector.

3. The method according to claim 2, wherein the recombinant vector is a transposon vector.

4. The method according to claim 2 or 3, produced by a method comprising: 1) a step of cleaving a recombinant vector having a nucleotide sequence arranged in the order of a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence, wherein the two first restriction enzyme recognition sequences are arranged such that the cleavage sites of the two first restriction enzymes are on the opposite side of the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence, with the first restriction enzyme; and 2) a step of ligating a double-stranded nucleic acid comprising a polymorphic base in the target sequence and its 5'- and 3'-flanking sequences and their complementary sequences.

5. The method according to any one of claims 1 to 3, wherein the sequence to be introduced further comprises a selectable marker.

6. The method according to any one of claims 1 to 3, wherein the mutation is a polymorphism.

7. The method according to any one of claims 1 to 3, wherein the analysis is a companion diagnosis.

8. The method according to any one of claims 1 to 3, wherein the calibration reference substance is used in the validation of a companion diagnostic agent.

9. Genomic DNA produced by the method according to any one of claims 1 to 3.

10. A cell having one or more copies of genomic DNA into which a mutated target sequence and an internal standard sequence have been introduced.

11. The cell according to claim 10, wherein the chromosome number is diploid.

12. A method for analyzing a mutation in a target sequence, the method comprising: 1) a step of subjecting genomic DNA into which a mutated target sequence and an internal standard sequence have been introduced to an amplification reaction; and 2) a step of calculating the copy numbers of the introduced internal standard sequence and different internal standard sequences inherent in the genomic DNA.

13. The method according to claim 12, further comprising a step of calculating the variant allele frequency (VAF) based on the copy number.

14. The method according to claim 13, wherein the VAF is a percentage of a value calculated from the following formula: (Copy number of the introduced internal standard array - Copy number of a different internal standard array inherent in genomic DNA) / Copy number of the introduced internal standard array 15. The method according to any one of claims 12 to 14, which is used for evaluating the sensitivity of a reagent for detecting a mutation.

16. The method according to claim 15, wherein the evaluation of the sensitivity is used as an index for validating the reagent for detecting the mutation.

17. The method according to any one of claims 12 to 14, wherein the amplification reaction is carried out by PCR.

18. The method according to claim 17, wherein the PCR is droplet digital PCR.