Recombinant vector for use in production of target sequence having polymorphic nucleotide incorporated therein

Abstract

The present invention provides a recombinant vector for use in the production of a target sequence having a polymorphic nucleotide incorporated therein. The recombinant vector has a nucleotide 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, corresponding to a region that includes the polymorphic nucleotide in the target sequence and 5'-side and 3'-side flanking sequences of the polymorphic nucleotide, and the sequences complementary thereto. The two first restriction enzyme recognition sequences are arranged such that two first restriction enzyme cleavage sites are present on the side opposite to the second restriction enzyme recognition sequence. The first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence. The second restriction enzyme recognition sequence is different from the first restriction enzyme recognition sequence.

Description

Recombinant vector for generating a target sequence incorporating a polymorphic base

[0001] The present invention broadly relates to a recombinant vector for producing a target sequence incorporating a polymorphic base.

[0002] Known methods for introducing a target gene mutation into a genome include a method for introducing a target gene into a genome using a viral vector and a method for introducing a target gene into a genome using a transposon system. In either method, construction of a recombination vector carrying the respective gene mutation is essential. PCR-based methods are widely used to introduce mutations into vectors (Non-Patent Document 1).

[0003] Karin L Heckman & Larry R Pease. Gene splicing and mutagenesis by PCR-driven overlap extension. NATURE PROTOCOLS. 2007, 2(4) 924-932.

[0004] In conventional PCR-based methods, the entire sequence or a portion of the sequence of a mutated vector is synthesized using a polymerase. This can lead to PCR errors, and after mutagenesis, the entire sequence or PCR-amplified sequence, as well as the integration site, must be verified, resulting in increased time and expense. Furthermore, the primers used in PCR-based methods must be highly purified, further increasing time and expense for primer synthesis. Therefore, there is a need for a simpler, more cost-effective method for introducing mutations into vectors.

[0005] As a result of extensive research to solve the above problems, the present inventors have discovered a method for producing a recombinant vector having a desired mutation without using conventional PCR-based methods.

[0006] That is, the present application encompasses the following inventions. [1] A recombinant vector for preparing a target sequence incorporating a polymorphic base, 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 a first restriction enzyme recognition sequence, corresponding to a region in the target sequence consisting of the polymorphic base, its 5'- and 3'-flanking sequences, and their complementary sequences; the two first restriction enzyme recognition sequences are arranged so that the two first restriction enzyme cleavage sites 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. [2] The recombinant vector according to [1], in which the two first restriction enzyme recognition sequences are spaced at least three bases apart. [3] The recombinant vector according to [1] or [2], in which the polymorphic base and its 5'- and 3'-flanking sequences have 10 to 200 base pairs. [4] The recombinant vector according to any one of [1] to [3], which is a transposon-based vector. [5] Type IIS restriction enzymes include AlwI, AlwXI, Alw26I, BbsI, BbvI, BbvII, BcefI, BccI, BcgI, BciVI, BinI, BmrI, BpmI, BsaI, B seRI, BsgI, BsmAI, BsmBI, BspMI, BsrDI, BstF5I, Earl, Eco31I, Eco57I, Esp3I, Esp3I, FauI, FokI, GsuI, H The recombinant vector according to any one of [1] to [4], wherein the target sequence is selected from the group consisting of gaI, HinGUII, HphI, Ksp632I, MboII, MmeI, Mn1I, NgoVIII, PaqCI, PleI, PsrI, RleAI, SapI, SfaNI, TaqII, Tth111II, AcuI, BsmI, BsrI, BsmFIBseMII, BspQI, and BtgZI. [6] The recombinant vector according to any one of [1] to [5], wherein the target sequence is derived from genomic DNA. [7] The recombinant vector according to any one of [1] to [6], wherein the recombinant vector further comprises a reference sequence. [8] The recombinant vector according to [7], wherein the reference sequence is an internal standard sequence derived from genomic DNA.[9] 1) A recombinant vector for preparing a target sequence incorporating a polymorphic base, the vector having a base sequence arranged in the order of a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and the first restriction enzyme recognition sequence, corresponding to a region in the target sequence consisting of the polymorphic base and its 5' and 3' flanking sequences and their complementary sequences, the two first restriction enzyme recognition sequences being arranged so that the two first restriction enzyme cleavage sites are located 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, 2) a first nucleic acid sequence to be inserted into the restriction enzyme recognition site, the first nucleic acid sequence including the polymorphic base and its 5' and 3' flanking sequences, and 3) a second nucleic acid sequence to be inserted into the restriction enzyme recognition site, the second nucleic acid sequence including a complementary strand of the first nucleic acid sequence of 2).

[10] A kit according to [9], for producing a gene as a calibration standard material.

[11] The kit according to

[10] , wherein the gene serving as the calibration standard material is genomic DNA.

[12] The kit according to any one of [9] to

[11] , wherein the recombinant vector further comprises an internal standard sequence derived from genomic DNA.

[13] The kit according to any one of [9] to

[12] , wherein the first nucleic acid sequence of 2) and the second nucleic acid sequence of 3) form a double strand.

[14] A method for producing a recombinant vector for generating a target sequence containing a polymorphic base, the method comprising: 1) a step of 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 a first restriction enzyme recognition sequence, the two first restriction enzyme recognition sequences being arranged so that the two cleavage sites for the first restriction enzyme 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) a 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'-flanking sequences, and their complementary sequences.

[15] The method of

[14] , further comprising a step of annealing to prepare a double-stranded nucleic acid.

[16] The method of

[14] or

[15] , wherein the double-stranded nucleic acid has a sequence complementary to the cleavage sequence with a first restriction enzyme.

[17] The method of any one of

[14] to

[16] , further comprising a step of digesting with a second restriction enzyme.

[0007] The present invention allows for the preparation of recombinant vectors carrying desired mutations without using conventional PCR-based methods.

[0008]

[0033] Figure 1 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. This figure 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. This figure shows a schematic diagram of a KRAS-G12A cassette vector. The top panel shows the results of colony direct PCR performed on Escherichia coli colonies transformed with the ligation reaction solution, after ligating a KRAS-G12X / G13X cassette vector treated with BsmBI-v2 with an annealed double-stranded DNA oligo. The bottom panel shows the results of treating the PCR product of the colony direct PCR with NotI. The KRAS-A59X / Q61X cassette vector treated with BsmBI-v2 was ligated to an annealed double-stranded DNA oligo, and the ligation reaction solution was used to transform Escherichia coli colonies, and the results of colony direct PCR were then shown.

[0009] 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.

[0010] (Method for producing a recombinant vector for preparing a target sequence containing a polymorphic base) In a first embodiment, there is provided a method for producing a recombinant vector for preparing a target sequence containing a polymorphic base, the method comprising: 1) a step of 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 a 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 located 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) a 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'-flanking sequences, and their complementary sequences.

[0011] As used herein, the term "polymorphic base" refers to one or more bases in which a base substitution, deletion, or insertion has occurred. The term "polymorphic base" can also be used as a concept that includes mutations. 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 appropriately set depending on the purpose. The type of mutation is not particularly limited, but examples include missense mutation, nonsense mutation, frameshift mutation, and silent mutation.

[0012] 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 to the gene sequence to be recombined. The gene sequence to be recombined is preferably a genomic sequence. The recombinant vector is not particularly limited as long as it can incorporate the gene of interest into the host's genes, particularly the genome, 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. A transposon vector refers to a vector having a region called a transposon that contains a gene encoding a transferase, and among transposon vectors, the Tol2 transposon vector is preferred.

[0013] Although not intended to be limiting, this embodiment enables the preparation of a vector into which a mutation has been introduced easily, quickly, and inexpensively without using conventional PCR-based methods. Because recombinant vectors prepared according to this embodiment are free of the possibility of PCR errors, the introduction of the desired mutation can be confirmed by examining only the sequence near the mutation introduction site. Furthermore, while highly purified primers used in conventional PCR-based methods require time and expense for synthesis, this embodiment does not require such primers, thereby reducing the time and cost required for vector preparation. Furthermore, by introducing mutations into genomic DNA using the recombinant vector prepared according to this embodiment, it is possible to easily, quickly, and inexpensively prepare genomic DNA as a calibration reference material that can be used to evaluate the sensitivity of gene mutation diagnostic agents. This embodiment provides a method for producing an extremely useful recombinant vector.

[0014] Step of cleaving with a first restriction enzyme The method in this embodiment includes a step of cleaving, with a 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 a first restriction enzyme recognition sequence are arranged in this order.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In one embodiment, the number of bases making up 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.

[0022] 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.

[0023] A recombinant vector containing a base sequence in which the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence are arranged as described above preferably has only two cleavage sites by the first restriction enzyme. That is, it is preferable that the recombinant vector has only two first restriction enzyme recognition sequences.

[0024] The base sequence in which the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence are arranged as described above is cleaved with the first restriction enzyme. Reaction conditions such as 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.

[0025] 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.

[0026] Step of ligating to double-stranded nucleic acid The method of this embodiment includes a step of ligating a double-stranded nucleic acid containing a region consisting of a polymorphic base and its 5'- and 3'-adjacent sequences in a target sequence with their complementary sequences.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031]

[0043] This embodiment may further include a step of annealing the polymorphic base and its 5'- and 3'-adjacent sequences in the target sequence with their complementary sequences. The annealing step is preferably performed before the ligation step.

[0032] 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. In this embodiment, 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, the nucleic acids 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.

[0033] According to the method of this embodiment, a site 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 a recombinant vector, and a double-stranded nucleic acid containing a polymorphic base is ligated, thereby producing a recombinant vector containing a polymorphic base.

[0034] Transformation Using Ligation Reaction Solution After the ligation step, it is preferable to carry out transformation using the ligation reaction solution in order to select and purify recombinant vectors containing the polymorphic base.

[0035] Transformation may be performed by any method known to those skilled in the art. Examples of transformation methods include electroporation and heat shock. The transformed host is also referred to as a transformant. Selection of transformants, growth of transformants, induction of expression of the target protein, and recovery of hosts expressing the target protein may also be performed by any method known to those skilled in the art. Transformants can be obtained, for example, by culturing a mixture of an expression vector and host competent cells in a medium containing an antibiotic corresponding to the selection marker encoded by the expression vector, and selecting the formed colonies. While the medium varies depending on the host cell, when the host is Escherichia coli, LB medium is an example of a suitable medium. Plate medium is preferred for selecting transformants. Culture conditions are appropriately adjusted, for example, at a temperature of approximately 30°C to 37°C and for a culture time of approximately 12 hours to overnight.

[0036] Selection of Recombinant Vector with Double-Stranded Nucleic Acid Inserted Whether the recombinant vector has been cleaved with the first restriction enzyme and the double-stranded nucleic acid has been inserted can be confirmed by subjecting the formed colonies to a PCR reaction and qualitatively analyzing the result. Primers used in the PCR reaction can be, for example, primers that amplify a region containing the insertion site of the double-stranded nucleic acid. Whether the double-stranded nucleic acid has been inserted can be confirmed by qualitatively analyzing the PCR amplification product (PCR product) to determine whether a PCR amplicon of the desired length has been generated. Qualitative analysis of the PCR product can be performed, for example, by electrophoresis, which separates the PCR product based on molecular weight. For example, it is preferable to directly use the formed colonies as templates in the PCR reaction (colony direct PCR), but nucleic acids extracted from the colonies may also be used as templates.

[0037] Selection of Recombinant Vector Having Inserted Double-Stranded Nucleic Acid Containing Polymorphic Base Recombinant vectors containing double-stranded nucleic acids include those in which, after a recombinant vector is cleaved 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 is inserted instead of the double-stranded nucleic acid containing the polymorphic base, and then self-ligated.Furthermore, those that are not cleaved at all by the first restriction enzyme and those that are cleaved at only one site by the first restriction enzyme are also included.

[0038] Therefore, by treating the PCR product in which it has been confirmed that a double-stranded nucleic acid has been inserted with a second restriction enzyme, it is possible to confirm whether a double-stranded nucleic acid containing a polymorphic base has been inserted into the recombinant vector.

[0039] A PCR product derived from a recombinant vector into which a double-stranded nucleic acid having 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, excised by a first restriction enzyme, rather than a double-stranded nucleic acid containing a polymorphic base, is inserted and self-ligated, is cleaved by the second restriction enzyme, whereas a PCR product derived from a recombinant vector into which a double-stranded nucleic acid containing a polymorphic base has been inserted is not cleaved by the second restriction enzyme.

[0040] After treatment with the second restriction enzyme, qualitative analysis is performed to confirm whether or not cleavage by the second restriction enzyme occurs, thereby making it possible to confirm whether or not the double-stranded nucleic acid containing the polymorphic base has been inserted into the recombinant vector.

[0041] For PCR products that have not been cleaved by the second restriction enzyme and in which insertion of a double-stranded nucleic acid containing a polymorphic base has been confirmed, it is preferable to amplify the region near the inserted double-stranded nucleic acid containing the polymorphic base and subject the amplified product to sequence analysis, thereby finally confirming that the double-stranded nucleic acid containing the polymorphic base has been inserted.

[0042] Colonies in which the insertion of the double-stranded nucleic acid containing the polymorphic base has been confirmed are grown by culture, and the target recombinant vector containing the polymorphic base is purified from the culture. It is preferable to amplify the region of the target recombinant vector obtained by purification, near the region where the double-stranded nucleic acid containing the polymorphic base has been inserted, and subject the amplified region to sequence analysis, thereby finally confirming that the target recombinant vector containing the polymorphic base has been produced.

[0043] When a recombinant vector containing a polymorphic base is prepared by conventional PCR, PCR errors may occur, and therefore, whether or not the desired recombinant vector has been prepared must ultimately be determined by sequencing the entire vector. However, the method of the present embodiment does not use PCR, and the region containing the polymorphic base is inserted by ligation, so that whether or not the desired recombinant vector has been prepared can be confirmed by sequencing only the region near the inserted region.

[0044] By the method of this embodiment, a recombinant vector containing a polymorphic base is produced, and the recombinant vector can be used to incorporate the polymorphic base into a nucleic acid, thereby producing a target sequence containing the polymorphic base. Thus, this embodiment provides a method for producing a recombinant vector for producing a target sequence containing a polymorphic base. In this embodiment, the method for producing a recombinant vector for producing a target sequence containing a polymorphic base may be a method for producing a recombinant vector for introducing a target sequence containing a polymorphic base into a nucleic acid.

[0045] The nucleic acid into which a polymorphic base is incorporated by the recombinant vector produced by the method of this embodiment refers to any nucleic acid such as DNA, genomic DNA, plasmid DNA, cDNA, or RNA, with genomic DNA being preferred. When the nucleic acid into which a polymorphic base is incorporated by the recombinant vector is genomic DNA, the target sequence in this embodiment is preferably derived from the genomic DNA into which the polymorphic base is incorporated. In other words, it is preferred that the sequence other than the polymorphic base is derived from the sequence of the genomic DNA into which the polymorphic base is incorporated.

[0046] As used herein, "genomic DNA" refers to the DNA that constitutes all of the genetic information (genome) contained in a cell.

[0047] If the recombinant vector produced by the method of this embodiment is a viral vector, the polymorphic base is incorporated into the nucleic acid by infecting a host with the viral vector; if the recombinant vector is a non-viral vector, the recombinant vector is introduced into a host by transformation; or if the recombinant vector is a transposon vector, the polymorphic base is incorporated into the nucleic acid by using a transposon system.

[0048] Nucleic acids incorporating polymorphic bases can be used as calibration standards. As used herein, the term "calibration standard" refers to a substance that serves as a reference when evaluating analytical methods, diagnostic agents, or calibrating devices. For example, to evaluate the sensitivity of a companion diagnostic agent, such as a gene mutation diagnostic agent, a gene containing a mutation is used as the calibration standard, and the mutation can be introduced using a recombinant vector containing the mutation.

[0049] Therefore, the method of this embodiment may be a method for producing a recombinant vector for producing a gene as a calibration reference material. By the method of this embodiment, a recombinant vector containing a mutation is produced, and a gene having the mutation as a calibration reference material is produced by the recombinant vector containing the mutation.

[0050] Preferably, the gene used as the calibration standard is genomic DNA.

[0051] When the sensitivity of a gene mutation diagnostic agent is evaluated using a calibration reference material, the sensitivity is evaluated by the VAF (variant allele frequency, mutant gene frequency) specified for the calibration reference material. Therefore, a VAF must be specified for each calibration reference material. Furthermore, for each mutant gene targeted by the diagnostic agent, a calibration reference material with a specified VAF for the mutant gene is required. For example, when sensitivity is evaluated using a calibration reference material with a specified VAF of 1%, if a mutation can be detected by testing the calibration reference material with the specified VAF with the gene mutation diagnostic agent whose sensitivity is to be evaluated, the sensitivity (VAF) of the gene mutation diagnostic agent can be evaluated as 1% or higher.

[0052] 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, as measured by validated droplet digital PCR. When using the copy number of a mutant gene, a validated droplet digital PCR reagent is required for each mutant gene.

[0053] When the gene serving as the calibration reference material is genomic DNA, in order to easily determine the VAF of the calibration reference material, the recombinant vector produced by the method of this embodiment preferably contains a reference sequence derived from the genomic DNA serving as the calibration reference material. The reference sequence is preferably an internal standard sequence derived from the genomic DNA serving as the calibration reference material, and the reference sequence, such as the internal standard sequence, is preferably incorporated into the calibration reference material together with the polymorphic base by the recombinant vector.

[0054] As used herein, the term "internal standard sequence" refers to any gene sequence derived from genomic DNA into which a polymorphic base is incorporated by a recombinant vector, which is different from the target sequence in the genomic DNA. 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.

[0055] By incorporating a reference sequence such as an internal standard sequence into a calibration reference material together with a polymorphic base, it becomes possible to accurately determine the VAF of the calibration reference material using the copy number of the reference sequence such as an internal standard sequence, without using the copy number of the mutant gene. In other words, regardless of the type of mutant gene the calibration reference material contains, it becomes possible to determine the VAF of the calibration reference material using the copy number of the reference sequence such as an internal standard sequence. By including a reference sequence such as an internal standard sequence derived from the calibration reference material in the recombinant vector, a validated droplet digital PCR reagent for the internal standard gene can be used in common for calibration reference materials containing each mutant gene, making it possible to easily determine the VAF of multiple calibration reference materials. Therefore, the method of this embodiment may also be a method for producing a recombinant vector for easily determining the VAF of a calibration reference material. A method for calculating the VAF using an internal standard gene when the calibration reference material is genomic DNA is described in detail below.

[0056]

[0023] The internal standard sequence introduced by the recombinant vector together with the target sequence containing the polymorphic base is referred to herein 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 target sequence containing the polymorphic base and the first internal standard sequence are introduced is genomic DNA having an unmutated wild-type target sequence and the first internal standard sequence. Therefore, when one copy each of the target sequence containing the polymorphic base and the first internal standard sequence is 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 target sequence containing the polymorphic base, and two copies of the first internal standard sequence.

[0057] Therefore, the copy number of the target sequence containing the polymorphic base in the genomic DNA used as the calibration reference 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 target sequence containing the polymorphic base. 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).

[0058] [Formula 1] [Copy number of target sequence containing polymorphic base]=[Copy number of first internal standard sequence after gene introduction]−[Copy number of first internal standard sequence before gene introduction]

[0059] 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 introduction of the target sequence and internal standard sequence containing the polymorphic base are equal. Even after the introduction of the target sequence and internal standard sequence containing the polymorphic base, the copy number of the second internal standard sequence does not change (Equation 2).

[0060] [Formula 2] [Number of copies of first internal standard sequence before gene introduction] = [Number of copies of second internal standard sequence]

[0061] Therefore, from [Equation 1] and [Equation 2], the copy number of the target sequence containing the polymorphic base in the genomic DNA used as the calibration reference material produced by the method of the present 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 target sequence containing the polymorphic base and the first internal standard sequence have been introduced (Equation 3).

[0062] [Formula 3] [Number of copies of target sequence containing polymorphic base] = [Number of copies of first internal standard sequence after gene transfer] - [Number of copies of second internal standard sequence]

[0063] 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 target sequence and internal standard sequence containing the polymorphic base are introduced (Equation 4).

[0064] [Formula 4] [Copy number of wild-type target sequence] = [Copy number of second internal standard sequence]

[0065] Furthermore, VAF is generally calculated by the calculation formula [Formula 5] using the copy number of the target sequence containing the polymorphic base.

[0066] [Formula 5] VAF (%) = {[copy number of target sequence containing polymorphic base] / ([copy number of target sequence containing polymorphic base] + [copy number of wild-type target sequence])} * 100

[0067] Therefore, from [Equation 3] to [Equation 5], the VAF of genomic DNA as a calibration reference substance can be calculated using the copy number of the first internal standard sequence and the copy number of the second internal standard sequence (Equation 6). [Equation 6] VAF (%) = {([Copy number of the first internal standard sequence after gene introduction] - [Copy number of the second internal standard sequence]) / ([Copy number of the first internal standard sequence after gene introduction] - [Copy number of the second internal standard sequence] + [Copy number of the second internal standard sequence])} * 100 = {([Copy number of the first internal standard sequence after gene introduction] - [Copy number of the second internal standard sequence]) / [Copy number of the first internal standard sequence after gene introduction]} * 100

[0068] Genomic DNA is preferably derived from cells with a diploid chromosome number. However, when the chromosome number 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 number of chromosomes carrying each of the first internal standard sequence, the second internal standard sequence, and the target sequence is the same.

[0069] Therefore, by incorporating the internal standard sequence together with the target sequence containing the polymorphic base into the calibration reference substance using the recombinant vector, it becomes possible to calculate the VAF of the calibration reference substance by using the copy numbers of two types of internal standard genes without using the copy number of the mutant gene. That is, it becomes possible to accurately calculate the VAF of the calibration reference substance without preparing a validated droplet digital PCR reagent for each mutant gene. Since the validated droplet digital PCR reagents for two types of internal standard genes can be commonly used for calibration reference substances having each mutant gene, it becomes possible to easily calculate the VAFs of a plurality of calibration reference substances.

[0070] The aspect of the method for producing a recombinant vector for preparing a target sequence containing a polymorphic base, which is the first embodiment, is also applicable to the second and third embodiments described below.

[0071] (Recombinant vector for preparing a target sequence incorporating a polymorphic base) In a second embodiment, there is provided a recombinant vector for preparing a target sequence incorporating a polymorphic base, 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 a first restriction enzyme recognition sequence, corresponding to a region consisting of the polymorphic base in the target sequence and its 5' and 3' flanking sequences, and their complementary sequences; the two first restriction enzyme recognition sequences are arranged so that the cleavage sites of the two first restriction enzymes 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.

[0072] 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, corresponding to a region consisting of a polymorphic base in a target sequence, its 5'- and 3'-flanking sequences, and their complementary sequences, refers to a recombinant vector having a nucleotide sequence in which the polymorphic base in the target sequence and its 5'- and 3'-flanking sequences are replaced with 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. In other words, this refers to a recombinant vector in which the polymorphic base and its 5'- and 3'-flanking sequences in the target sequence are excised from a recombinant vector containing the target sequence, and 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 is inserted into the excised region. The polymorphic base and its 5'- and 3'-flanking sequences may be the same length as or different from the 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.

[0073] Using the recombinant vector of this embodiment, a recombinant vector containing a polymorphic base is produced by the method of the first embodiment. The recombinant vector containing a polymorphic base incorporates the polymorphic base into a nucleic acid, allowing a target sequence containing the polymorphic base to be produced. Thus, this embodiment provides a recombinant vector for producing a target sequence incorporating a polymorphic base. The recombinant vector of this embodiment may be a recombinant vector for producing a recombinant vector containing a polymorphic base.

[0074] When the nucleic acid into which the polymorphic base is incorporated is a calibration standard, the recombinant vector of this embodiment may be a recombinant vector for producing a gene as the calibration standard. When the calibration standard is genomic DNA, the recombinant vector of this embodiment preferably contains a reference sequence derived from the calibration standard. The reference sequence may be an internal standard sequence.

[0075] (Kit Comprising a Recombinant Vector for Preparing a Target Sequence Incorporating a Polymorphic Base and a Nucleic Acid Sequence) In a third embodiment, there is provided a kit comprising: 1) a recombinant vector for preparing a target sequence incorporating a polymorphic base, 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 the first restriction enzyme recognition sequence, corresponding to a region in the target sequence consisting of the polymorphic base and its 5'- and 3'-flanking sequences, and their complementary sequences; the two first restriction enzyme recognition sequences are arranged so that the two first restriction enzyme cleavage sites 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; 2) a first nucleic acid sequence to be inserted into the restriction enzyme recognition site, the first nucleic acid sequence comprising the polymorphic base and its 5'- and 3'-flanking sequences; and 3) a second nucleic acid sequence to be inserted into the restriction enzyme recognition site, the second nucleic acid sequence comprising a complementary strand of the first nucleic acid sequence of 2).

[0076] In the kit of this embodiment, the first nucleic acid sequence of 2) and the second nucleic acid sequence of 3) may be provided in a double-stranded state. The double-stranded structure can be formed by annealing.

[0077] The 1) recombinant vector, 2) first nucleic acid sequence, and 3) second nucleic acid sequence provided as the kit of this embodiment may be provided together with any buffer, enzyme, etc. For example, they may be provided together with a first restriction enzyme or ligase, or together with a DNA or RNase inhibitor, TE buffer, etc.

[0078] In the kit of this embodiment, the recombinant vector (1), the first nucleic acid sequence (2), and the second nucleic acid sequence (3) may be provided in the same container, or each may be provided in a separate container. Alternatively, only one of the recombinant vector (1), the first nucleic acid sequence (2), and the second nucleic acid sequence (3) may be provided in a separate container. When the recombinant vector (1), the first nucleic acid sequence (2), and / or the second nucleic acid sequence (3) are provided in separate containers, these containers may be provided together in a single box or the like.

[0079] The kit of this embodiment can produce a recombinant vector containing a polymorphic base by the method of the first embodiment, by reacting the recombinant vector of 1) with a first restriction enzyme to cleave it, and ligating it with a double-stranded sequence consisting of the first nucleic acid sequence of 2) and the second nucleic acid sequence of 3). The recombinant vector containing the polymorphic base incorporates the polymorphic base into a nucleic acid, allowing a target sequence containing the polymorphic base to be produced. Thus, this embodiment provides a kit for producing a target sequence incorporating a polymorphic base. The kit of this embodiment may be a kit for producing a recombinant vector containing a polymorphic base.

[0080] When the nucleic acid into which the polymorphic base is incorporated is a calibration standard, the kit of this embodiment may be a kit for producing a gene as the calibration standard. When the calibration standard is genomic DNA, the recombinant vector of 1) in the kit of this embodiment preferably contains a reference sequence derived from the calibration standard. The reference sequence may be an internal standard sequence.

[0081] The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples.

[0082] <Restriction enzyme treatment and purification of cassette vector> A partial sequence of a gene containing an exon and intron region was replaced with a 20-mer sequence having two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3'), and the KRAS-G12X / G13X cassette vector (Example 1: SEQ ID NO: 1) and the KRAS-A59X / Q61X cassette vector (Example 2: SEQ ID NO: 3) were inserted into a Tol2 vector. 2), KRAS-K117X / A146X cassette vector (Example 3: SEQ ID NO: 3), NRAS-G12X / G13X cassette vector (Example 4: SEQ ID NO: 4), NRAS-A59X / Q61X cassette vector (Example 5: SEQ ID NO: 5), NRAS-K117X / A146X cassette vector (Example 6: SEQ ID NO: 6), and BRAF-V600X cassette vector (Example 7: SEQ ID NO: 7) were prepared. In the name of the cassette vector, for example, "G12X / G13X" means that the cassette vector is for creating a vector having a gene in which the sequence corresponding to the 12th glycine and / or the 13th glycine has been mutated to a sequence corresponding to any amino acid (X). The same applies to other cassette vectors. The prepared cassette vectors of SEQ ID NOS: 1 to 7 were digested with BsmBI-v2 (New England BioLabs, Cat: R0739L) at 55°C for 1 hour. The digested products were electrophoretically separated on a 0.5% agarose / TAE gel and stained with SYBR Gold staining reagent (Thermo Fisher Scientific, Cat: S11494) for 10 minutes to detect nucleic acids. The gel region containing the desired cleavage product was excised and purified using a QIAquick Gel Extraction Kit (QIAGEN, Cat: 28704).

[0083] Here, the KRAS-G12X / G13X cassette vector (SEQ ID NO: 1) is a Tol2 vector having a partial sequence of the KRAS gene (a sequence in which Exon 2 is sandwiched between a 500-mer portion of Intron 1 and a 500-mer portion of Intron 2), in which a 22-mer sequence containing 8-mers before and after the 12th and 13th glycine codons has been replaced with a 20-mer sequence having two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3'). An example of a KRAS-G12A cassette vector is shown in Figure 3.

[0084] The KRAS-A59X / Q61X cassette vector (SEQ ID NO: 2) is a Tol2 vector having a partial sequence of the KRAS gene (a sequence in which Exon 3 is sandwiched between a 500-mer portion of Intron 2 and a 500-mer portion of Intron 3), in which a 21-mer sequence containing 6-mers before and after the codons of the 59th and 61st amino acids has been replaced with a 20-mer sequence having two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3').

[0085] The KRAS-K117X / A146X cassette vector (SEQ ID NO: 3) is a Tol2 vector having a partial sequence of the KRAS gene (a sequence in which Exon 4 is sandwiched between a 500-mer portion of Intron 3 and a 500-mer portion of Intron 4), in which a 90-mer sequence including the codons of the 117th and 146th amino acids has been replaced with a 20-mer sequence having two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3').

[0086] The NRAS-G12X / G13X cassette vector (SEQ ID NO: 4) is a Tol2 vector having a partial sequence of the NRAS gene (a sequence in which Exon 2 is sandwiched between a 500-mer portion of Intron 1 and a 500-mer portion of Intron 2), in which a 22-mer sequence containing 8-mers before and after the 12th and 13th glycine codons has been replaced with a 20-mer sequence having two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3').

[0087] The NRAS-A59X / Q61X cassette vector (SEQ ID NO: 5) is a Tol2 vector having a partial sequence of the NRAS gene (a sequence in which Exon 3 is sandwiched between a 500-mer portion of Intron 2 and a 500-mer portion of Intron 3), in which a 21-mer sequence containing 6-mers before and after the codons of the 59th and 61st amino acids has been replaced with a 20-mer sequence having two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3').

[0088] The NRAS-K117X / A146X cassette vector (SEQ ID NO: 6) is a Tol2 vector having a partial sequence of the NRAS gene (a sequence in which Exon 4 is sandwiched between a 500-mer portion of Intron 3 and a 500-mer portion of Intron 4), in which a 90-mer sequence including the codons of the 117th and 146th amino acids has been replaced with a 20-mer sequence having two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3').

[0089] The BRAF-V600X cassette vector (SEQ ID NO: 7) is a Tol2 vector having a partial sequence of the BRAF gene (a sequence in which Exon 15 is sandwiched between a 500-mer portion of Intron 14 and a 500-mer portion of Intron 15), in which a 20-mer sequence including an 8-mer before and a 9-mer after the codon of valine at position 600 has been replaced with a 20-mer sequence having two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3').

[0090] Annealing Reaction of DNA Oligonucleotides Containing Each Variant: TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0) and 5 M NaCl were mixed at a ratio of 49:1, and 10 μL of this mixture was added to 5 μL of two 200 μM DNA oligos (Example 1: SEQ ID NOS: 8-31, Example 2: SEQ ID NOS: 32-51, Example 3: SEQ ID NOS: 52-61, Example 4: SEQ ID NOS: 62-85, Example 5: SEQ ID NOS: 86-117, Example 6: SEQ ID NOS: 118-127, Example 7: SEQ ID NOS: 128-141) whose sequences are complementary to each other except for the sticky end to be formed (total volume 20 μL). The prepared DNA oligo mixture was heat denatured at 95°C for 5 minutes and then cooled to room temperature at a rate of 1°C per minute, thereby annealing the two DNA oligos to form double-stranded DNA oligos. The mixture was stored on ice or in a refrigerator set at 4°C until use.

[0091] <Double-stranded DNA oligo ligation reaction> The restriction enzyme-treated, gel-purified cassette vector and the annealed double-stranded DNA oligo were mixed at a molar ratio of 1:3, and an equal amount of Ligation High Ver. 2 (TOYOBO, Cat: LGK-201) to this nucleic acid mixture was added. After mixing, the mixture was allowed to react at 16°C for 30 minutes.

[0092] <Transformation> The ligation reaction solution was transformed into Competent high DH5α (TOYOBO, Cat: DNA-903). The transformed E. coli was plated on an ampicillin-containing LB plate and cultured overnight at 37°C to form E. coli colonies.

[0093] <Colony Direct PCR> An appropriate amount of E. coli single colony formed by transformation was added to 1x EmeraldAmp FastOCR Master Mix (Takara Bio, Cat: RR300A) containing 200 nM each of the two primers of SEQ ID NOs: 141 and 142. 30 cycles of PCR reaction were performed, each cycle consisting of 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 75 seconds. The purified products were separated by electrophoresis on a 1.0% agarose / TAE gel and stained with SYBR Gold staining reagent for 10 minutes to detect nucleic acids. For samples in which PCR amplicons of the desired length were generated, insertion of annealed double-stranded DNA oligos was confirmed by restriction enzyme digestion.

[0094] <Confirmation of insertion of target DNA oligo by restriction enzyme digestion of colony direct PCR product> The colony direct PCR product obtained in the above <Colony Direct PCR> section was digested with restriction enzyme NotI-HF (New England BioLabs, Cat: R3189S) (37°C, 1 hour). The digested product was separated by electrophoresis on a 1.0% agarose / TAE gel and stained with SYBR Gold staining reagent for 10 minutes to detect nucleic acids. If restriction enzyme digestion of the PCR product did not occur, the product was subjected to direct sequencing to determine the DNA sequence.

[0095] <Direct Sequencing of PCR Product> The concentration of the colony direct PCR product obtained in the above section <Colony Direct PCR> 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: 141 and 142) used in the colony direct PCR.

[0096] <Large-scale purification and sequence confirmation of constructed vector> Colonies that were confirmed to have the desired sequence in the above section <Direct sequencing of PCR product> were cultured in LB medium, and pDNA was purified from the resulting E. coli pellet using Hispeed Plasmid Midi Kit (QIAGEN, Cat: 12643). Sequence analysis was performed at Eurofins Genomics using two primers, SEQ ID NOs: 141 and 142.

[0097] Example 1 <Results 1> A restriction enzyme-treated, gel-purified cassette vector (KRAS-G12X / G13X cassette vector) prepared according to the experimental method was ligated with annealed double-stranded DNA oligos (each DNA oligo was 28 mer in length) and transformed, resulting in the formation of multiple colonies. Colony direct PCR revealed that amplicons of the desired length were obtained in all of the 24 selected colonies (Figure 4, top panel), and NotI treatment suggested that the desired annealed double-stranded DNA oligos had been inserted in 23 colonies (Figure 4, bottom panel). All colonies that were successfully sequenced were confirmed to have the desired sequence incorporated. Because recombination efficiency was extremely high, four single colonies from each of the other G12X / G13X variants (11 variants in total) were selected and analyzed, and the desired recombination was confirmed in 41 of the 44 colonies. After large-scale purification of the constructed vector, sequence analysis using pDNA confirmed that the desired sequence had been incorporated.

[0098] Example 2 <Results 2> A restriction enzyme-treated, gel-purified cassette vector (KRAS-A59X / Q61X cassette vector) prepared according to the experimental method was ligated with annealed double-stranded DNA oligos (each DNA oligo was 27 mer in length) and transformed, resulting in the formation of multiple colonies. Colony direct PCR showed that amplicons of the desired length were obtained in all of the four selected colonies (Figure 5). Direct sequencing of the obtained amplicons confirmed the desired recombination in 38 of the 40 colonies. Sequence analysis using pDNA after large-scale purification of the constructed vector confirmed the integration of the desired sequence.

[0099] Example 3 <Result 3> A restriction enzyme-treated, gel-purified cassette vector (KRAS-K117X / A146X cassette vector) prepared according to the experimental method was ligated with annealed double-stranded DNA oligos (each DNA oligo was 96 mer in length) and transformed, resulting in the formation of multiple colonies. Four colonies from each vector were selected and sequenced, confirming the desired recombination in 16 of the 20 colonies. Although the recombination success rate decreased as the size of the incorporated double-stranded DNA oligo increased, a recombination efficiency of 80% was observed. After large-scale purification of the constructed vector, sequence analysis using pDNA confirmed that the desired sequence had been incorporated.

[0100] Example 4 <Results 4> A restriction enzyme-treated, gel-purified cassette vector (NRAS-G12X / G13X cassette vector) prepared according to the experimental method was ligated with annealed double-stranded DNA oligos (each DNA oligo was 28 mer in length) and transformed, resulting in the formation of multiple colonies. Four colonies from each vector were selected and sequenced, and the desired recombination was confirmed in 20 of the 48 colonies. After large-scale purification of the constructed vector, sequence analysis using pDNA confirmed that the desired sequence had been incorporated.

[0101] Example 5 <Results 5> A restriction enzyme-treated, gel-purified cassette vector (NRAS-A59X / Q61X cassette vector) prepared according to the experimental method was ligated with annealed double-stranded DNA oligos (each DNA oligo was 27 mer in length) and transformed, resulting in the formation of multiple colonies. Four colonies from each vector were selected and sequenced, confirming the desired recombination in 60 of the 64 colonies. After large-scale purification of the constructed vector, sequence analysis using pDNA confirmed that the desired sequence had been incorporated.

[0102] Example 6 <Results 6> A restriction enzyme-treated, gel-purified cassette vector (NRAS-K117X / A146X cassette vector) prepared according to the experimental method was ligated with annealed double-stranded DNA oligos (each DNA oligo was 96 mer in length) and transformed, resulting in the formation of multiple colonies. Four colonies from each vector were selected and sequenced, confirming the desired recombination in 8 of the 20 colonies. As sequence errors were observed in all colonies analyzed for K117N, 12 more colonies were selected and sequenced, confirming the desired recombination in 7 colonies. After large-scale purification of the constructed vector, sequence analysis using pDNA confirmed the integration of the desired sequence.

[0103] Example 7 <Results 7> A restriction enzyme-treated, gel-purified cassette vector (BRAF-V600X cassette vector) prepared according to the experimental method was ligated with annealed double-stranded DNA oligos (each DNA oligo was 26 mer in length) and transformed, resulting in the formation of multiple colonies. Four colonies from each vector were selected and sequenced, confirming the desired recombination in 24 of the 27 colonies. After large-scale purification of the constructed vector, sequence analysis using pDNA confirmed that the desired sequence had been incorporated.

[0104] The base sequences used in this example are shown in Table 1. The sequences of the cassette vectors of SEQ ID NOs: 1 to 7 show only the cassette sequence portion. For example, the KRAS-G12X / G13X cassette vector of SEQ ID NO: 1 is a partial KRAS gene sequence (a sequence in which Exon 2 is sandwiched between a 500-mer portion of Intron 1 and a 500-mer portion of Intron 2), and the 22-mer sequence containing 8-mers before and after the 12th and 13th glycine codons has been replaced with a 20-mer sequence containing two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3'), as shown in Table 1. SEQ ID NOs: 2 to 6 are also shown in Table 1, similar to SEQ ID NO: 1.

[0105]

Claims

1. A recombinant vector for producing a target sequence incorporating polymorphic bases, wherein a base sequence is arranged in the order of a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence corresponding to the polymorphic bases in the target sequence and the 5'- and 3'-flanking sequences thereof and the regions consisting of their complementary sequences, and the two first restriction enzyme recognition sequences are arranged such that the cleavage sites of the two first restriction enzymes are present on the side opposite to 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.

2. The recombinant vector according to claim 1, wherein the two first restriction enzyme recognition sequences are arranged at least 3 bases apart.

3. The recombinant vector according to claim 1 or 2, wherein the polymorphic bases and the 5'- and 3'-flanking sequences thereof have 10 to 200 base pairs.

4. The recombinant vector according to claim 1 or 2, which is a transposon vector.

5. The recombinant vector according to claim 1 or 2, wherein the IIS-type restriction enzyme is selected from the group consisting of 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, Esp3I, FauI, FokI, GsuI, HgaI, HinGUI, HphI, Ksp632I, MboII, MmeI, Mn1I, NgoVIII, PaqCI, PleI, PsrI, RleAI, SapI, SfaNI, TaqII, Tth111II, AcuI, BsmI, BsrI, BsmFI, BseMII, BspQI, and BtgZI.

6. The recombinant vector according to claim 1 or 2, wherein the target sequence is derived from genomic DNA.

7. The recombinant vector according to claim 6, wherein the recombinant vector further contains a reference sequence.

8. The recombinant vector according to claim 7, wherein the reference sequence is an internal standard sequence derived from genomic DNA.

9. 1) A recombinant vector for preparing a target sequence incorporating polymorphic bases, the recombinant vector having a nucleotide sequence in which a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence are arranged in this order corresponding to the polymorphic bases in the target sequence, the adjacent sequences on the 5' side and 3' side thereof, and the regions consisting of their complementary sequences, and the two first restriction enzyme recognition sequences are arranged such that the cleavage sites of the two first restriction enzymes are present on the side opposite to the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence being an IIS-type 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; 2) A first nucleic acid sequence inserted into the restriction enzyme recognition site, the first nucleic acid sequence containing polymorphic bases, and the adjacent sequences on the 5' side and 3' side thereof; 3) A second nucleic acid sequence inserted into the restriction enzyme recognition site, the second nucleic acid sequence containing the complementary strand of the first nucleic acid sequence of 2). A kit containing these components.

10. The kit according to claim 9 for producing a gene as a calibration reference substance.

11. The kit according to claim 10, wherein the gene as a calibration reference substance is genomic DNA.

12. The kit according to claim 11, wherein the recombinant vector further contains an internal standard sequence derived from genomic DNA.

13. The kit according to any one of claims 9 to 12, wherein the first nucleic acid sequence of 2) and the second nucleic acid sequence of 3) form a double strand.

14. A method for producing a recombinant vector for preparing a target sequence containing polymorphic bases, the method including: 1) A step of cleaving a recombinant vector having a nucleotide sequence in which a first restriction enzyme recognition sequence, a second restriction enzyme recognition sequence, and a first restriction enzyme recognition sequence are arranged in this order, and the two first restriction enzyme recognition sequences are arranged such that the cleavage sites of the two first restriction enzymes are present on the side opposite to the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence being an IIS-type 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, with a first restriction enzyme; 2) A step of ligating a double-stranded nucleic acid containing the polymorphic bases in the target sequence, the adjacent sequences on the 5' side and 3' side thereof, and the regions consisting of their complementary sequences.

15. The method according to claim 14, further including a step of annealing to prepare a double-stranded nucleic acid.

16. The method according to claim 14 or 15, wherein the double-stranded nucleic acid has a sequence complementary to the cleavage sequence by the first restriction enzyme.

17. The method according to claim 14 or 15, further comprising the step of digesting with a second restriction enzyme.

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