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WO2025143215A1 - Vecteur recombinant destiné à être utilisé dans production de séquence cible ayant un nucléotide polymorphe incorporé - Google Patents

Vecteur recombinant destiné à être utilisé dans production de séquence cible ayant un nucléotide polymorphe incorporé Download PDF

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WO2025143215A1
WO2025143215A1 PCT/JP2024/046377 JP2024046377W WO2025143215A1 WO 2025143215 A1 WO2025143215 A1 WO 2025143215A1 JP 2024046377 W JP2024046377 W JP 2024046377W WO 2025143215 A1 WO2025143215 A1 WO 2025143215A1
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sequence
restriction enzyme
enzyme recognition
recognition sequence
recombinant vector
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裕嗣 久保
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Denka Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • 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 amino acid sequence is selected from the group consisting of gaI, HinGUII, HphI, Ksp632I, MboII, MmeI, Mn1I, NgoVIII, PaqCI, PleI, PsrI, RleAI, SapI, SfaNI, TaqII, Tth111II
  • the kit according to [9] for producing a gene as a calibration standard material [11] The kit according to [10], wherein the gene 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.
  • a method for producing a recombinant vector for generating a target sequence containing a polymorphic base comprising the steps of: 1) 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, the two first restriction enzyme recognition sequences are arranged such that the cleavage sites for the two first restriction enzymes are on opposite sides of the second restriction enzyme recognition sequence; a step of cleaving the recombinant vector with a first restriction enzyme, the first restriction enzyme recognition sequence being a type IIS restriction enzyme recognition sequence and the second restriction enzyme recognition sequence being a different restriction enzyme recognition sequence from the first restriction enzyme recognition sequence; 2) ligating a double-stranded nucleic acid including a region consisting of a polymorphic base in a target sequence and its 5'- and 3'-adjacent sequences and their complementary sequences; A method comprising: [15] The method according to [14], further comprising
  • FIG. 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 is a schematic diagram showing 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.
  • a schematic diagram of the KRAS-G12A cassette vector is shown. The upper panel shows the results of colony direct PCR performed on E.
  • a "polymorphic base” refers to one or more bases in which a base substitution, deletion, or insertion has occurred. Furthermore, “polymorphic base” can 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 set appropriately depending on the purpose.
  • the type of mutation is not particularly limited, but examples include missense mutations, nonsense mutations, frameshift mutations, and silent mutations.
  • the present embodiment provides a method for producing a highly useful recombinant vector.
  • the first restriction enzyme (which may be one or two) is a type IIS restriction enzyme.
  • a type IIS restriction enzyme generally refers to a restriction enzyme characterized in that the restriction enzyme recognition sequence and the cleavage site are separated. Among type IIS restriction enzymes, type IIS restriction enzymes that have all of their cleavage sites on one side, away from the restriction enzyme recognition sequence, are preferred. In addition, the recognition sequence of a type IIS restriction enzyme does not have to be a palindrome.
  • the two first restriction enzyme recognition sequences may be distinguished as the former being the first restriction enzyme recognition sequence A and the latter being the first restriction enzyme recognition sequence B.
  • the second restriction enzyme recognition sequence on the 5' side may be distinguished as the 5' side first restriction enzyme recognition sequence
  • the second restriction enzyme recognition sequence on the 3' side may be distinguished as the 3' side first restriction enzyme recognition sequence.
  • the first restriction enzyme recognition sequences are preferably arranged at least 3 bases apart, and more preferably at least 6 bases apart.
  • the number of bases between the first restriction enzyme recognition sequences is selected from the group consisting of 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases and 20 bases.
  • the 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 may have a linker sequence having any number of bases.
  • the 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; or in the order of a first restriction enzyme recognition sequence, a linker, a second restriction enzyme recognition sequence, a linker, and a first restriction enzyme recognition sequence.
  • they 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, and a linker; or they 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, and a linker; or they 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, and a linker.
  • 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.
  • the two first restriction enzyme cleavage sites are arranged so that they are located on the opposite side to the second restriction enzyme recognition sequence.
  • the first and second restriction enzyme recognition sequences are arranged so that the underlined cleavage site (where the bond between the two underlined bases is cleaved) is 5'- NN NNNN-(first first restriction enzyme recognition sequence)-(second restriction enzyme recognition sequence)-(second first restriction enzyme recognition sequence) -NN NNNN-3', and in the complementary strand, it is 3'-NNNN NN- (first first restriction enzyme recognition sequence)-(second restriction enzyme recognition sequence)-(second first restriction enzyme recognition sequence)-NNNN -5'.
  • FIG. 1 A schematic diagram of the arrangement of the first and second restriction enzyme recognition sequences 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.
  • N represents any base, but is a part of the target gene sequence near the site where a mutation is to be inserted.
  • 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. In other words, it is preferable that the recombinant vector has only two first restriction enzyme recognition sequences.
  • 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 a first restriction enzyme
  • the site in the 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 excised.
  • Step of ligating to double-stranded nucleic acid 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, and their complementary sequences.
  • 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 reaction with the ligase, can be appropriately determined by those skilled in the art. Commercially available ligation reagents may also be used.
  • the ligation step is preferably followed by a first restriction enzyme cleavage step.
  • a double-stranded nucleic acid including a region consisting of a polymorphic base in a target sequence, its 5'-side and 3'-side adjacent sequences, and their complementary sequences is inserted and ligated at a site where 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 is cut by the first restriction enzyme treatment.
  • FIG. 2 shows a schematic diagram of a case where a double-stranded nucleic acid is inserted and ligated after the recombinant vector shown as an example in FIG.
  • the double-stranded nucleic acid to be ligated has a 3'- or 5'-overhanging end that forms a complementary strand with the 5'- or 3'-overhanging end. If the cleavage sequence generated 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.
  • Recombinant vectors into which double-stranded nucleic acid is inserted include those into which, after a recombinant vector is cleaved with a first restriction enzyme, a double-stranded nucleic acid having a base sequence arranged 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 a polymorphic base, and which are then self-ligated.Furthermore, those which are not cleaved at all by the first restriction enzyme and those which are cleaved at only one site by the first restriction enzyme are also included.
  • double-stranded nucleic acid containing a polymorphic base double-stranded nucleic acid having a base sequence cut out by a first restriction enzyme, 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, and the PCR product derived from the self-ligated recombinant vector is cleaved by the second restriction enzyme.
  • the PCR product derived from the recombinant vector into which double-stranded nucleic acid containing a polymorphic base has been inserted is not cleaved by the second restriction enzyme.
  • PCR products that were not cleaved by the second restriction enzyme and in which insertion of a double-stranded nucleic acid containing a polymorphic base was confirmed, it is preferable to amplify the area near the region in which the double-stranded nucleic acid containing the polymorphic base was inserted and subject the product to sequence analysis to finally confirm that the double-stranded nucleic acid containing the polymorphic base was inserted.
  • Colonies in which it has been confirmed that the double-stranded nucleic acid containing the polymorphic base has been inserted are grown in culture, and the desired recombinant vector containing the polymorphic base is purified from the culture. It is preferable to amplify the region of the desired recombinant vector obtained by purification near the region where the double-stranded nucleic acid containing the polymorphic base has been inserted, and subject the region to sequence analysis to finally confirm that the desired recombinant vector containing the polymorphic base has been produced.
  • the nucleic acid into which the 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, RNA, etc., and among these, genomic DNA is preferred.
  • 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.
  • the polymorphic base is incorporated into the nucleic acid by infecting the host with the viral vector; if the recombinant vector is a non-viral vector, the recombinant vector is introduced into the host by transformation; and if the recombinant vector is a transposon vector, the polymorphic base is incorporated into the nucleic acid by using a transposon system.
  • Nucleic acids incorporating polymorphic bases can be used as calibration standards.
  • calibration reference material refers to a material that serves as a reference for evaluating an analytical method or diagnostic agent, or for calibrating an apparatus.
  • a companion diagnostic agent such as a gene mutation diagnostic agent
  • a gene having a mutation is used as the calibration reference material, and the mutation can be introduced using a recombinant vector containing the mutation.
  • the method of this embodiment may be a method for producing a recombinant vector for producing a gene as a calibration reference material.
  • a recombinant vector containing a mutation is produced, and a gene having a mutation is produced as a calibration reference material by the recombinant vector containing the mutation.
  • the gene used as the calibration standard is preferably genomic DNA.
  • the sensitivity of a gene mutation diagnostic agent is evaluated using a calibration standard substance
  • the sensitivity is evaluated by the VAF (mutant allele frequency, mutant gene frequency) specified for the calibration standard substance. Therefore, it is necessary that a VAF be specified for each calibration standard substance.
  • a calibration standard substance is required in which the VAF of the mutant gene is specified. For example, when the 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 a gene mutation diagnostic agent whose sensitivity is to be evaluated, the sensitivity (VAF) of this gene mutation diagnostic agent can be evaluated as being 1% or more.
  • a known method for determining the VAF of a calibration reference material is, for example, to use the copy number of a mutant gene in the calibration reference material measured by validated droplet digital PCR.
  • a validated droplet digital PCR reagent is required for each mutant gene.
  • the recombinant vector produced by the method of this embodiment 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 it is preferable that the reference sequence, such as the internal standard sequence, is incorporated into the calibration reference material together with the polymorphic base by the recombinant vector.
  • 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.
  • the internal standard sequence is a gene sequence in 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 may be sequences derived from different chromosomes. There is no need to select a sequence such as a housekeeping gene, which is constantly expressed in cells and has small expression fluctuations, as the internal standard sequence. Examples of internal standard sequences include the PTEN gene, the AGO1 gene, the TMEM11 gene, and the ERBB2 gene.
  • the method of this embodiment may be a method of 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.
  • the internal standard sequence introduced by the recombinant vector together with the target sequence containing the polymorphic base is referred to as the first internal standard sequence in this specification. Since 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.
  • a target sequence containing a polymorphic base and a first internal standard sequence each of which is a copy
  • 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.
  • 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, which is 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).
  • any internal standard sequence that is present in genomic DNA and is different from the first internal standard sequence is referred to as a second internal standard sequence.
  • the first internal standard sequence and the second internal standard sequence may be sequences derived from different gene loci or different chromosomes.
  • 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 target sequence containing the polymorphic base and the internal standard sequence are introduced are equal. Even after the target sequence containing the polymorphic base and the internal standard sequence are introduced, the copy number of the second internal standard sequence does not change (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 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 target sequence containing the polymorphic base and the first internal standard sequence have been introduced (Equation 3).
  • 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).
  • VAF is generally calculated using the number of copies of the target sequence containing the polymorphic base, according to the formula [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
  • the kit of this embodiment may be a kit for producing a gene as the calibration reference material.
  • the recombinant vector 1) in the kit of this embodiment preferably contains a reference sequence derived from the calibration reference material.
  • the reference sequence may be an internal standard sequence.
  • the digestion 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.
  • SYBR Gold staining reagent Thermo Fisher Scientific, Cat: S11494
  • the gel region containing the cleavage product of interest was excised and purified using a QIAquick Gel Extraction Kit (QIAGEN, Cat: 28704).
  • 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 containing 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').
  • the NRAS-G12X/G13X cassette vector (SEQ ID NO:4) is a Tol2 vector that has a partial sequence of the NRAS gene (a sequence in which Exon2 is sandwiched between a 500mer portion of Intron1 and a 500mer portion of Intron2), and is a sequence in which a 22mer sequence containing 8mers before and after the 12th and 13th glycine codons has been replaced with a 20mer sequence containing two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3').
  • the NRAS-A59X/Q61X cassette vector (SEQ ID NO:5) is a Tol2 vector that has a partial sequence of the NRAS gene (a sequence in which Exon3 is sandwiched between a 500mer portion of Intron2 and a 500mer portion of Intron3), and is a sequence in which a 21mer sequence containing 6mers before and after the codons of the 59th and 61st amino acids has been replaced with a 20mer sequence containing two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3').
  • the NRAS-K117X/A146X cassette vector (SEQ ID NO:6) is a Tol2 vector that has 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 containing the codons of the 117th and 146th amino acids has been replaced with a 20-mer sequence containing two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3').
  • the BRAF-V600X cassette vector (SEQ ID NO: 7) is a Tol2 vector having a partial BRAF gene sequence (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 600th valine codon has been replaced with a 20-mer sequence having two BsmBI sites (5'-CGTCTC-3') separated by a NotI site (5'-GCGGCCGC-3').
  • Annealing reaction of DNA oligos 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 ⁇ L of this mixture was added with 5 ⁇ L of two types of 200 ⁇ M 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 ⁇ L).
  • the prepared DNA oligo mixture was heat denatured at 95° C. for 5 minutes, and cooled to room temperature at a rate of 1° C. per minute to anneal the two types of DNA oligos to form double-stranded DNA oligos.
  • the mixture was stored on ice or in a refrigerator set at 4° C. until use.
  • ⁇ Direct sequencing of PCR products> The concentration of the colony direct PCR product obtained in the above ⁇ Colony Direct PCR> section was determined using 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 two types of primers (SEQ ID NOs: 141 and 142) used in the colony direct PCR.
  • Example 2 The 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 length was 27 mer) and transformed, resulting in the formation of multiple colonies.
  • amplicons of the desired length were obtained in all of the four selected colonies ( Figure 5).
  • the desired recombination was confirmed in 38 of the 40 colonies.
  • sequence analysis using pDNA after large-scale purification of the constructed vector it was confirmed again that the desired sequence had been incorporated.
  • Example 4 ⁇ Result 4> The 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 length was 28 mer) and transformed, resulting in the formation of multiple colonies. Four colonies from each 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.
  • Example 5 The 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 length was 27 mer) and transformed, resulting in the formation of multiple colonies. Four colonies from each 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.
  • Example 6 The 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 length was 96 mer) and transformed, resulting in the formation of multiple colonies. Four colonies were selected from each group and sequenced, confirming the desired recombination in eight of the 20 colonies. As for K117N, sequence errors were observed in all analyzed colonies, so 12 colonies were further selected and sequenced, confirming the desired recombination in seven colonies. After large-scale purification of the constructed vector, sequence analysis using pDNA confirmed that the desired sequence had been incorporated.
  • Example 7 The 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 length was 26 mer) and transformed, resulting in the formation of multiple colonies. Four colonies were selected from each group 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.
  • the base sequences used in this example are shown in Table 1.
  • 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 having 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.

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Abstract

La présente invention concerne un vecteur recombinant destiné à être utilisé dans la production d'une séquence cible ayant un nucléotide polymorphe incorporé. Le vecteur recombinant a une séquence nucléotidique dans laquelle une première séquence de reconnaissance d'enzyme de restriction, une seconde séquence de reconnaissance d'enzyme de restriction et une autre première séquence de reconnaissance d'enzyme de restriction sont agencées, dans cet ordre, correspondant à une région qui comprend le nucléotide polymorphe dans la séquence cible et des séquences flanquantes côté 5' et 3' du nucléotide polymorphe et les séquences complémentaires à celle-ci. Les deux premières séquences de reconnaissance d'enzyme de restriction sont agencées de telle sorte que deux premiers sites de clivage d'enzyme de restriction sont présents sur le côté opposé à la seconde séquence de reconnaissance d'enzyme de restriction. La première séquence de reconnaissance d'enzyme de restriction est une séquence de reconnaissance d'enzyme de restriction de type IIS. La seconde séquence de reconnaissance d'enzyme de restriction est différente de la première.
PCT/JP2024/046377 2023-12-28 2024-12-27 Vecteur recombinant destiné à être utilisé dans production de séquence cible ayant un nucléotide polymorphe incorporé Pending WO2025143215A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020025561A1 (en) * 2000-04-17 2002-02-28 Hodgson Clague Pitman Vectors for gene-self-assembly
GB2465986A (en) * 2008-12-04 2010-06-09 Angeletti P Ist Richerche Bio Method of generating diversity in polynucleotide sequences
JP2020527938A (ja) * 2017-06-14 2020-09-17 テクニスチェ ユニベルシタト ドレスデン デザイナーｄｎａ組換え酵素を利用したゲノムを遺伝的に改変するための方法及び手段
JP2022501025A (ja) * 2018-09-19 2022-01-06 ザ・ユニバーシティ・オブ・ホンコンThe University Of Hong Kong 改良されたハイスループットコンビナトリアル遺伝子改変システムおよび最適化されたＣａｓ９酵素変異体

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020025561A1 (en) * 2000-04-17 2002-02-28 Hodgson Clague Pitman Vectors for gene-self-assembly
GB2465986A (en) * 2008-12-04 2010-06-09 Angeletti P Ist Richerche Bio Method of generating diversity in polynucleotide sequences
JP2020527938A (ja) * 2017-06-14 2020-09-17 テクニスチェ ユニベルシタト ドレスデン デザイナーｄｎａ組換え酵素を利用したゲノムを遺伝的に改変するための方法及び手段
JP2022501025A (ja) * 2018-09-19 2022-01-06 ザ・ユニバーシティ・オブ・ホンコンThe University Of Hong Kong 改良されたハイスループットコンビナトリアル遺伝子改変システムおよび最適化されたＣａｓ９酵素変異体

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "2-8. Checking the inserted DNA fragment using restriction enzymes", BIOFUNCTIONAL MATERIALS ENGINEERING EXPERIMENT II, 14 September 2014 (2014-09-14), pages 1 - 3, XP093329286, Retrieved from the Internet <URL:https://web.archive.org/web/20170914181101/https://www.cc.kochi-u.ac.jp/~tatataa/tech2/gene/check.html> [retrieved on 20250218] *
HAISU WAN; YONGWEN LI; YU FAN; FANRONG MENG; CHEN CHEN; QINGHUA ZHOU;: "A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning", ANALYTICAL BIOCHEMISTRY, vol. 420, no. 2, 17 September 2011 (2011-09-17), Amsterdam, NL , pages 163 - 170, XP028111228, ISSN: 0003-2697, DOI: 10.1016/j.ab.2011.09.019 *
Restriction Enzyme Aat II, 日本ジェネティクス[online], 18 August 2023, Internet<URL: https://n-genetics.com/products/detail/FG-AatII/document/#tab>, [retrieved on 18 February 2025], (NIPPON GENETICS CO., LTD.) *

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