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WO2005066346A1 - Amorce pour amplification d'acide nucleique, ensemble d'amorces pour amplification d'acide nucleique et methode de depistage du cancer associee - Google Patents

Amorce pour amplification d'acide nucleique, ensemble d'amorces pour amplification d'acide nucleique et methode de depistage du cancer associee Download PDF

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WO2005066346A1
WO2005066346A1 PCT/JP2004/014741 JP2004014741W WO2005066346A1 WO 2005066346 A1 WO2005066346 A1 WO 2005066346A1 JP 2004014741 W JP2004014741 W JP 2004014741W WO 2005066346 A1 WO2005066346 A1 WO 2005066346A1
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codon
gene
nucleic acid
mutation
acid amplification
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Takeshi Nagasaka
Nagahide Matsubara
Noriaki Tanaka
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • Primer for nucleic acid amplification primer set for nucleic acid amplification, and method for detecting cancer using the same
  • the present invention relates to a method for determining the presence or absence of a mutation at codon 599 of the BRAF gene (hereinafter abbreviated as “mutation” as appropriate), which is used in the field of clinical laboratory tests for cancer, and a nucleic acid used for the method.
  • the present invention relates to an amplification primer, a nucleic acid amplification primer set, and a reagent kit for determining the presence or absence of a mutation in BRAF gene codon 599.
  • the present invention further relates to the above method combined with the detection of a mutation in the KRAS gene, a nucleic acid amplification primer, a nucleic acid amplification primer set, and a reagent kit for use in the method.
  • RAS gene which encodes the RAS protein
  • the RAS gene has been mutated in various human tumors. It is known that when a mutation occurs in the RAS gene, intracellular phosphorylation is abnormally enhanced, and the regulatory functions such as cell differentiation, growth, and proliferation are impaired. Mutations in the RAS gene have been reported to occur in 15% of all human tumors.
  • the BRAF gene which is located downstream of the RAS gene and encodes the BRAF protein, is an oncogene activated by the RAS gene. If a mutation occurs in the BRAF gene, intracellular phosphorylation abnormally increases, It is known that regulatory functions such as cell differentiation, growth, and proliferation are impaired. Mutations in the BRAF gene have been reported to occur in 70% of melanomas and 10% of colorectal cancers. Thus, mutations in the RAS gene and BRAF gene are presumed to be closely related to human tumor formation.
  • V599E conversion of valine to glutamic acid
  • Non-Patent Document 3 a technique described in Non-Patent Document 3 has been known as a method for detecting a mutation at codon 12 of the KRAS gene with high sensitivity and good reproducibility.
  • Non-Patent Document 1 Rajagopalan, ⁇ ⁇ , Bardelli, A., Lengauer, C., Kinzler, K.W., Vogelstein,
  • Non-Patent Document 2 Yuen, S.T., Davies, H., Chan, T.L., Ho, J.W., Bignell, G.R., Cox,
  • Non-Patent Document 3 "Oncogene” 1991 Jun, 6 (6), 1079-1083 Therefore, specific mutations such as codon 599 of the BRAF gene, codon 12 and codon 13 of the KRAS gene can be efficiently performed at low cost. In addition, there is a need for a method of detecting with high accuracy.
  • an object of the present invention is to provide a method for simply and accurately detecting a specific mutation of the specific gene as described above, particularly a mutation of codon 599 of the BRAF gene.
  • Another object of the present invention is to provide a nucleic acid amplification primer, a nucleic acid amplification primer set, and a BRAF gene codon 599 mutation detection reagent kit for use in this test method.
  • Another object of the present invention is to provide a method for detecting a gene mutation which is useful for diagnosing cancer such as colorectal cancer and predicting the possibility of its onset or progression.
  • the present inventors acted on the BRAF gene, Amplification of the BRAF gene using a primer set for nucleic acid amplification containing the primers for nucleic acid amplification into which the BRAF gene has been introduced by the normal PCR method, or by nested PCR method, semi-nested PCR method, or double PCR method It has been found that the mutation of codon 599 can be easily and accurately detected, and the presence or absence of the mutation of codon 599 of the BRAF gene can be determined easily and accurately.
  • the primer set for nucleic acid amplification containing the primer for nucleic acid amplification if there is a mutation in codon 599 of the BRAF gene, the amplification product of the BRAF gene in the primer set for nucleic acid amplification is converted to a specific restriction enzyme. Does not recognize, and if there is no mutation at codon 599 of the BRAF gene, the specific restriction enzyme recognizes the BRAF gene amplification product of the nucleic acid amplification primer set.
  • the primer set for nucleic acid amplification generates an amplification product of the BRAF gene having a different sequence depending on the presence or absence of the mutation at codon 599 of the BRAF gene, so that the specific restriction enzyme generates restriction enzyme fragments of different lengths. I do. Therefore, by detecting the restriction enzyme fragment using the restriction fragment length polymorphism, the mutation at codon 599 of the BRAF gene can be detected, and the presence or absence of the mutation at codon 599 of the BRAF gene can be determined.
  • reaction mixture treated with the specific restriction enzyme is subjected to nucleic acid amplification of the BRAF gene using the second primer set for nucleic acid amplification, and the amplified product of the BRAF gene is subjected to the specific restriction enzyme treatment.
  • a second step of treating with the specific restriction enzyme is subjected to nucleic acid amplification of the BRAF gene using the second primer set for nucleic acid amplification, and the amplified product of the BRAF gene is subjected to the specific restriction enzyme treatment.
  • the restriction enzyme does not recognize the amplification product of the primer set for nucleic acid amplification, and if there is no mutation in codon 599 of the BRAF gene, The restriction enzyme recognizes the amplification product of the primer set. Then, the presence or absence of the mutation at codon 599 of the BRAF gene As a result, the primer set for nucleic acid amplification generates an amplification product of the BRAF gene having a different sequence, so that the specific restriction enzyme generates restriction enzyme fragments having different lengths. Therefore, the presence or absence of the mutation at codon 599 of the BRAF gene can be determined by detecting the restriction enzyme fragment using the restriction fragment length polymorphism.
  • a mismatch different from the mismatch of the nucleic acid amplification primer set used in the first step is used. Is used.
  • the restriction enzyme recognizes a site other than codon 599 of the BRAF gene regardless of the presence or absence of the mutation of codon 599 in the second step.
  • restriction enzyme fragments of different lengths generated in the first step due to the presence or absence of the mutation at codon 599 of the BRAF gene will be amplified.
  • a large amount of amplification products of different lengths are produced due to the presence or absence of the mutation at codon 599 of the BRAF gene. Therefore, a restriction enzyme fragment obtained by digesting this amplification product with a restriction enzyme is converted into a restriction enzyme fragment.
  • the detection sensitivity is higher than when only the first step is performed. Is relatively low, and cells or tissues derived from the patient's blood, serum, serum, feces, semen, saliva, sputum, cerebrospinal fluid, etc. are collected to determine the presence or absence of codon 599 mutations in the BRAF gene can do. Therefore, it is possible to make a minimally invasive diagnosis (the burden on the patient is small).
  • the method of the present invention for detecting a mutation at codon 599 of the BRAF gene comprises:
  • the restriction enzyme Btsl does not recognize the amplification product of the BRAF gene by the primer set for nucleic acid amplification, and if there is no mutation in codon 599 of the BRAF gene, the nucleic acid Amplification products obtained by using the first primer set for nucleic acid amplification by which the restriction enzyme Btsl recognizes the amplification product of the BRAF gene by the amplification primer set are cut with the restriction enzyme Btsl,
  • the amplified product of the BRAF gene by the first primer set for nucleic acid amplification is subjected to a second nucleic acid amplification with a type II, whereby the codon 599 of the BRAF gene is obtained.
  • a method for detecting a mutation at codon 599 of the BRAF gene characterized by amplifying an amplification product having a mutation at a higher rate.
  • nucleic acid amplification product in which no mutation at codon 599 of the BRAF gene is observed (that is, a normal nucleic acid amplification product) is cleaved by the restriction enzyme.
  • the nucleic acid amplification step in the third step only the gene having a mutation at codon 599 of the BRAF gene will be amplified, thereby strongly amplifying even a small amount of the gene having a mutation at codon 599 of the BRAF gene. Become.
  • the detection sensitivity is higher in the case where the second step is performed than in the case where only the first step is performed.
  • the cells and tissues derived from the patient's blood, knee fluid, serum, feces, semen, saliva, sputum, cerebrospinal fluid, etc. are collected to determine the presence or absence of mutations in codon 599 of the BRAF gene. Judgment ability S can. Therefore, it is possible to make a minimally invasive diagnosis (the burden on the patient is small).
  • a method for determining mutations in codons 12 and 13 of the KRAS gene using the nucleic acid amplification product obtained in the third step includes:
  • the nucleic acid amplification product obtained in the third step is typically represented by the SSCP method, the WAVE method, or the method of judging a mutation using an oligo chip.
  • the presence or absence of a mutation in the BRAF gene codon 599 is determined with high accuracy using the BRAF gene codon 599 mutation detection reagent kit containing the above-described nucleic acid amplification primer set, a restriction enzyme, and a DNA polymerase. Can be.
  • a nucleic acid amplification primer, a nucleic acid amplification primer set, and a BRAF gene codon 599 mutation detection reagent kit for use in the detection method, the mutation of codon 599 of the BRAF gene
  • the detection can be performed easily and accurately, and the mutation at codon 599 of the BRAF gene can be detected accurately to determine the presence or absence of the mutation.
  • primers for nucleic acid amplification and a detection method can be similarly designed for codons 12 and 13 of the KRAS gene described in detail for the BRAF gene. However, two mismatches are introduced into the primer for the KRAS gene.
  • the primer for nucleic acid amplification containing two specific mismatches or the primer set for nucleic acid amplification containing the primer for nucleic acid amplification if there is a change in codon 12 of the KRAS gene, If the amplification product of the KRAS gene by the nucleic acid amplification primer set 1 is not recognized by the specific first restriction enzyme (for example, Mval) and there is a mutation in codon 13 of the KRAS gene, the specific first nucleic acid If the amplification product of the KRAS gene by the amplification primer set is not recognized by a specific second restriction enzyme (for example, Bgll) and there is no mutation in codon 12 or codon 13 of the KRAS gene, the specific Specific first and second restriction enzymes (Mval and Bgll) recognize the amplification product of the KRAS gene by the nucleic acid amplification primer set (1).
  • the specific first restriction enzyme for example, Mval
  • Bgll specific second restriction enzyme
  • the specific first nucleic acid amplification primer set generates an amplification product of the KRAS gene having a different sequence depending on the presence or absence of a mutation at codons 12 and 13 of the KRAS gene.
  • One restriction enzyme generates restriction enzyme fragments of different lengths based on the presence or absence of a mutation at codon 12 of the KRAS gene, and the specific second restriction enzyme generates a restriction fragment at codon 13 of the KRAS gene. Based on the presence or absence, different length restriction fragments are generated.
  • mutation of codon 12 and codon 13 of the KRAS gene can be detected by one operation, and codon 12 and codon 13 of the KRAS gene can be detected.
  • the presence or absence of the mutation can be determined in a smaller number of nucleic acid amplification steps.
  • First step a step of performing nucleic acid amplification of the KRAS gene using a first nucleic acid amplification primer set;
  • the second step a step of treating the KRAS gene amplification product obtained in the first step with the specific restriction enzymes (Mval and Bgll);
  • KR When the amplification products of the KRAS gene having different restriction enzyme fragment lengths generated due to the presence or absence of the codon 12 mutation in the AS gene are amplified and treated with the restriction enzyme Bgll in the second step, the KRAS gene Amplification products with different restriction enzyme fragment lengths, which are generated depending on the presence or absence of the mutation at codon 13, are amplified.
  • the detection sensitivity is higher than when only the first step is performed. Therefore, in addition to directly collecting cancer cells and tissues, Cells and tissues derived from the patient's blood, sperm fluid, serum, feces, semen, saliva, sputum, cerebrospinal fluid, etc., from which the proportion of cells is relatively low, are collected and analyzed for codons 12 and 13 of the KRAS gene. S ability to determine the presence or absence of mutation. Therefore, it is possible to make a minimally invasive diagnosis (the burden on the patient is small).
  • a typical method is to use the SSCP method or an oligo chip to determine the mutation of the nucleic acid amplification product obtained in the third step.
  • the mutation of codon 12 and codon 13 of the KRAS gene is detected.
  • the method of the present invention may be performed before or after each step for detecting a mutation at codon 599 of the BRAF gene, or simultaneously or in parallel therewith.
  • the specific first restriction enzyme does not recognize the amplification product of the KRAS gene by the first nucleic acid amplification primer set, and the mutation occurs at codon 13 of the KRAS gene.
  • the specific second restriction enzyme does not recognize the amplification product of the KRAS gene by the first nucleic acid amplification primer set, and there must be no mutation in codon 12 or codon 13 of the KRAS gene.
  • the amplification product of the KRAS gene by the first nucleic acid amplification primer set can be used for the first nucleic acid amplification such that it recognizes both the specific first restriction enzyme and the specific second restriction enzyme.
  • the amplification product obtained using the primer set is cleaved with the first and second restriction enzymes, 2)
  • the amplification product having a mutation at codon 12 and / or codon 13 of the KRAS gene is obtained by performing amplification of the KRAS gene by the first nucleic acid amplification primer set into a type II and performing second nucleic acid amplification.
  • the method may further include a step of comparing the result with the result regarding the presence or absence of a mutation at codons 12 and 13 of the KRAS gene.
  • the kit of the present invention the primer for nucleic acid amplification for use in the method, the primer set for nucleic acid amplification, and the above-described primer set for nucleic acid amplification, a restriction enzyme, and a DNA polymerase are provided. According to this, the presence or absence of mutations at codons 12 and 13 of the KRAS gene as well as mutations at codon 599 of the BRAF gene can be accurately determined with fewer operations.
  • FIG. 1 is a schematic diagram showing an outline of a method for detecting a mutation at codon 599 of the BRAF gene according to the present invention.
  • FIG. 2 is a diagram showing detection of a mutation at codon 599 of the BRAF gene according to the present invention.
  • FIG. 3 is a schematic diagram showing an outline of the method for detecting mutations at codons 12 and 13 of the KRAS gene according to the present invention.
  • FIG. 4 is a diagram showing detection of mutations at codons 12 and 13 of the KRAS gene according to the present invention.
  • FIG. 5 (A), (B), (C), and (D) constitute one table.
  • 21 cases of BRAF A summary of the results for the sample with the mutation and 72 samples with the KRAS mutation is shown. Black squares indicate promoter methylation and blank squares indicate no promoter methylation.
  • the present invention acts on the BRAF gene and introduces a mismatch into the nucleotide sequence.
  • the primer set for amplifying the nucleic acid amplifies the gene based on a base sequence without the mutation.
  • a primer for example, in the sequence containing the BRAF gene shown in SEQ ID NO: 5, a sequence in which adenine (A) at base number 425 is substituted with guanine (G) is used, and the substituted base is used.
  • primers for nucleic acid amplification prepared with a base sequence that does not include base numbers 428 to 430, including the base sequence that includes the base number.
  • the amplification product of the gene has a different base sequence.
  • the amplification product of the gene when the specific codon of the gene has a mutation does not recognize a specific restriction enzyme, and the amplification product of the gene when the codon of the gene has no mutation is not recognized.
  • the specific restriction enzyme recognizes a specific palindrome of the amplification product. Then, if the specific codon of the gene has a mutation, the amplification product of the gene will have one fragment by the restriction enzyme, but if the specific codon of the gene has a mutation, The amplified product of the gene has two fragments due to restriction enzymes.
  • the total fragment length of these two fragments is almost equal to the length of the above one fragment.
  • Btsl can be used as the restriction enzyme.
  • the presence or absence of a mutation at a specific codon of this gene can be determined.
  • the gene when the gene is amplified using a primer set for nucleic acid amplification (primer set for nucleic acid amplification) containing the primer for nucleic acid amplification, if there is a mutation in a specific first codon of the gene, The primer set for nucleic acid amplification amplifies the gene based on the nucleotide sequence with the mutation. Further, when the gene is amplified using a primer set for nucleic acid amplification containing the primer for nucleic acid amplification, if the specific second codon of the gene has a mutation, the primer set for nucleic acid amplification is Then, the gene is amplified based on the base sequence with the mutation.
  • primer set for nucleic acid amplification primer set for nucleic acid amplification
  • the set of primers for nucleic acid amplification is based on the nucleotide sequence without the mutation. Try to amplify the gene.
  • the amplification product of the gene has a different base sequence.
  • a primer for example, in the sequence containing the KRAS gene shown in SEQ ID NO: 11, adenine (A) at base number 466 is replaced with cytosine (C) and guanine (G) at base number 467 is used.
  • a primer for nucleic acid amplification prepared by using a sequence in which is substituted with cytosine (C) and comprising a base sequence containing the two substituted bases and excluding base numbers 470 to 475.
  • the specific first restriction enzyme is not recognized for the amplification product of the gene when the specific first codon of the gene has a mutation, and the specific first codon of the gene is not recognized.
  • the specific first restriction enzyme will recognize the specific first palindrome structure of the amplified product
  • the number of fragments of the amplification product of the gene by the restriction enzyme is one, but the first characteristic of the gene is If there is a mutation at a certain codon, the fragment of the amplification product of that gene
  • a specific second restriction enzyme is not recognized for an amplification product of a gene when the specific second codon of the gene has a mutation, and the specific specific codon of the gene is not recognized.
  • the specific second restriction enzyme will recognize the specific second palindrome structure of the amplification product
  • two mutations at a specific first codon and a specific second codon of this gene can be detected in one operation, and two mutations at a specific first codon and a specific second codon of this gene can be detected.
  • the presence or absence of a mutation at a location can be determined with a single operation.
  • the amplification product of the gene will have two restriction fragments, but if the specific codon of the gene is not mutated, There are three fragments of the gene amplification product due to restriction enzymes. At this time, the total fragment length of these three fragments is almost equal to the above two fragment lengths.
  • a mismatch is introduced into one nucleic acid amplification primer of the second nucleic acid amplification primer set. This time, with or without the mutation of the specific first codon of the gene, the specific first restriction enzyme is different from the specific first one place in the amplification product. A mismatch is introduced to recognize the palindrome structure at each point.
  • a mismatch is introduced into one of the third nucleic acid amplification primer sets in the third nucleic acid amplification primer set. This time, regardless of the presence or absence of the mutation of the specific second codon of the gene, the specific second restriction enzyme is different from the specific second one in the amplification product in another one. A mismatch is introduced to recognize the palindrome structure of.
  • the specific first codon of the gene has a mutation
  • the number of fragments of the amplification product of the gene by the specific first restriction enzyme becomes three
  • the specific first codon of the offspring is not mutated, the amplification product of the gene will have two fragments with the specific first restriction enzyme. At this time, the total fragment length of these three fragments is almost equal to the above two fragment lengths.
  • FIG. 1 One example of a method for detecting a mutation at codon 599 of the BRAF gene is shown in FIG.
  • the region containing codon 599 of the BRAF gene is amplified with a primer set for nucleic acid amplification consisting of two primers for nucleic acid amplification ( Figure 1 (a)).
  • 599S is a nucleic acid amplification primer represented by SEQ ID NO: 2, and the 18851-th power of the complementary strand of the BRAF gene is also the 18878-th site (the 1713-th to 171426-th sites of the original strand of the BRAF gene). ).
  • SEQ ID NO: 1 shows the 18601st base sequence at the 18900th position of the complementary chain of this BRAF gene.
  • the first base of the primer for nucleic acid amplification represented by SEQ ID NO: 2 binds to position 18878 of the complementary strand of the BRAF gene
  • the 28th base of the primer for nucleic acid amplification represented by SEQ ID NO: 2 Binds to position 18851 of the complementary strand of the BRAF gene.
  • This 599S does not recognize the restriction enzyme Btsl (B.T.I.S'Wan) for the amplification product of the gene when the codon 599 of the BRAF gene has a mutation, and It is designed so that the restriction enzyme Btsl recognizes one specific palindrome of the amplified product of the gene when there is no mutation in Don 599.
  • the base corresponding to the 18852th base of the complementary strand of the BRAF gene (corresponding to 171425th base of the original strand of the BRAF gene) is guanine (the 27th base of the primer of SEQ ID NO: 2). Has been converted to.
  • 599wtAS is a nucleic acid amplification primer represented by SEQ ID NO: 3, and the 18747th position of the complementary strand of the BRAF gene also corresponds to the 18772th position (the 171505th position of the original strand of the BRAF gene). Et 171530 th (M).
  • the first base of the nucleic acid amplification primer represented by SEQ ID NO: 3 is BRAF It binds to the site corresponding to position 18772 of the complementary strand of the gene, and the base position 28 of the primer for nucleic acid amplification represented by SEQ ID NO: 3 binds to the site corresponding to position 18747 of the complementary strand of the BRAF gene.
  • the linkage of nucleotides (bases) by 599S is performed from upstream to downstream, and the linkage of nucleotides (bases) by 599wtAS is performed from downstream to upstream. 171399th power, etc. 171530th part is amplified.
  • 599S is a primer for amplifying nucleic acid represented by SEQ ID NO: 2 described above, and is located at position 18851 of the complementary strand of the BRAF gene at position 18878 (corresponding to the site from position 171399 to position 171426 of the BRAF gene) To join.
  • 599mtAS is a nucleic acid amplification primer represented by SEQ ID NO: 4, and corresponds to a site corresponding to the 18749th position of the complementary strand of the BRAF gene and the 18774th position (the 171503th position of the original strand of the BRAF gene). 171528th position).
  • the first base of the nucleic acid amplification primer represented by SEQ ID NO: 4 binds to a site corresponding to position 18774 of the complementary strand of the BRAF gene
  • the nucleic acid amplification primer represented by SEQ ID NO: 2 The 26th base binds to the site corresponding to 18749 in the complementary strand of the BRAF gene.
  • the restriction enzyme Btsl is designed so that the amplified product of the BRAF gene recognizes one palindrome structure different from the specific one described above. Have been. Specifically, of the 599mtAS, the base corresponding to the 18764th base of the complementary strand of the BRAF gene is converted to guanine (the 11th base of the primer of SEQ ID NO: 4). Ligation of nucleotides (bases) by 599S is performed from upstream to downstream, and ligation of nucleotides (bases) by 599mtAS is performed from downstream to upstream, and the BRAF gene is amplified by a PCR reaction.
  • restriction enzyme length polymorphism RFLP
  • a 78 bp restriction enzyme fragment was detected in a sample without mutation, and a 112 bp fragment was detected in a sample with mutation.
  • the presence or absence of a mutation in codon 599 of the BRAF gene can be determined based on this difference (FIG. 2 (a)).
  • FIG. 3 shows an example of a method for detecting a mutation at codon 12 and a mutation at codon 13 of the KRAS gene by a single operation.
  • a region containing codons 12 and 13 of the KRAS gene represented by SEQ ID NO: 6 (160 bases from the 454th base to 613th base of the KRAS gene, from the 1st base of codon 1 to the 1st base of codon 54 ) Is amplified with a set of nucleic acid amplification primers consisting of two nucleic acid amplification primers (Fig. 3 (a)).
  • 12 & 13SP are primers for nucleic acid amplification represented by SEQ ID NO: 7, and correspond to the 486th to 486th positions of the KRAS gene, that is, the codons of the KRAS gene represented by SEQ ID NO: 6.
  • a region corresponding to the fourth to 33rd sequence of the region containing 12 and codon 13 (actually, the fourth to 33rd sequence of the region containing codon 12 and codon 13 of the KRAS gene represented by SEQ ID NO: 6)
  • the amplification product of the gene when there is a mutation in codon 12 of the KRAS gene It does not recognize the restriction enzyme Mval, and b) the restriction enzyme Bgll (B.J.I.) for gene amplification products when there is a mutation in codon 13 of the KRAS gene.
  • the specific first one palindrome of the amplification product The structure (the 488th CCWGGZGGWCC of the KRAS gene and the 488th CCWGGZGGWCC) is designed to be recognized by the restriction enzyme Mval, and the specific second single palindrome structure of the amplified product (the 482th power of the KRAS gene)
  • the 492nd GCCNNNNNGGCZCGGNNNNNCCG is designed to be recognized by the restriction enzyme Bgll.
  • the base corresponding to the 484th base of the KRAS gene is converted into cytosine (the 28th base of the primer of SEQ ID NO: 7), and the 483rd base of the KRAS gene is converted. Is converted to cytosine (the 27th base of the primer of SEQ ID NO: 7).
  • the difference in the amplification product due to the presence or absence of the mutation at codon 12 can be identified by Mval (or BstNI (BS'T'N'Wan)). Differences in amplification products due to presence or absence can be identified by Bgll.
  • WMAS is a primer for nucleic acid amplification represented by SEQ ID NO: 8, and is located at positions 549 to 576 of the KRAS gene, that is, at positions 96 to 123 of the KRAS gene represented by SEQ ID NO: 6. Binds to the site (actually, the site of the 123rd sequence from the 96th position of the KRAS gene region represented by SEQ ID NO: 6; the first base of SEQ ID NO: 8 is represented by SEQ ID NO: 6 And the base at position 28 in SEQ ID NO: 8 binds to position 96 in the KRAS gene region represented by SEQ ID NO: 6).
  • nucleotides (bases) by 12 & 13SP Ligation of nucleotides (bases) by 12 & 13SP is performed from upstream to downstream, and ligation of nucleotides (bases) by WildAS is performed from downstream to upstream, and the KRAS gene is amplified by a PCR reaction.
  • one restriction enzyme fragment produced by treatment with the restriction enzyme Mval (or BstNI) when there is a mutation in codon 12 of the KRAS gene, and a mutation in codon 12 of the KRAS gene The two restriction enzyme fragments without the primer set are amplified using a primer set for nucleic acid amplification different from the above-mentioned primer set for nucleic acid amplification (corresponding to the "second nucleic acid amplification primer set") (Fig. 3 (d)).
  • 12 & 13SP is a primer for nucleic acid amplification represented by the aforementioned SEQ ID NO: 7, a site corresponding to the 486th position from the 457th position of the KRAS gene, that is, a codon of the KRAS gene represented by the SEQ ID NO: 6.
  • Site corresponding to the 4th to 33rd sequences of the region containing 12 and codon 13 (actually the 4th sequence of the region containing codons 12 and 13 of the KRAS gene represented by SEQ ID NO: 6) Site of the complementary strand).
  • 12mtAS is a primer for nucleic acid amplification represented by SEQ ID NO: 9, and is located at positions 549 to 576 of the KRAS gene, that is, the 96th position 123rd position of the KRAS gene represented by SEQ ID NO: 6.
  • the restriction enzyme Mval (or BstNI) is designed to recognize one palindrome structure different from the specific first one.
  • the base corresponding to the 103rd base of the KRAS gene represented by SEQ ID NO: 6 was converted to cytosine (the 21st base of the primer of SEQ ID NO: 8).
  • the base corresponding to the 104th base of the KRAS gene is converted to cytosine (the 20th base of the primer of SEQ ID NO: 8).
  • nucleotides (bases) by 12 & 13SP Ligation of nucleotides (bases) by 12 & 13SP is performed from upstream to downstream, ligation of nucleotides (bases) by 12mtAS is performed from downstream to upstream, and the KRAS gene is amplified by a PCR reaction.
  • the amplification product of the gene having a mutation in codon 12 of the KRAS gene had 99bp and 21bp. Restriction enzyme fragments are generated, and three restriction enzyme fragments of 29 bp, 60 bp and 21 bp are generated for the amplified product of the KRAS gene without codon 12 mutation (Fig. 3 (f )).
  • one restriction enzyme fragment produced by treatment with the restriction enzyme Bgll and having a mutation at codon 13 of the KRAS gene The two restriction enzyme fragments are amplified with a nucleic acid amplification primer set different from the above-described nucleic acid amplification primer set (equivalent to the “third nucleic acid amplification primer set”) (FIG. 3 (e)).
  • 12 & 13SP is a nucleic acid amplification primer represented by SEQ ID NO: 7 described above, and corresponds to the 486th position to the 457th position of the KRAS gene, that is, the codon of the KRAS gene represented by SEQ ID NO: 6.
  • a region corresponding to the fourth to 33rd sequence of the region containing 12 and codon 13 (actually, the fourth to 33rd sequence of the region containing codon 12 and codon 13 of the KRAS gene represented by SEQ ID NO: 1) (The site of the complementary strand of the sequence).
  • 13mtAS is a primer for nucleic acid amplification represented by SEQ ID NO: 5, and is located at positions 549 to 576 of the KRAS gene, that is, at position 96 to 123 of the KRAS gene represented by SEQ ID NO: 6.
  • the 96th position of the KRAS gene region represented by SEQ ID NO: 6 is also the site of the 123rd sequence, and the first base of SEQ ID NO: 8 is the KRAS represented by SEQ ID NO: 6). It binds to position 123 in the gene region, and the 28th base in SEQ ID NO: 8 binds to position 96 in the KRAS gene region represented by SEQ ID NO: 6), regardless of the presence or absence of codon 13 mutation in the KRAS gene.
  • the restriction enzyme Bgll is designed to recognize one palindrome structure different from the specific second one.
  • the base corresponding to the 113th base of the KRAS gene represented by SEQ ID NO: 6 was converted to guanine (the 11th base of the primer of SEQ ID NO: 10),
  • the base corresponding to the 105th base of the KRAS gene represented by 6 is converted to guanine (the 19th base of the primer of SEQ ID NO: 10), and the 103rd base of the KRAS gene represented by SEQ ID NO: 6 is converted.
  • the base corresponding to is converted to cytosine (the 21st base of the primer of SEQ ID NO: 10).
  • nucleotides (bases) by 12 & 13SP Ligation of nucleotides (bases) by 12 & 13SP is performed from upstream to downstream, ligation of nucleotides (bases) by 13mtAS is performed from downstream to upstream, and the KRAS gene is amplified by a PCR reaction.
  • the primer for nucleic acid amplification in the present invention can be synthesized by a known method.
  • a solid phase chemical synthesis method such as a phosphate triester method, a phosphate amidite method, and a phosphate group site-protection method can be used.
  • it can be synthesized using an oligonucleotide synthesizer (Expedite Model 8909, manufactured by Applied Biosystem).
  • a method for detecting mutations at codon 599 of the BRAF gene and, optionally, codons 12 and 13 of the KRAS gene can be performed by preparing a sample, extracting a gene (genomic DNA), and It consists of amplification, cleavage of the amplified product with restriction enzymes, and detection of gene mutation.
  • the human sample used in the test method of the present invention is not particularly limited as long as it contains a gene encoding a BRAF protein (BRAF gene) and, in some cases, a KRAS gene.
  • BRAF gene a gene encoding a BRAF protein
  • KRAS gene a gene encoding a BRAF protein
  • Specific examples include tissues collected from a living body, such as cancer tissues such as colorectal cancer removed by surgery, biopsy materials (biopsy tissues) used for endoscopy, etc. before surgery, and surgical specimens. It is preferably used in terms of effective use of the sample.
  • blood, knee fluid, serum, feces, semen, saliva, sputum, cerebrospinal fluid, or a reaction solution in which the BRAF gene is amplified by a nucleic acid amplification reaction using these are also examples of the sample.
  • DNA to be subjected to the detection method of the present invention can be obtained by crushing tissue from a human sample as described above using a blender, and then extracting the DNA by a known gene extraction method such as the phenol-chloroform method. Such DNA can be used as a test sample.
  • a known method can be used as a BRAF gene amplification method, and examples thereof include a PCR method, a NASBA method, and a LAMP method.
  • the PCR method Is used.
  • PCR for amplifying a nucleic acid by PCR can be a nested PCR method.
  • Nested PCR is a method in which a two-step PCR is performed using an outer primer and an inner primer. Set primers inside and perform the next PCR.
  • PCR is a technique for amplifying a specific fragment based on the specificity of two primer pairs facing each other at appropriate intervals.However, mis-splicing sometimes occurs due to the similar sequence of primers, and amplification along with amplification of the target sequence. Non-specific amplification occurs.
  • nested PCR is performed using the PCR product containing this non-specific fragment as type III, the probability of the presence of a sequence similar to the nested primer in the non-specific fragment is extremely low. Only the target sequence can be successfully picked up from the "sea of noise" of the amplification. Therefore, the nested PCR method is an effective method in the case where the background is easily generated, or in the case of PCR.
  • a normal PCR method may be used, and in addition to nested PCR, semi-nested PCR and dub / le PCR may be used.
  • the amplification product by the nucleic acid amplification primer set including the nucleic acid amplification primer is not recognized, and If there is no mutation at a specific codon, a specific restriction enzyme that recognizes the amplification product is used.
  • the amplification product of the BRAF gene amplification is treated with the restriction enzyme Btsl.
  • the optimal temperature of Btsl is around 37 ° C.
  • the amplification product obtained by the KRAS gene amplification is treated with a restriction enzyme Mval to detect a mutation at codon 12.
  • the optimum temperature of Mval is around 37 ° C.
  • treat with restriction enzyme Bgll For detection of codon 13 mutation, treat with restriction enzyme Bgll.
  • the optimum temperature of Bgll is around 37 ° C.
  • the fragment treated with the restriction enzyme is detected using restriction fragment length polymorphism or the like. (Mutation detection reagent 'mutation detection reagent kit)
  • the present invention also includes a mutation detection reagent and a mutation detection reagent kit used in the method for detecting a mutation at codon 599 of the BRAF gene.
  • the mutation detection reagent include any of the reagents used in the method of the present invention, such as a nucleic acid amplification primer that amplifies a region containing codon 599 of the BRAF gene, a DNA polymerase, an exonuclease, and a label for detecting a nucleic acid. It may be.
  • the reagents and kits of the present invention can also include a mutation detection reagent and a mutation detection reagent kit used in the method for detecting mutations at codons 12 and 13 of the KRAS gene.
  • mutation detection reagents include any reagents used in the method of the present invention, such as nucleic acid amplification primers for amplifying a region containing codon 12 and codon 13 of the KRAS gene, DNA polymerase, exonuclease, and a label for nucleic acid detection. It can be any of.
  • the mutation detection reagent kit used in the method for detecting a mutation at codon 599 of the BRAF gene uses at least two or more of all the reagents used in the detection method of the present invention as a kit. Should be fine.
  • DNA labeled with a fluorescent label may be included in the kit.
  • the kit of the present invention preferably comprises a nucleic acid amplification primer set for amplifying at least a region containing codon 599 of the BRAF gene of the present invention (and optionally a nucleic acid for amplifying a region containing codon 12 and codon 13 of the KRAS gene). Examples including amplification primer sets)
  • Genomic genes were extracted and purified from cancerous and normal mucous membranes obtained from surgery for colorectal cancer patients.
  • the mutation at codon 599 was introduced by introducing a mismatch that converts the 599S primer, which corresponds to A (adenine), located 3 bases upstream of codon 599 of the BRAF gene, to G (guanine).
  • A adenine
  • G guanine
  • the first restriction enzyme treatment (primary restriction enzyme treatment) was performed with the restriction enzyme Btsl.
  • the primers 599S (SEQ ID NO: 2) and 599mtAS (SEQ ID NO: 4) in FIG. 1 were used:
  • 599S SEQ ID NO: 2
  • 599mtAS SEQ ID NO: 4
  • the above restriction enzyme fragment treated with the restriction enzyme was detected by restriction fragment length polymorphism.
  • 78 bp and 112 bp fragments were detected in the left lane of 540, and it was determined that a codon 599 mutation was present. In the remaining 5 lanes, only the 78 bp fragment was detected, and the codon 599 mutation was detected. It was determined to be none.
  • the presence or absence of the codon 599 mutation in the BRAF gene can be accurately determined by detecting the mutation at codon 599 in the BRAF gene.
  • a region containing codons 12 and 13 (bases 470 to 475 of the sequence shown in SEQ ID NO: 11) of the KRAS gene represented by SEQ ID NO: 11 For nucleic acid amplification comprising two primers for nucleic acid amplification Amplify with the primer set (Fig. 3 (a)).
  • the primers 12 & 13SP used this time are primers for nucleic acid amplification represented by 5'-ACTGAATATAAACTTGTGGTA GTTGGCCCT (SEQ ID NO: 7).
  • Sequences in which adenine (A) is replaced with cytosine (C) and guanine (G) having base number 467 is replaced with cytosine (C) are positions 440 to 469.
  • This primer is used for a) when the restriction enzyme Mval is not recognized for the amplification product of the gene when codon 12 of the KRAS gene has a mutation, and b) when the codon 13 of the KRAS gene has a mutation.
  • the amplification product of the gene does not recognize the restriction enzyme Bgll, and c) the amplification product of the gene when there is no mutation in codon 12 or codon 13 of the KRAS gene.
  • the first specific palindrome (the 467th force and the 471st CCWGG / GGWCC of SEQ ID NO: 11) is designed to be recognized by the restriction enzyme Mval, and the second specific It is designed so that the restriction enzyme Bgll recognizes the palindrome structure at the position (465th position 475th position G CCNNNNNGGC / CGGNNNNNCCG of SEQ ID NO: 11).
  • WildAS AACAAGATTTACCTCTATTGTTGGATCA (SEQ ID NO: 8). This is the complementary strand of the 532nd sequence and the 559th sequence of the nucleotide sequence shown in SEQ ID NO: 11.
  • the gene amplification product obtained by the primary PCR is used for detecting mutations at codons 12 and 13 respectively, and the first restriction enzyme treatment (primary Restriction enzyme treatment was performed, and the first restriction enzyme treatment (primary restriction enzyme treatment) was performed with Bgll to detect mutations at codon 13.
  • primers of 12 & 13SP and 13mtAS (SEQ ID NO: 10) were used:
  • 12 & 13SP SEQ ID NO: 7
  • 12mtAS SEQ ID NO: 9
  • restriction enzyme fragment treated with the restriction enzyme was detected by restriction enzyme fragment length polymorphism.
  • the DNA used as type I in this experiment was extracted from tumor tissue and corresponding normal mucosa samples from 234 patients who underwent therapeutic surgery. Samples of both tumor and normal mucosal tissues were stored at -80 ° C and DNA was extracted by standard procedures including proteinase K digestion and phenolic chloroform extraction.
  • Tumor staging was based on the Duke's specification.
  • FIG. 5 shows a summary of the results.
  • the 234 cases examined could be divided into the following subgroups: the group with a mutation in the BRAF gene (BRAF-mt), the group with a mutation in the KRAS gene (KRAS-mt), and these genes Group without any mutation (Wt).
  • Table 3 shows these three groups and the relevant clinicopathological features, including MSI status.
  • the samples examined for mutations in the BRAF and KRAS genes contained MINT1, MINT2, MINT31, CACNA1G, pl6 INK4a , pl4 ARF , COX2, DAPK and MGMT, and the 5 'and 3' regions of the hMLHl promoter region. Methylation at 9 loci The status was assessed and the results were assessed based on whether the CRC in question had a BRAF or KRAS mutation (Table 3).
  • Table 3 shows the statistical results for a 3 ⁇ 2 qualitative correlation table. This correlation table highlights the fact that there are differences between wild type tumors for BRAF mutations, KRAS mutations and both genes. The specific differences described above were evident by examining the proportions in this table, and these differences were confirmed to be statistically significant by individual two-gnore comparison using a 2 ⁇ 2 table.
  • the presence or absence of mutations in the BRAF and KRAS genes is detected by the method of the present invention.
  • MINT1, MINT2, MINT31, CACNA1G, pl6, pl4, COX2, DAPK, and MGMT the level of methyl chloride in the 5 ′ and 3 ′ regions of the hMLH1 promoter region.
  • the likelihood of cancer development, its type, site, and the like can be predicted.
  • the production of the nucleic acid amplification primer, the nucleic acid amplification primer set, and the kit for detecting the mutation of codon 599 of the BRAF gene (and the mutation of codon 12 and codon 13 of the KRAS gene) of the present invention are carried out in the pharmaceutical industry and in the pharmaceutical industry. It can be used in the field of technology.
  • a method for detecting a mutation at codon 599 (and mutations at codons 12 and 13 of the KRAS gene) of the BRAF gene of the present invention, a primer for nucleic acid amplification, a primer set for nucleic acid amplification, and a BRAF gene for use in this detection method The codon 599 mutation (and KRAS gene codon 12 and codon 13 mutation) detection kits can be usefully used in the medical industry.

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Abstract

L'invention concerne une méthode permettant de détecter rapidement et avec une haute précision une mutation quelconque du codon 599 du gène BRAF. L'invention concerne également, en vue d'une utilisation dans cette méthode, une amorce pour amplification d'acide nucléique, un ensemble d'amorces pour amplification d'acide nucléique, ainsi qu'une trousse de réactif pour la détection d'une mutation du codon 599 du gène BRAF. L'invention se rapporte en outre à une méthode destinée à détecter une mutation génétique en combinaison avec la détection d'une mutation quelconque du codon 12 et du codon 13 du gène KRAS, une amorce utilisée dans cette méthode, etc.
PCT/JP2004/014741 2003-12-26 2004-10-06 Amorce pour amplification d'acide nucleique, ensemble d'amorces pour amplification d'acide nucleique et methode de depistage du cancer associee Ceased WO2005066346A1 (fr)

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WO2013181125A2 (fr) 2012-05-29 2013-12-05 Abbott Laboratories, Inc. Méthode de conception d'amorces, méthode de détection de polymorphismes mononucléotidiques (snp), méthode de distinction des snp, et amorces associées, oligonucléotides détectable, et kits
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CRUZ F. III. ET AL.: "Absence of BRAF and KRAS mutations in uveal melanoma", CANCER RES., vol. 63, no. 18, September 2003 (2003-09-01), pages 5761 - 5766, XP002984264 *
KIMURA E.T. ET AL.: "High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillarx thyroid carcinoma", CANCER RES., vol. 63, no. 7, April 2003 (2003-04-01), pages 1454 - 1457, XP002984265 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013181125A2 (fr) 2012-05-29 2013-12-05 Abbott Laboratories, Inc. Méthode de conception d'amorces, méthode de détection de polymorphismes mononucléotidiques (snp), méthode de distinction des snp, et amorces associées, oligonucléotides détectable, et kits
US10077474B2 (en) 2012-05-29 2018-09-18 Abbott Molecular, Inc. Method of designing primers, method of detecting single nucleotide polymorphisms (SNPs), method of distinguishing SNPs, and related primers, detectable oligonucleotides, and kits
EP3604552A1 (fr) 2012-05-29 2020-02-05 Abbott Molecular Inc. Procédé de conception d'apprêts, procédé de détection de polymorphismes de nucléotide unique (snp), procédé de distinction de snp et apprêts associés, oligonucléotides détectables et kits
CN108998534A (zh) * 2018-09-05 2018-12-14 遵义医学院 一种kras基因突变检测的新方法

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