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WO2006112141A1 - Procede et kit de determination de base - Google Patents

Procede et kit de determination de base Download PDF

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Publication number
WO2006112141A1
WO2006112141A1 PCT/JP2006/303265 JP2006303265W WO2006112141A1 WO 2006112141 A1 WO2006112141 A1 WO 2006112141A1 JP 2006303265 W JP2006303265 W JP 2006303265W WO 2006112141 A1 WO2006112141 A1 WO 2006112141A1
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WIPO (PCT)
Prior art keywords
primer
base
complementary
oligonucleotide
homologous
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PCT/JP2006/303265
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English (en)
Japanese (ja)
Inventor
Yoshihiro Soya
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Toyobo Co Ltd
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Toyobo Co Ltd
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Priority to JP2006520622A priority Critical patent/JP4650420B2/ja
Publication of WO2006112141A1 publication Critical patent/WO2006112141A1/fr
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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
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the present invention relates to the detection or detection of mutations, substitutions, polymorphisms, insertions or deletions using oligonucleotides that amplify regions containing mutations, DNA polymerases, and oligonucleotides that specifically amplify mutant nucleic acid sequences.
  • the present invention relates to a determination method and a base determination kit.
  • the present invention is particularly useful for diagnosis of genetic diseases and examination of diseases caused by point mutations. Mutations include both acquired gene mutations and SNPs.
  • nucleic acid sequence mutation means having a nucleotide sequence different from the wild type. It is known that gene mutations can cause disease or show resistance to drugs. Therefore, elucidation of variations in these nucleic acid sequences is clinically important, and routine phenotyping is particularly recommended for psychiatric patients and volunteers in clinical research (Non-patent document 1 and Non-patent document 1). 2). In addition, a nucleic acid sequence analysis method for detecting each genotype following identification of the causative mutant gene is desired.
  • nucleic acid sequencing methods can detect and identify nucleotide polymorphisms contained in nucleic acid sequences.
  • this method is very extensive because it involves the preparation of vertical nucleic acids, DNA polymerase reaction, polyacrylamide gel electrophoresis, and analysis of nucleic acid sequences. It takes effort and time. In addition, it is possible to save labor by using a recent automatic sensor, but there is a problem that an expensive device is required.
  • a gene amplification method such as a PCR (polymerase chain reaction) method (see Patent Document 1 and Patent Document 2) has been conventionally used.
  • Methods for detecting point mutations in genes are known.
  • the amplified gene fragment is treated with a restriction enzyme that cleaves a specific nucleic acid sequence, and a method of determining the size of the resulting fragment (PCR-RFLP) can be used.
  • PCR-RFLP a method of determining the size of the resulting fragment
  • the specificity of the primer used was used.
  • Alelle specific amplification method is used as one of a pair of oligonucleotides used in the gene amplification method.
  • a wild-type oligonucleotide that is completely complementary to the end region of the wild-type gene amplification region and a mutant-type gene are used as one of a pair of oligonucleotides used in the gene amplification method.
  • a mutant oligonucleotide that is completely complementary to the end region of the amplification region is used.
  • Mutant oligonucleotides become nucleotides that are complementary to the nucleotide with the expected point mutation at the 3 'end.
  • the sample gene is wild-type, the ability to amplify nuclear acid when using a wild-type oligonucleotide.
  • the oligonucleotide ends at the 3rd end of the sample gene. Since it is not complementary to the corresponding nucleotide! /, (Mismatch), no extension reaction occurs and nucleic acid amplification does not occur.
  • the sample gene is a mutant type, on the contrary, amplification does not occur when the wild type oligonucleotide is used, and amplification occurs when the mutant type oligonucleotide is used.
  • the sample gene is a wild type or a mutant type by investigating whether or not amplification is possible when each oligonucleotide is used, and thereby, point mutations in the sample gene can be determined. Can be identified.
  • the amplified product was electrophoresed on an agarose gel, stained with a nucleic acid-specific binding fluorescent reagent such as ethidium bromide, and then irradiated with UV to amplify the amplified nucleic acid. The presence or absence of can be detected.
  • Patent Document 1 Japanese Patent Publication No. 4-67960
  • Patent Document 2 Japanese Patent Publication No. 4-67957
  • Non-patent literature 1 Gram and Brsen, European onsensus Conference on Pharmacogen etics. Commission of the European Communities, Luxembourg, 1990, pp. 87-96
  • Non-patent literature 2 Balant et al., Eur. J. Clin. Pharmacol. Tsuji, pp. 551-554, (1989) Brief description of the drawings
  • FIG.1 Positional relationship between at least two types of oligonucleotide primers for base determination and oligonucleotide primers (B, C)
  • FIG. 7 PCR analysis of bcr-abl chimeric gene abl gene ATP binding region (T315I) mutation gene analysis results
  • Figure 8 Analysis of 681 (G ⁇ A) single nucleotide polymorphism of CYP2C19 gene by PCR
  • FIG. 10 Analysis of 1075 (A ⁇ C) single nucleotide polymorphism of CYP2C9 gene by PCR method.
  • FIG. 11 Detection of EGFR 19 exon 19 Del E746-A750 deletion mutation.
  • FIG. 14 Detection result of human EGFR gene exon 19 Del L747-T751del P753 deletion mutation
  • FIG. 15 Detection result of human EGFR gene exon 19 Del L747-A750 substitution P deletion mutation
  • An object of the present invention is to solve the above-mentioned problems and to detect a mutation, substitution, single nucleotide polymorphism, insertion or deletion in a nucleic acid sequence clearly and reproducibly, and It is to provide reagents for this purpose.
  • the present invention is based on a method for determining at least one type of base site on a nucleic acid sequence contained in a sample solution. As a result, at least one type of base determination oligonucleotide containing the base site is determined.
  • Primer (A) is a sense primer or an antisense primer, and is composed of a sense primer (B) that is homologous to the upstream region of the base site to be judged and an antisense primer (C) that is complementary to the downstream region.
  • B sense primer
  • C antisense primer
  • Primer (A) is a sense primer and judged! /, A combination of a sense primer (B) homologous to the downstream region of the base site and an antisense primer (C) complementary to the downstream region (However, primer (C) is downstream of primer (B));
  • Primer (A) is an antisense primer, and judged! /, A sense primer (B) that is homologous to the upstream region of the base site, and an antisense primer (C) that is complementary to the upstream region. Combination (however, primer (C) is downstream from primer (B));
  • a gene amplification reaction is performed, and the amplification product (for example, electrophoresis, electrophoresis, detection method using hypri, fluorescence detection method, melting curve analysis method, thermal elution chromatogram method)
  • the amplification product for example, electrophoresis, electrophoresis, detection method using hypri, fluorescence detection method, melting curve analysis method, thermal elution chromatogram method
  • the present invention has been completed by finding that the presence of a base to be determined can be easily determined by detection). That is, the present invention has the following constitutional power.
  • a base determination method comprising a step of performing a reaction
  • the primers (A), (B) and (C) are selected from any combination of the following (1) to (3):
  • Primer (A) is a sense primer or an antisense primer, and is composed of a sense primer (B) that is homologous to the upstream region of the base site to be judged and an antisense primer (C) that is complementary to the downstream region.
  • B sense primer
  • C antisense primer
  • Primer (A) is a sense primer and judged! /, A combination of a sense primer (B) homologous to the downstream region of the base site and an antisense primer (C) complementary to the downstream region (However, primer (C) is downstream of primer (B)); (3) Primer (A) is an antisense primer, and judged! /, A sense primer (B) that is homologous to the upstream region of the base site, and an antisense primer (C) that is complementary to the upstream region. Combination (however, primer (C) is downstream of primer (B)).
  • the base determination method according to item 1 wherein the determined! /, The base site contains a mutation, substitution, single nucleotide polymorphism, insertion or deletion.
  • Item 1 or 2 characterized in that the second base from the 3 ′ end of the oligonucleotide primer for base determination (A) is the same as or complementary to the predicted base of the base site 2.
  • the Tm value of the oligonucleotide primer for base determination (A) is determined! /, At least having a sequence complementary or homologous to the upstream and Z or downstream region of the base site.
  • Item 6 The base determination method according to any one of Items 1 to 5, which is higher than Tm values of two types of oligonucleotide primers (B, C).
  • oligonucleotide primer (A) for base determination was judged! Item 1, characterized in that it is higher than the concentration when using at least two types of oligonucleotide primers (B, C) having sequences complementary or homologous to the upstream and Z or downstream regions of the base site.
  • the base determination method in any one of -6.
  • a base determination method comprising a step of performing a gene amplification reaction using at least one type of oligonucleotide probe (D), wherein the primers (A), (B) and (C) are as follows: Selected from any combination of (1) to (3):
  • Primer (A) is a sense primer or an antisense primer, and is composed of a sense primer (B) that is homologous to the upstream region of the base site to be judged and an antisense primer (C) that is complementary to the downstream region.
  • B sense primer
  • C antisense primer
  • Primer (A) is a sense primer and judged! /, A combination of a sense primer (B) homologous to the downstream region of the base site and an antisense primer (C) complementary to the downstream region (However, primer (C) is downstream of primer (B));
  • Primer (A) is an antisense primer, and judged! /, A sense primer (B) that is homologous to the upstream region of the base site, and an antisense primer (C) that is complementary to the upstream region. Combination (however, primer (C) is downstream of primer (B)).
  • a base determination method comprising a step of performing a gene amplification reaction using at least one type of oligonucleotide probe (E) that is homologous or complementary to a nucleic acid sequence containing at least one base different from the nucleic acid sequence comprising ,
  • the primers (A), (B) and (C) are selected from any combination of the following (1) to (3):
  • Primer (A) is a sense primer or an antisense primer, and is composed of a sense primer (B) that is homologous to the upstream region of the base site to be judged and an antisense primer (C) that is complementary to the downstream region.
  • B sense primer
  • C antisense primer
  • Primer (A) is a sense primer and judged! /, A combination of a sense primer (B) homologous to the downstream region of the base site and an antisense primer (C) complementary to the downstream region (However, primer (C) is downstream of primer (B));
  • Primer (A) is an antisense primer, and judged! /, A sense primer (B) that is homologous to the upstream region of the base site, and an antisense primer (C) that is complementary to the upstream region. Combination (however, primer (C) is downstream of primer (B)).
  • the label is any one selected from a group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance.
  • a base judging kit comprising at least one base judging oligonucleotide primer (A) containing the base site to be judged (A) and oligonucleotide primers (B) and (C),
  • the primers (A), (B) and (C) are selected from any combination of the following (1) to (3):
  • Primer (A) is a sense primer or an antisense primer, and is complementary to a sense primer (B) that is homologous to the upstream region of the base site to be determined and complementary to the downstream region.
  • B sense primer
  • C antisense primer
  • Primer (A) is a sense primer and judged! /, A combination of a sense primer (B) homologous to the downstream region of the base site and an antisense primer (C) complementary to the downstream region (However, primer (C) is downstream of primer (B));
  • Primer (A) is an antisense primer, and judged! /, A sense primer (B) that is homologous to the upstream region of the base site, and an antisense primer (C) that is complementary to the upstream region. Combination (however, primer (C) is downstream of primer (B)).
  • the base determination kit according to item 17 further comprising at least one type of oligonucleotide probe (D) that is homologous or complementary to a nucleic acid sequence containing the base site.
  • E oligonucleotide probe
  • the base determination kit according to any one of items 17 to 19, further comprising a DNA polymerase.
  • the present invention provides a method that can clearly and easily determine the presence of a specific base in a sample nucleic acid. Since the method of the present invention does not require complicated operations as in the conventional methods, the results can be obtained quickly and easily with good reproducibility.
  • a chromosome or a fragment thereof containing a specific base site related to a mutation, substitution, single nucleotide polymorphism, insertion or deletion contained in a sample is a target nucleic acid containing a base site responsible for information on the gene of interest. If it is, it will not be restrict
  • the target nucleic acid include an Alu sequence, an exon of a gene encoding a protein, an intron, and a promoter. More specifically, genes related to various diseases including genetic diseases, drug metabolism, lifestyle-related diseases (hypertension, diabetes, etc.) can be mentioned.
  • the nucleic acid sequence may be simply referred to as a nucleic acid.
  • a sequence in which a specific base exists is sometimes referred to as a mutant type, and a sequence that does not have a specific type as a wild type.
  • a mutant type is one in which at least one, preferably one nucleotide of a wild-type base sequence is point-mutated and replaced with another nucleotide, or a part of a wild-type nucleic acid is inserted or deleted.
  • the base site to be determined may be in another chromosome or may be in a distant position in the same gene or may be adjacent.
  • the primers (A) to (C) may be used at the same time to carry out a gene amplification reaction. You may go.
  • the gene amplification reaction is performed in the presence of primers (A) to (C).
  • Probe (D) and probe) can be used in combination with primers (A) to (C) or separately.
  • the primers used in the present invention are amplified by PCR, NASBA, LCR, SDA, RCA, TMA, LAMP and ICAN methods, which are known amplification methods, so that an amplification product is obtained so as to contain at least one base to be determined. Design to be.
  • the primer chain length is preferably 9 to 35 bases, preferably 11 to 30 bases, more preferably 15 to 28 bases, and even more preferably 18 to 25 bases.
  • Base site to be determined includes “mutation”, “substitution”, “single nucleotide polymorphism”, “insertion”, “deletion” and the like. Based on these determinations, a base determination oligonucleotide primer (A) including a base site may be set.
  • the “base site to be determined” means a site having “mutation”, “substitution”, “single nucleotide polymorphism”, “insertion” or “deletion”. It is not always necessary for the primer (A) to contain an insertion sequence or deletion sequence. Before and after the occurrence of “mutation”, “substitution”, “single nucleotide polymorphism”, “insertion” or “deletion”. It may contain a base site.
  • the forward primer commonly used by the trader is a sense primer
  • the reverse primer is an antisense primer. It may be written as one.
  • an oligonucleotide primer (A) for base determination is set so as to include a base after mutation, or the same or complementary base as the base after substitution. You just have to decide.
  • the first or second base from the 3 ′ end of the oligonucleotide primer for base determination (A) is the same as or complementary to the predicted base of “mutation” or “substitution”. Good.
  • the second base from the 3 ′ end of the oligonucleotide primer for base determination (A) is the same as or complementary to the base expected to be “mutated” or “substituted”.
  • At least one of the 3rd to 5th bases from the 3 ′ end of the oligonucleotide primer for base determination (A) is not complementary or homologous to the nucleic acid sequence having a cage shape, but is replaced with a base. Is preferred. When an extension from this primer (A) is confirmed, it is judged that there is “mutation” or “substitution”.
  • the oligonucleotide primer (A) for base determination should be set so as to contain the same or complementary base as the mutant base!
  • the first or second base from the third end of the oligonucleotide primer for base determination (A) is the same as or complementary to the predicted base variant of the “single nucleotide polymorphism”.
  • the second base from the 3 ′ end of the oligonucleotide primer for base determination (A) is the same or complementary base as the expected base variant of “single nucleotide polymorphism”. Good.
  • At least one of the 3rd to 5th bases from the 3rd and 3rd ends of the oligonucleotide primer for base determination (A) is not complementary or homologous to the hooked nucleic acid sequence! Is preferred.
  • the oligonucleotide primer (A) for base determination may be set so as to include the same or complementary base as the wild type base! / ⁇ .
  • the first or second base from the third end of the oligonucleotide primer for base determination (A) is the same as or complementary to the wild type of the predicted base of the “single nucleotide polymorphism” Good.
  • the second base from the 3 ′ end of the oligonucleotide primer for base determination (A) is the same or complementary salt as the wild type of the expected base of the “single nucleotide polymorphism”.
  • At least one of the 3rd to 5th bases from the 3rd and 3rd ends of the oligonucleotide primer for base determination (A) is not complementary or homologous to the nucleic acid sequence in a cage shape, but is replaced with a base. Preferred.
  • an extension of the primer (A) force is confirmed to determine this wild type, it is determined that there is at least a wild type in the polymorphism analysis.
  • an oligonucleotide primer for base determination (A) set to contain the same or complementary base as the mutant base, and a complementary base with the wild type base A base determination oligonucleotide primer (A) set to contain a base or the same base may be used simultaneously.
  • the oligonucleotide primer (A) for judging the mutant type is used as a sense primer
  • the oligonucleotide primer (A) for judging the wild type is used as an antisense primer.
  • the oligonucleotide primer (A) for judging the mutant type is used as an antisense primer
  • the oligonucleotide primer (A) for judging the wild type is used as a sense primer.
  • an extension product with only the oligonucleotide primer (A) force for judging the mutant homozygous and wild type is found.
  • the extension product from both the oligonucleotide primer (A) for judging the wild type homozygous, mutant type and wild type it can be judged to be heterozygous.
  • the corresponding oligonucleotide primer (A) can be used.
  • one is a sense primer and the other is an antisense primer as in the combination of the mutant type and the wild type.
  • the base determination oligonucleotide primer (A) contains the same or complementary base or base sequence as the base sequence and does not contain the same or complementary base or base sequence as the inserted base or base sequence. ) Can be set! / When the extension from this primer (A) is confirmed, it is judged that there is no “insertion”.
  • the oligonucleotide primer (A) for base determination is set so as to include the same or complementary base or base sequence as the base or base sequence to be “inserted”. May be.
  • the 3 to 1 bases of the primer (A) are designed so that they do not coincide with the base or base sequence downstream of the base when it is not “inserted” or the base sequence.
  • a base or base sequence that is the same as or complementary to the base sequence or base sequence upstream or downstream of the predicted site of "deletion” or a base sequence, and is missing. You may set the oligonucleotide primer for base determination (A) so that it does not contain the same or complementary base or base sequence as the base or base sequence to be lost! /. When an extension from this primer (A) is confirmed, it is judged that there is a “deletion”.
  • an oligonucleotide primer for base determination (A) is used so as to include the same or complementary base or base sequence as the base or base sequence to be deleted. It may be set. In this case, it is preferable that the 1 to 5 bases at the 3 ′ end of the primer (A) are designed so as not to coincide with the base or base sequence downstream of the “deleted base” or base sequence. When an extension from this primer (A) is confirmed, it is judged that there is no “deletion”.
  • the Tm value of the oligonucleotide primer for base determination (A) was determined, and at least two kinds of oligonucleotide primers (B, B) having sequences complementary or homologous to the upstream and Z or downstream regions of the base site. It is preferably higher than the Tm value of C).
  • the Tm value of the oligonucleotide primer is the temperature at which 50% of the double-stranded DNA molecules that have hybridized are released, and the calculation method is not particularly limited as long as it is a known method. It is obtained by any one of the neighbor method, the Wallace method, and the GC% method, and preferably satisfies the characteristics of the present invention.
  • the sequences of the oligonucleotide primers (A), (B), and (C) to be used are determined by comparing the Tm values calculated by the method.
  • Particularly preferred Tm values are those calculated by the Nearest neighbor method.
  • the difference in Tm value is not particularly limited as long as it is 0.5 ° C or more, but it is preferably 1 ° C or more.
  • the concentration at the time of using the oligonucleotide primer for determination (A) was determined to improve the detection sensitivity.
  • the concentration is preferably higher than the concentration when using at least two types of oligonucleotide primers (B, C) having a sequence complementary or homologous to the region.
  • the concentration of the oligonucleotide primer at the time of use is not particularly limited, but it is preferably used in the range of 0.5 to 50 M, particularly preferably 1 to 20 / ⁇ M.
  • the concentration of the oligonucleotide primer for base determination is the concentration of at least two oligonucleotide primers (B, C) having sequences complementary or homologous to the upstream and Z or downstream regions of the base site to be determined. Higher is preferred.
  • the difference in concentration is not particularly limited, and can be appropriately selected within a range higher than 1-fold and lower than 100-fold depending on the sequence of the oligonucleotide primer.
  • At least one type of oligonucleotide primer for base judgment (A) containing the base site to be judged (A) and oligonucleotide primers (B) and (C)
  • Primer (A) is a sense primer or an antisense primer, and is composed of a sense primer (B) that is homologous to the upstream region of the base site to be judged and an antisense primer (C) that is complementary to the downstream region.
  • B sense primer
  • C antisense primer
  • Primer (A) is a sense primer and judged! /, A combination of a sense primer (B) homologous to the downstream region of the base site and an antisense primer (C) complementary to the downstream region (However, primer (C) is downstream of primer (B));
  • Primer (A) is an antisense primer, and judged! /, A sense primer (B) that is homologous to the upstream region of the base site, and an antisense primer (C) that is complementary to the upstream region. Combination (however, primer (C) is downstream from primer (B));
  • Formed from a primer (A, B, C) with a combination selected from A set is required. For example, if there are two base sites to be judged, two sets of the primers ( ⁇ , ⁇ , C) are usually required.
  • the oligonucleotide primers (B, C) can be used in common for two or more base sites to be determined.
  • the determined oligonucleotide primer for base determination ( ⁇ ) containing the base site may be prepared for each of two or more base sites to be determined.
  • the base determination Bases can be determined by using only two types of oligonucleotide primers ( ⁇ ), either wild type or mutant type.
  • the two types of primers may be in the same direction (both sense primers or both antisense primers) or in different directions (one is a sense primer and the other is an antisense primer).
  • each judgment place may be selected as desired.
  • the amplification products are designed so that the lengths of the amplification products are different.
  • the presence of a plurality of bases for example, 2 to: LOO positions, preferably 2 to 50 positions, more preferably 2 to 20 positions, particularly 2 to 10 positions
  • LOO positions for example, 2 to: LOO positions, preferably 2 to 50 positions, more preferably 2 to 20 positions, particularly 2 to 10 positions
  • the presence of a specific base can be determined regardless of the length of the amplification product chain.
  • the number of "insertion” or “deletion” bases that can be detected by the present invention is not necessarily one, and two or more arbitrary Number of, eg 2 to: LO It may be 00, especially 2 to 500.
  • the “inserted” or “deleted” bases are scattered (discontinuous) and constitute a series of “insertions” or “deletions”, then one set of primers (A, (B, C) Detectable force If there is no mutual relationship between the “insertion” or “deletion” sites, two or more primer ( A, B, C) set can be used.
  • At least two types of oligonucleotide primers (B, C) having a sequence complementary or homologous to the upstream and ⁇ or downstream regions of the base site determined for use in the present invention are the primers ( ⁇ ).
  • the primers ( ⁇ ) are the primers ( ⁇ ).
  • Primer ( ⁇ ) is a sense primer or an antisense primer, and a sense primer ( ⁇ ) that is homologous to the upstream region of the base site to be judged and an antisense primer (C) that is complementary to the downstream region Combination;
  • Primer ( ⁇ ) is a sense primer and determined! /, A combination of a sense primer ( ⁇ ) that is homologous to the downstream region of the base site and an antisense primer (C) that is complementary to the downstream region (However, primer (C) is downstream of primer ( ⁇ ));
  • Primer ( ⁇ ) is an antisense primer, and judged! /, A sense primer ( ⁇ ) that is homologous to the upstream region of the base site and an antisense primer (C) that is complementary to the upstream region. Combination (however, primer (C) is downstream of primer ( ⁇ ));
  • the primer ( ⁇ ) is used as the oligonucleotide primer (B C) (where primer ( ⁇ ⁇ ⁇ ⁇ ⁇ ) is sandwiched between primer ( ⁇ ) and primer (C)) (see 1 in Figure 1), primer ( ⁇ ) and primer (C) May be outside (see 2 and 3 in Figure 1).
  • X and X ′ indicate the sites for which bases are to be determined.
  • the right primer indicates the forward primer
  • the left primer indicates the reverse primer.
  • primer ( ⁇ ) and ( ⁇ ′) simply illustrate at least two types of oligonucleotide primers for base determination ( ⁇ ⁇ ), and are not limited to two types.
  • primer ( ⁇ ) has an example of the same direction (both sense primer or both antisense primer). In Fig. 1, 1 shows different directions (one is a sense primer and the other is an antisense primer). It may be ima).
  • the primer (A) is located between the oligonucleotide primers (B, C) (the primer (A) Is located between the primer (B) and the primer (C).
  • the primer (A) has 1 to 4 types.
  • primer (A) can use 2 or 3 types, and there is a possibility to have a base of the base site strength to be determined
  • the primer (A) can be used in 3 or 4 types.
  • an oligonucleotide primer for base determination designed to contain the same or complementary base or base sequence as the base or base sequence to be “deleted” and the prediction of “deletion” Including a base or base sequence that is the same or complementary to the base or base sequence upstream and downstream of the site to be deleted, and does not include a base or base sequence that is the same or complementary to the base or base sequence to be deleted
  • Two types of designed base determination oligonucleotide primers (A) may be used simultaneously.
  • it is sufficient to determine the presence of a nucleotide or nucleotide sequence that is predicted to be deleted use at least one primer (A).
  • an oligonucleotide primer for base determination designed to contain the same or complementary base or base sequence as the base or base sequence to be “inserted”, and the expected site of “insertion” It is designed to include the same or complementary bases or base sequences as the upstream and downstream bases or base sequences, and the same or complementary bases or base sequences as the inserted bases or base sequences. Two types of base primer (A) may be used at the same time. In addition, since it is necessary to determine the presence of a base or a base sequence that is expected to be inserted, use at least one primer (A).
  • the primers (A), (B), and (C) are selected from any of the following combinations (1) to (3):
  • primer (A) is a sense primer or an antisense primer A combination of a sense primer (B) homologous to the upstream region of the desired base site and an antisense primer (C) complementary to the downstream region;
  • Primer (A) is a sense primer and judged! /, A combination of a sense primer (B) homologous to the downstream region of the base site and an antisense primer (C) complementary to the downstream region (However, primer (C) is downstream of primer (B));
  • Primer (A) is an antisense primer, and judged! /, A sense primer (B) that is homologous to the upstream region of the base site, and an antisense primer (C) that is complementary to the upstream region. Combination (however, primer (C) is downstream of primer (B)).
  • Primer (A) is a sense primer or an antisense primer, and is composed of a sense primer (B) that is homologous to the upstream region of the base site to be judged and an antisense primer (C) that is complementary to the downstream region.
  • B sense primer
  • C antisense primer
  • Primer (A) is a sense primer and judged! /, A combination of a sense primer (B) homologous to the downstream region of the base site and an antisense primer (C) complementary to the downstream region (However, primer (C) is downstream of primer (B));
  • Primer (A) is an antisense primer and was judged! /, A sense primer (B) homologous to the upstream region of the base site, and an antisense primer complementary to the upstream region ( Combination with C) (provided that primer (C) is downstream of primer (B)); primer (A, B, C) with a combination selected from either
  • the present inventors have found that a non-specific amplification reaction does not occur unexpectedly, and the presence and location of the base to be determined can be easily identified by the length of the amplified product.
  • the present invention will be described by way of examples. That is, in the method using the nucleic acid amplification method using the oligonucleotide primers (B and C) described in (1) above, the oligonucleotide primers (B) and ( It is recognized that the sequence between C) is specifically amplified. In this reaction, when at least one type of base determination oligonucleotide primer (A) containing the base site to be determined is added,
  • the nucleic acid sequence having the target base is also specifically amplified by the oligonucleotide primer for base determination (A) and the oligonucleotide primer (B) or (C), It is easy to recognize that in addition to the amplification products described above, another amplification product can be obtained.
  • the nucleic acid fragment that is specifically amplified by the sequence sandwiched between the oligonucleotide primers (B) and (C) is As a result, a non-specific amplification reaction by the oligonucleotide primer for base determination (A) occurs, and as a result, it is expected that another amplification product is obtained in addition to the amplification product described above. It was. In this case, since two or more kinds of amplified nucleic acid fragments are always obtained regardless of the presence of a specific base, two kinds of oligonucleotides having sequences complementary or homologous to the upstream and downstream regions of the base site. In the presence of primers (B and C) and oligonucleotide primers (A) for base determination, it was considered difficult to determine specific bases (PCR
  • the combination of the determined oligonucleotide primer (A) containing the base site and the oligonucleotide primer (B, C) described above is the “determined! /, Base site” “mutation”, “ “Substitution”, “single nucleotide polymorphism”, “insertion” or “deletion” can be easily determined.
  • the detection method will be described with reference to the drawings, but the present invention is not limited to this.
  • X represents a “mutation” or “substitution” base.
  • Right primer indicates forward primer and left primer indicates reverse primer.
  • oligonucleotide primer (A) for base determination so that it contains the base after mutation or the same base as the base after substitution. This is a case where a combination of a sense primer (B) homologous to the upstream region of the base site and an antisense primer (C) complementary to the downstream region is selected.
  • the oligonucleotide primer for base determination (A) is set so as to include the base after the mutation or a base complementary to the base after the substitution, and (1) This is a case where a combination of a sense primer (B) homologous to the upstream region of the desired base site and an antisense primer (C) complementary to the downstream region is selected.
  • oligonucleotide primer (A) for base determination so that it contains the same base as the base after the mutation or the base after the substitution, and (2) I want to make a determination.
  • a combination of a sense primer (B) that is homologous to the downstream region of the base site and an antisense primer (C) that is complementary to the downstream region (provided that primer (C) is downstream of primer (B)) Is selected.
  • the oligonucleotide primer (A) for base determination was set so as to include a base after mutation or a base complementary to the base after substitution, and (3) was determined.
  • a combination of a sense primer (B) homologous to the upstream region of the base site and an antisense primer (C) complementary to the upstream region (provided that primer (C) is downstream of primer (B)) ) Is selected.
  • primer (A, B) If an amplification product of primer (A, B) is obtained, it is judged that there is “mutation” or “substitution”.
  • X and Y represent different bases.
  • Right primer is forward primer
  • the left direction shows the reverse primer.
  • (A) and ( ⁇ ') indicate different nucleotide determination oligonucleotide primers ( ⁇ ).
  • an oligonucleotide primer ( ⁇ ) for base determination is set so that it contains the same base as the base of X, and salt is added so that it contains a base complementary to the base of ⁇ .
  • X is a mutant type and ⁇ is a wild type
  • only the amplification products of primers (A, C) can be obtained.
  • primer ( ⁇ ', ⁇ ) If the amplification product is obtained, ⁇ (mutant) and ⁇ (wild type) will be judged as hetero.
  • X is a mutant type and ⁇ is another mutant type
  • an amplification product of primer (A, C) is obtained, it can be determined that it is a mutation type to X, and the amplification product of primer ( ⁇ ') If it is obtained, it can be judged that it is a mutant form of cocoon.
  • oligonucleotide primer ( ⁇ ) for base determination to include a base complementary to the base of ⁇ , and (1) determine! /, Upstream region of the base site
  • a combination of a sense primer ( ⁇ ) that is similar to the above and an antisense primer (C) that is complementary to the downstream region is selected.
  • an oligonucleotide primer for base determination ( ⁇ ) is set so that it contains the same base as the base of X, and (1) sense homologous to the upstream region of the base site to be determined. This is the case where a combination of the primer ( ⁇ ) and the antisense primer (C) complementary to the downstream region is selected.
  • X is a mutant type and ⁇ is a wild type, if only the amplification product of primer (A, C) is obtained, if only the amplification product of ⁇ ⁇ (mutation type) homo, primer ( ⁇ , C) is obtained, If the amplification product of ⁇ (wild type), primer (A, C) and primer ( ⁇ , C) is obtained, it is judged as X (mutant) and ⁇ (wild type) heterozygous.
  • an oligonucleotide primer for base determination ( ⁇ ⁇ ) is set so that it contains the same base as the base of X, and (2) the determination is homologous to the downstream region of the base site.
  • the combination of the sense primer ( ⁇ ) and the antisense primer (C) complementary to the downstream region is selected (provided that primer (C) is downstream of primer ( ⁇ )) .
  • X is a mutant type and Y is a wild type
  • if an amplification product of primer (A, C) is obtained it is judged that there is at least a mutant type to X. If only the amplification products of primers (B, C) are obtained, it is judged that there is at least no mutation to X.
  • the oligonucleotide primer for base determination (A) is set so as to include a base complementary to the base of Y, and (3) the determined! /, Upstream region of the base site A combination of a sense primer (B) similar to the above and an antisense primer (C) complementary to the upstream region (provided that primer (C) is downstream of primer (B)) It is a case.
  • X is a mutant type and Y is a wild type, it is judged that there is at least a wild type if an amplification product of primer (A, B) is obtained. If only the amplification products of primers (B, C) are obtained, it is judged that there is at least no wild type.
  • the base or base sequence to be inserted includes the base or base sequence upstream or downstream of the expected site (i) of the “insertion” or the same base sequence or base sequence.
  • a base or base sequence that is complementary to a base sequence or a base sequence upstream or downstream of the predicted site (i) of “insertion” or a base sequence is inserted.
  • the base judgment mode is such that it does not contain a base sequence or base sequence complementary to the base sequence ( ⁇ ).
  • primer (A, B) If the amplification product of primer (A, B) cannot be obtained, it is judged that there is no “insertion”.
  • the base or base sequence to be inserted includes the base or base sequence upstream or downstream of the expected site (i) of the “insertion” or the same base sequence or base sequence.
  • An oligonucleotide primer for base determination (A) is set so as not to contain the same base or base sequence as ( ⁇ ), (2) a sense primer (B) that is homologous to the downstream region of the determined base site, This is a case where a combination of a downstream region and a complementary antisense primer (C) (where primer (C) is downstream of primer (B)) is selected. If the amplification product of primer (A, C) cannot be obtained, it is judged that there is no “insertion”.
  • a base or base sequence that is complementary to a base sequence or a base sequence upstream or downstream of the predicted site (i) of “insertion” or a base sequence is inserted.
  • primer (A, B) If the amplification product of primer (A, B) cannot be obtained, it is judged that there is no “insertion”.
  • the base or base sequence that includes the same base or base sequence as the base or base sequence upstream or downstream of the expected site (i) of “insertion” and is inserted ( ⁇ )
  • primer (A, B) If an amplification product of primer (A, B) is obtained, it is judged that there is “insertion”.
  • the base or base sequence that includes the same base or base sequence as the base or base sequence upstream or downstream of the expected site (i) of “insertion” and is inserted ( ⁇ )
  • the oligonucleotide detector for base determination (A) is set so as to include the same base or base sequence as in (2), and (2) the sense primer (B) homologous to the downstream region of the determined base region and the downstream region This is the case where a combination with a complementary antisense primer (C) (where primer (C) is downstream of primer (B)) is selected. If an amplification product of primer (A, C) is obtained, it is judged that there is “insertion”.
  • a base or base sequence that is complementary to a base sequence or a base sequence upstream or downstream of the predicted site (i) of “insertion” or a base sequence is inserted.
  • a combination of an upstream region and a complementary antisense primer (C) (provided that primer (C) is downstream of primer (B)). If an amplification product of primer (A, B) is obtained, it is judged that there is “insertion”.
  • DDDDDDDD indicates a base or base sequence that is predicted to be deleted. “D” indicates not the base but the site after “deletion”. Right primer is forward primer The left direction is the reverse primer.
  • the base or base sequence that contains the same base or base sequence as the base or base sequence upstream and downstream of the predicted site of “deletion” and that is to be deleted “DDDDDDDDDD”
  • A Complement to the downstream region with the sense primer (B) that is homologous to the upstream region of the base site to be determined. This is the case when a combination with a typical antisense primer (C) is selected.
  • a base or base sequence that includes a base or base sequence that is complementary to a base or base sequence upstream and downstream of the predicted site of “deletion” and that is to be deleted is “
  • the oligonucleotide primer (A) for base determination is set so that it does not contain a base or base sequence complementary to ⁇ DDDDDDDD '', and (1) a homologous center in the upstream region of the base site determined.
  • a combination of a primer (B) and an antisense primer (C) complementary to the downstream region is selected.
  • the base or base sequence that contains the same base or base sequence as the base or base sequence upstream and downstream of the predicted site of “deletion” and that is to be deleted “DDDDDDDDDD”
  • the oligonucleotide primer for base determination (A) is set so that it does not contain the same base or base sequence as (1), and (2) the detected sense primer (B) that is homologous to the downstream region of the base site and the downstream region This is the case when a combination with a complementary antisense primer (C) (provided that primer (C) is downstream of primer (B)) is selected. If an amplification product of primers (A, C) is obtained, it is judged that there is a “deletion”.
  • a base or base sequence complementary to the base or base sequence to be deleted (DDDDDDDD) is included, and 1 to 5 bases at the 3 ′ end are bases to be “deleted”.
  • set a base judgment oligonucleotide primer (A) that does not match the base or base sequence downstream of the base sequence, and (1) judged! /, Homologous to the upstream region of the base site This is the case where a combination of a sense primer (B) and an antisense primer (C) complementary to the downstream region is selected.
  • the same base or base sequence as the “deletion” base or base sequence (DDDDDDDD) is included, and 1 to 5 bases at the 3 ′ end may be “deleted” bases or base sequences.
  • the same base or base sequence as the “deletion” base or base sequence (DDDDDDDD) is included, and 1 to 5 bases at the 3 ′ end are the bases or bases to be “deletion”
  • a base or base sequence complementary to the base or base sequence to be deleted (DDDDDDDD) is included, and 1 to 5 bases at the 3 ′ end are bases to be “deleted”.
  • a base determination oligonucleotide primer (A) that does not match the base sequence or base sequence downstream of the base sequence is set, and (3) the sense is homologous to the upstream region of the base site. This is the case where a combination of primer (B) and antisense primer (C) complementary to the upstream region (provided that primer (C) is downstream of primer (B)) is selected.
  • whether the amplification reaction is correct or not can also be determined at a time.
  • an amplification product from the extension reaction from the primer (A) cannot be obtained, an amplification product is obtained from at least the combination of the primer (B) and the primer (C).
  • the base was determined according to the present invention and no amplification product was obtained, it indicates that the amplification reaction itself was unsuccessful. This is because, in the conventional determination method, if no amplification product was obtained, there was no mutation, substitution, single nucleotide polymorphism, insertion or deletion, or the amplification reaction was unsuccessful. This overcomes the problem of being unable to discriminate between the powers of a person.
  • oligonucleotide probe (D) that is homologous or complementary to the nucleic acid sequence containing the base site, it contains the base site (wild type or mutant) and is homologous or complementary to the nucleic acid sequence of the product. Oligonucleotides having a typical nucleotide sequence are widely included.
  • At least one kind of homologous or complementary oligonucleotide probe (E) having a sequence different from the nucleic acid sequence containing the base site by at least one base is more preferably at least one of the oligonucleotide probe (D) and the oligonucleotide probe (D).
  • Examples include oligonucleotides having different bases (preferably 1 base).
  • the probe is used as a labeled probe. Sign The probe is designed to hybridize to newly generated sequences! Therefore, it is possible to efficiently detect newly generated extension products by extension reactions that are not affected by existing nucleic acids. can do.
  • As the label of the probe the same label as that of the primer is used. When both the primer and the probe are labeled, different labels can be preferably used.
  • the TaqMan (registered trademark) is an oligonucleotide probe pre-labeled with a fluorescent dye and a polymerase having 5 'nuclease activity
  • a single nucleotide mutation (polymorphism) is performed with PCR reaction.
  • the sequence can be specified by performing a melting curve analysis using an oligonucleotide probe.
  • the obtained nucleic acid fragment can be detected by fluorescence using an intercalator such as S YBR (registered trademark) Greenl or ethylene bromide, and the amplified fragment can be detected by electrophoresis. good.
  • Oligonucleotide probes that are substantially homologous or complementary to the point mutation site may be immobilized on a water-insoluble simple substance.
  • the water-insoluble carrier include synthetic polymers, biological polymers, metals, and glass. That is, the PCR product is bound on the carrier by adding the PCR product that has been previously made single-stranded to the oligonucleotide probe immobilized on the water-insoluble carrier.
  • the sequence of the PCR product can be specified by detecting the label.
  • Oligonucleotide probes may be arranged in an array on these water-insoluble carriers and used as a DNA array.
  • the oligonucleotide primer can be basically extended by using a conventional method.
  • the target nucleic acid is made to react with four types of deoxynucleoside triphosphates (dNTP) and DNA polymerase on a chromosome or fragment thereof containing a specific nucleotide polymorphism site denatured into a single strand.
  • Oligonucleotide stretches as a saddle type. The extension reaction is
  • Nucleic acid amplification methods include PCR, NASBA (Nucleic acid sequence-based amplification method; Nature No. 350, 91 (1991)), LCR (International Publication No. 89/12696, JP-A-2-2934) ), SDA (Strand
  • the PCR method has a three-step cycle of denaturation, annealing, and extension in the presence of a sample nucleic acid, four types of deoxynucleoside triphosphates, a pair of oligonucleotides, and a heat-resistant DNA polymerase.
  • This is a method of amplifying exponentially the region of the sample nucleic acid sandwiched between the pair of oligonucleotides by repeating.
  • each oligonucleotide is hybridized with a region on the complementary single-stranded sample nucleic acid in the subsequent annealing step, and each oligonucleotide is converted in the subsequent extension step.
  • a DNA strand complementary to each single-stranded sample nucleic acid that becomes a saddle shape is extended by the action of DNA polymerase to form double-stranded DNA. This single cycle amplifies one double-stranded DNA into two double-stranded DNAs.
  • the region of the sample DNA sandwiched between the pair of oligonucleotides is theoretically amplified 2 n times. Since the amplified DNA region exists in large quantities, it can be easily detected by methods such as electrophoresis. Therefore, using the gene amplification method, it is possible to detect even a very small amount of sample nucleic acid that could not be detected in the past, and this is a very widely used technology. .
  • the gene amplification method is PCR, it is determined! /, The presence of the base can be easily determined by the length of the amplified nucleic acid fragment obtained. That is, the amplified nucleic acid fragment obtained is an oligo having a sequence complementary or homologous to the upstream and downstream regions of the base site to be determined. Nucleotide primer (B, C) is judged to be one type of displacement force!
  • oligonucleotide primer (A) containing or deleted base, or upstream and downstream regions of the base site to be judged By detecting whether the amplified nucleic acid fragment is longer than the above-mentioned amplified nucleic acid fragment by an oligonucleotide primer (B, C) having a complementary or homologous sequence with a known method such as electrophoresis. The presence of the base can be determined.
  • the DNA polymerase used for PCR amplification is not particularly limited, and any DNA polymerase can be used.
  • DNA polymerase can be used.
  • DNA polymerases having no 5 'exonuclease activity include metathermic DNA polymerases derived from Pyrococcus foriosus (Pfo polymerase, WO92 / 09689, JP-A-5-328969), Thermus litoralis (Thermococcus
  • thermostable DNA polymerase derived from litoralis (Tli polymerase, JP-A-6-7160) Pyrococcus sp .
  • Metathermal DNA polymerase derived from KOD1
  • a combination of a sense primer ( ⁇ ) homologous to the upstream region of the base site to be determined and an antisense primer (C) complementary to the upstream region (provided that the primer ( In the case of combination with C), which is downstream of the primer ( ⁇ ) a DNA polymerase having exonuclease activity can be preferably used.
  • DNA polymerases with 5 'exonuclease activity include Taq polymerase, Tfl polymerase, Tth polymerase, Bst
  • DNA polymerase For example, DNA polymerase. These enzymes are merely examples, and polymerases having the same action are not limited thereto and can be used in the present invention.
  • the method for detecting the nucleic acid fragment (amplification product) obtained according to the present invention is not particularly limited, but includes electrophoresis, detection using hypri, fluorescence detection, melting curve. Existing methods such as analysis methods and thermal elution chromatogram methods can be used as appropriate.
  • Electrophoresis can be used as a very simple detection method. That is, the amplified nucleic acid fragment obtained was judged! /, The base to be judged as one of oligonucleotide primers (B, C) having a sequence complementary or homologous to the upstream and downstream regions of the base site. Oligonucleotide primer (B, C) having a sequence complementary to or homologous to the upstream and downstream regions of the base site determined by the amplified nucleic acid fragment by the nucleotide primer (A) for inclusion or deletion If the chain lengths of the amplified nucleic acid fragments mentioned above differ, they can be easily distinguished.
  • an amplified nucleic acid fragment obtained by the oligonucleotide primer for base determination (A) is obtained, at least one base is different from the wild-type sequence.
  • An elution chromatogram method can be used.
  • the sequence differs from the wild-type nucleic acid sequence by at least 2 bases. Therefore, the specificity in high-pri detection is extremely high.
  • the primer to be used may optionally be labeled.
  • a labeled probe is preferably used.
  • the label used can be selected from the group consisting of fluorescent chemical substances, luminophores, enzymes, fluorescent proteins, photoproteins, magnetic substances, and conductive substances.
  • the detection of the label is not particularly limited as long as it is a known method, but when the label is a fluorescent substance or fluorescent protein, light of a specific wavelength is irradiated to excite the fluorescent chemical substance or fluorescent protein. It is possible to measure the amount of fluorescence of a specific wavelength generated when converted to the ground state.
  • the fluorescent chemical substance used in these include FITC, FAM, TAMRA, Texas Red, VIC, Cy3, Cy5, HEX, and the like
  • examples of the fluorescent protein include GFP, YFP, and RFP.
  • the label is an enzyme, it can be measured by detecting the reaction product produced by adding the enzyme substrate.
  • the combination of enzyme and substrate used for these is alkaline phosphatase and para-trophyl-phosphate, CDP-star, AMPPD, DDAOphospate, BCIP-NBT, etc., peroxidase and TMB, Lurd-Light (Roche 'Light Diagnostics), SAT-1 (Dojinka) Etc.), combinations of diaphorase and NTB, various oxidases and substrates, combinations of various dehydrogenases and substrates, etc., as long as reaction products can be detected, they are not limited to these. It is.
  • measurement can be performed by detecting the magnetism. If the label is a conductive substance, it can be measured by detecting the current value.
  • the kit comprises a DNA polymerase, determined! /, And at least two oligonucleotide primers having sequences complementary or homologous to the upstream and downstream regions of the base site. (B, C) as long as it contains at least one base determination oligonucleotide primer (A) containing or deleted the base to be determined. In addition, it may contain 4 types of deoxynucleoside triphosphates.
  • Samples used in the assembly are biomaterials (blood, plasma, mucous membranes, nails, hair, body fluids, mucus, tissues or tissue fragments, etc.), environmental samples (river water, sludge, drainage, soil or air), or Examples include, but are not limited to, food (agricultural products, livestock products, processed foods, marine products, etc.), viruses, bacteria, or culture solutions thereof. These samples may be prepared by known methods. As a typical example, the phenol Z black mouth form extraction method (Biochi mica et Biophysica acta
  • the present invention provides means and kits that are effective in reducing detection errors such as poor amplification due to samples as well as economic effects such as halving the amount of labor required for detection and reagents. It becomes possible to do.
  • oligos 1 to 4 an oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 1 to 4 (hereinafter referred to as oligos 1 to 4) was synthesized by the phosphoramidite method. The synthesis was performed according to the manual, and the deprotection of each oligonucleotide was carried out overnight at 55 ° C with ammonia water. Oligonucleotide purification was carried out on a Perkin Elma OPC column.
  • Oligo 1 is a common sense primer (B)
  • oligo 2 is a common antisense primer (C)
  • both common primers are combined and used as an oligonucleotide for the amplification reaction.
  • Oligo 3 is a primer (A) for amplifying the (E255K) mutant gene in the abl gene ATP binding region of the bcr-a bl chimeric gene.
  • Oligo 4 is a primer (A) for amplifying the (T 3151) gene mutation in the abl gene ATP binding region of the bcr-abl chimeric gene.
  • RNA extracted from the leukemia cell line is adsorbed to a column (QIAampRNA Blood Mini Kit; QIAGEN), the column is washed, and then adsorbed
  • RNA was eluted with DEPC water, ciiethylpyrocar Donate treated distilled and deionized water. The concentration and purity of RNA were confirmed by the ratio of absorbance A260nmZA280nm.
  • RNA 2 ⁇ g of total RNA obtained above, 500 ng random primer, 200 units Reverse transcriptase (Superscript II; manufactured by GIBCO), 40 units of RNase inhibitor (GIB CO) and ImM
  • the PCR reaction begins at 94 ° C for 2 minutes, then 94 ° C for 15 seconds of denaturation, and 62.5 ° C—30 seconds of annealing and 68 ° C—30 seconds of DNA extension step. This was done by performing a total of 35 cycles.
  • the amplification reaction solution 51 was electrophoresed using a 10% acrylamide gel. The results are shown in FIG.
  • RNA extracted from the leukemia cell line was adsorbed onto a column (QIAampRNA Blood Mini Kit; manufactured by QIAGEN), the column was washed, and the adsorbed RNA was eluted with DEPC water (diet hylpyro carbonate treated distilled and deionizea water). The concentration and purity of RNA were confirmed by the ratio of absorbance A260nmZA280nm.
  • RNA obtained above 2 ⁇ g was added to 500 ng of random primer, 200 units of reverse transcriptase (Superscript II; manufactured by GIBCO), 40 units of RNase inhibitor (GIB CO) and ImM
  • the PCR reaction begins at 94 ° C for 2 minutes, then 94 ° C for 15 seconds of denaturation, and 62.5 ° C—30 seconds of annealing and 68 ° C—30 seconds of DNA extension step. This was done by performing a total of 35 cycles.
  • the amplification reaction solution 51 was electrophoresed using a 1% acrylamide gel.
  • Figure 7 shows the results.
  • Oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 5 to 8 were synthesized by the phosphoramidite method using a DNA synthesizer type 392 manufactured by Perkin Elma. The synthesis was carried out according to the manual, and the deprotection of each oligonucleotide was carried out overnight at 55 ° C with ammonia water. Oligonucleotide purification was carried out on a Perkin Elma OPC column. Or we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigmajinosis, etc.).
  • Oligo 5 is a common sense primer (B), and oligo 6 (SEQ ID NO: 6) is a common antisense primer (C). Both common primers are combined and used as an oligonucleotide for amplification reaction. Is done.
  • Oligo 7 is a primer (A) for amplifying the 681G gene (wild type) of the CYP2C19 gene.
  • Oligo 8 is a primer (A) for amplifying the 681A gene (mutant) of the CYP2C19 gene.
  • nucleotide polymorphism of the human CYP2C19 gene under the following conditions (681G ⁇ A) was analyzed.
  • the PCR reaction was first performed at 94 ° C for 2 minutes, followed by a denaturation step at 94 ° C for 15 seconds, and an annealing step at 60 ° C for 30 seconds and a DNA extension reaction step at 68 ° C for 30 seconds. This was done by performing a total of 35 cycles.
  • the amplification reaction solution 51 was electrophoresed using a 3% agarose gel. The results are shown in Fig. 8.
  • the amplified fragments obtained by the samples are different, so that the sequence can be easily determined.
  • CYP2C19 gene 636 (G ⁇ A) —Synthesis of oligonucleotides to detect nucleotide polymorphisms
  • oligos 9 to 12 an oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 9 to 12 (hereinafter referred to as oligos 9 to 12) was synthesized by the phosphoramidite method. The synthesis was performed according to the manual, and the deprotection of each oligonucleotide was carried out overnight at 55 ° C with ammonia water. Oligonucleotide purification was carried out on a Perkin Elma OPC column.
  • Oligo 9 is the common sense primer (A)
  • oligo 10 is the common antisense primer (B)
  • both common primers are combined and used as the oligonucleotide for the amplification reaction.
  • Oligo 11 is a primer for amplifying the 636G gene (wild type) of the CYP 2C19 gene.
  • Oligo 12 is a primer for amplifying the 636A gene (mutant) of the CYP2C19 gene.
  • nucleotide polymorphism of the human CYP2C19 gene under the following conditions (636G ⁇ A) was analyzed.
  • the PCR reaction begins with 94 ° C for 2 minutes, followed by a denaturation step at 94 ° C for 15 seconds, and annealing at 60 ° C for 30 seconds and a DNA extension reaction at 68 ° C for 30 seconds. This was done by performing a total of 35 cycles.
  • the amplification reaction solution 51 was electrophoresed using a 3% agarose gel. The results are shown in Fig. 9.
  • GG or G / G is a wild type homozygote
  • AZA is a mutant homozygote
  • GZA is heterozygous.
  • oligos 13 to 16 an oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 13 to 16 (hereinafter referred to as oligos 13 to 16) was synthesized by the phosphoramidite method.
  • oligos 13 to 16 an oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 13 to 16 (hereinafter referred to as oligos 13 to 16) was synthesized by the phosphoramidite method.
  • Oligo 13 is a common sense primer (B) and oligo 14 is a common antisense primer (C), and both common primers are combined and used as an oligonucleotide for the amplification reaction.
  • Oligo 15 is a primer (A) for amplifying the 1075A gene (wild type) of the CYP2C9 gene.
  • Oligo 16 is a primer (A) for amplifying the 1075C gene (mutant) of the CYP2C9 gene.
  • dNTPs, ImM MgS04, and KOD—plusDNApolymerase were added to milli-Q water to make the total amount 25 1.
  • the PCR reaction begins with 94 ° C for 2 minutes, followed by a denaturation step at 94 ° C for 15 seconds, and annealing at 60 ° C for 30 seconds and a DNA extension reaction at 68 ° C for 30 seconds. This was done by performing a total of 35 cycles.
  • the amplification reaction solution 51 was electrophoresed using a 3% agarose gel. The results are shown in Fig. 10. AA indicates wild type homology, CC indicates mutant type homology, and AC indicates heterogeneity. As the electrophoresis results show, the amplified fragments obtained by the samples are different, so that the sequence can be easily determined.
  • oligos 17 to 20 an oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 17 to 20 (hereinafter referred to as oligos 17 to 20) was synthesized by the phosphoramidite method.
  • oligos 17 to 20 an oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 17 to 20 (hereinafter referred to as oligos 17 to 20) was synthesized by the phosphoramidite method.
  • Oligo 17 is the exon 21 common sense primer (B), and oligo 18 is the exon 21 common antisense primer (C). Used. Oligo 19 is an exon 19 common sense primer, and oligo 20 is an EGFR gene exon 19
  • Human leukocyte power DNA solution (sample 1) extracted by the phenol / chloroform method, and human EGFR exon 19
  • a plasmid DNA solution containing the deletion mutant genes of I (Sample 4), Del L747-T751del P753 (Sample 5), Del L747-A750 substitution P (Sample 6) and a plasmid DNA solution containing the human EGFR exon 21 wild type gene
  • the mixed DNA solution and the plasmid DNA solution containing the human EGFR exon 19 wild type gene and the DNA solution mixed with the plasmid DNA solution containing the human EGFR exon 21 wild type gene (sample 7) as samples, The following reagents were added, and human EGFR gene deletion mutations were analyzed under the following conditions.
  • dNTPs, ImM MgS04, and KOD—plusDNApolymerase were added to milli-Q water to make the total amount 25 1.
  • the PCR reaction begins with 94 ° C for 2 minutes, followed by a denaturation step at 94 ° C for 15 seconds, and annealing at 60 ° C for 30 seconds and a DNA extension reaction at 68 ° C for 30 seconds. This was done by performing a total of 35 cycles.
  • the amplification reaction solution 5 ⁇ 1 was subjected to electrophoresis using 3% agarose gel. The results are shown in Fig. 11.
  • oligonucleotides to detect the exon 19 Del L747-S752 deletion mutation in the human EGFR gene Using a DNA synthesizer type 392 manufactured by Perkin Elma Co., Ltd., oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 17-19 and 21 (hereinafter referred to as oligos 17-19 and 21) were synthesized by the phosphoramidite method. did. The synthesis was performed according to the manual, and various oligonucleotides were deprotected with ammonia water at 55 ° C overnight. Oligonucleotide purification was performed on a Perkin Elma OPC column.
  • Oligo 17 is an exon 21 common sense primer (B), and oligo 18 is an exon 21 common antisense primer (C), and both common primers are combined and used as oligonucleotides for amplification reaction.
  • Oligo 19 is an exon 19 common sense primer, and oligo 21 is an EGFR gene exon 19
  • Human leukocyte power DNA solution (sample 1) extracted by the phenol / chloroform method, and human EGFR exon 19
  • a plasmid DNA solution containing the deletion mutant genes of I (Sample 4), Del L747-T751del P753 (Sample 5), Del L747-A750 substitution P (Sample 6) and a plasmid DNA solution containing the human EGFR exon 21 wild type gene
  • the mixed DNA solution and the plasmid DNA solution containing the human EGFR exon 19 wild type gene and the DNA solution mixed with the plasmid DNA solution containing the human EGFR exon 21 wild type gene (sample 7) as samples, The following reagents were added, and human EGFR gene deletion mutations were analyzed under the following conditions.
  • dNTPs, ImM MgS04, and KOD—plusDNApolymerase were added to milli-Q water to make the total amount 25 1.
  • the PCR reaction was first performed at 94 ° C for 2 minutes, followed by a denaturation step at 94 ° C for 15 seconds, and annealing at 60 ° C for 30 seconds and a DNA extension reaction at 68 ° C for 30 seconds. This process was performed for a total of 35 cycles.
  • the amplification reaction solution 51 was electrophoresed using a 3% agarose gel. The results are shown in Fig. 12.
  • oligos 17-19 and 22 an oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 17-19 and 22 (hereinafter referred to as oligos 17-19 and 22) was synthesized by the phosphoramidite method. did. The synthesis was performed according to the manual, and various oligonucleotides were deprotected with ammonia water at 55 ° C overnight. Oligonucleotide purification was performed on a Perkin Elma OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigmaji Anosys, etc.).
  • Oligo 17 is an exon 21 common sense primer (B), and oligo 18 is an exon 21 common antisense primer (C), and both common primers are combined and used as oligonucleotides for amplification reaction.
  • Oligo 19 is an exon 19 common sense primer, and oligo 22 is an EGFR gene exon 19
  • Human leukocyte power DNA solution (sample 1) extracted by the phenol / chloroform method, and human EGFR exon 19
  • a plasmid DNA solution containing the deletion mutant genes of I (Sample 4), Del L747-T751del P753 (Sample 5), Del L747-A750 substitution P (Sample 6) and a plasmid DNA solution containing the human EGFR exon 21 wild type gene
  • a plasmid DNA solution containing the human EGFR exon 21 wild type gene Mixed DNA solution and human EGFR exon 19 wild type Using the DNA solution (sample 7) mixed with the plasmid DNA solution containing the gene and the plasmid DNA solution containing the human EGFR exon 21 wild type gene as a sample, add the following reagents and the human EGFR gene under the following conditions: Deficient mutations were analyzed.
  • dNTPs, ImM MgS04, and KOD—plusDNApolymerase were added to milli-Q water to make the total amount 25 1.
  • the PCR reaction begins with 94 ° C for 2 minutes, followed by a denaturation step at 94 ° C for 15 seconds, and annealing at 60 ° C for 30 seconds and a DNA extension reaction at 68 ° C for 30 seconds. This was done by performing a total of 35 cycles.
  • the amplification reaction solution 51 was electrophoresed using a 3% agarose gel. The results are shown in Fig. 13.
  • oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 17 to 19 and 23 were synthesized by the phosphoramidite method. did. The synthesis was performed according to the manual, and various oligonucleotides were deprotected with ammonia water at 55 ° C overnight. Oligonucleotide purification was performed on a Perkin Elma OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigmaji Anosys, etc.).
  • Oligo 17 is an exon 21 common sense primer (B), and oligo 18 is an exon 21 common antisense primer (C), and both common primers are combined and used as oligonucleotides for amplification reaction.
  • Oligo 19 is an exon 19 common sense primer, and oligo 23 is an EGFR gene exon 19
  • Human leukocyte power DNA solution (sample 1) extracted by the phenol / chloroform method, and human EGFR exon 19
  • a plasmid DNA solution containing the deletion mutant genes of I (Sample 4), Del L747-T751del P753 (Sample 5), Del L747-A750 substitution P (Sample 6) and a plasmid DNA solution containing the human EGFR exon 21 wild type gene
  • the mixed DNA solution and the plasmid DNA solution containing the human EGFR exon 19 wild type gene and the DNA solution mixed with the plasmid DNA solution containing the human EGFR exon 21 wild type gene (sample 7) as samples, The following reagents were added, and human EGFR gene deletion mutations were analyzed under the following conditions.
  • dNTPs, ImM MgS04, and KOD—plusDNApolymerase were added to milli-Q water to make the total amount 25 1.
  • the PCR reaction begins with 94 ° C for 2 minutes, followed by a denaturation step at 94 ° C for 15 seconds, and annealing at 60 ° C for 30 seconds and a DNA extension reaction at 68 ° C for 30 seconds. This was done by performing a total of 35 cycles.
  • the amplification reaction solution 51 was electrophoresed using a 3% agarose gel. The results are shown in Fig. 14.
  • an oligonucleotide having a nucleotide sequence shown in SEQ ID NOs: 17 to 19 and 24 (hereinafter referred to as “O”) by the phosphoramidite method.
  • Rigo 17-19 and 24) were synthesized. The synthesis was performed according to the manual, and various oligonucleotides were deprotected with ammonia water at 55 ° C overnight. Oligonucleotide purification was performed on a Perkin Elma OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigmaji Anosys, etc.).
  • Oligo 17 is an exon 21 common sense primer (B), and oligo 18 is an exon 21 common antisense primer (C), and both common primers are combined and used as oligonucleotides for amplification reaction.
  • Oligo 19 is an exon 19 common sense primer, and oligo 24 is an EGFR gene exon 19
  • Human leukocyte power DNA solution (sample 1) extracted by the phenol / chloroform method, and human EGFR exon 19
  • a plasmid DNA solution containing the deletion mutant genes of I (Sample 4), Del L747-T751del P753 (Sample 5), Del L747-A750 substitution P (Sample 6) and a plasmid DNA solution containing the human EGFR exon 21 wild type gene
  • the mixed DNA solution and the plasmid DNA solution containing the human EGFR exon 19 wild type gene and the DNA solution mixed with the plasmid DNA solution containing the human EGFR exon 21 wild type gene (sample 7) as samples, The following reagents were added, and human EGFR gene deletion mutations were analyzed under the following conditions.
  • dNTPs, ImM MgS04, and KOD—plusDNApolymerase were added to milli-Q water to make the total amount 25 1.
  • the PCR reaction begins with 94 ° C for 2 minutes, followed by a denaturation step at 94 ° C for 15 seconds, and annealing at 60 ° C for 30 seconds and a DNA extension reaction at 68 ° C for 30 seconds. This was done by performing a total of 35 cycles. [0105] (Detection using electrophoresis)
  • the amplification reaction solution 51 was electrophoresed using a 3% agarose gel. The results are shown in Fig. 15.
  • oligos 17 to 19 and 21 to 24 an oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 17 to 19 and 21 to 24 (hereinafter referred to as oligos 17 to 19 and 21 to 24) was obtained by the phosphoramidite method. ) was synthesized. The synthesis was carried out according to the manual, and various oligonucleotides were deprotected with ammonia water at 55 ° C overnight. Oligonucleotide purification was carried out on a Perkin Elma OPC column. Or we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigmajinosis, etc.).
  • Oligo 17 is the Exon 21 common sense primer (B), and Oligo 18 is the Exon 21 common antisense primer (C). These two common primers are combined and used as an oligonucleotide for amplification reaction.
  • Oligo 19 is an exon 19 common sense primer, and oligos 21 to 24 are primers (A) for amplifying an exon 19-deficient mutant gene (mutant) of the EGFR gene.
  • Plasmid DNA solution containing each deletion mutant gene of L747-A750 substitution P (Sample 5) and DNA solution mixed with plasmid DNA solution containing human EGFR exon 21 wild type gene, and plasmid containing human EGFR exon 19 wild type gene DNA solution and human EGFR exon 2 1 DNA solution mixed with plasmid DNA solution containing wild-type gene (sample 6), and human white Using a DNA solution (sample 7) extracted from blood cells by the phenol'chloroform method as a sample, the following reagents were added and the human EGFR gene deletion mutation was analyzed under the following conditions.
  • dNTPs, ImM MgS04, and KOD—plusDNApolymerase were added to milli-Q water to make the total amount 25 1.
  • the PCR reaction begins with 94 ° C for 2 minutes, followed by a denaturation step at 94 ° C for 15 seconds, and annealing at 60 ° C for 30 seconds and a DNA extension reaction at 68 ° C for 30 seconds. This was done by performing a total of 35 cycles.
  • the amplification reaction solution 51 was electrophoresed using a 3% agarose gel. The results are shown in Fig. 16.
  • oligos 13, 14, 16, 25 oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 13, 14, 16, 25 (hereinafter referred to as oligos 13, 14, 16, 25) by the phosphoramidite method ) was synthesized.
  • the synthesis was carried out according to the manual, and various oligonucleotides were deprotected with ammonia water at 55 ° C overnight. Oligonucleotide purification was carried out on an OPC column from Parkin Elma. Or we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigmaji Anosys, etc.).
  • Oligo 13 is a common sense primer (B), oligo 14 is a common antisense primer (C), and both common primers are combined and used as an oligonucleotide for the amplification reaction.
  • Oligo 16 is a primer (A) for amplifying the 1075C gene (mutant) of the CYP2C9 gene.
  • Oligo 25 is a probe that contains a detection site. Be labeled.
  • the total amount was adjusted to 25 / z l by adding to milli-Q water.
  • the PCR reaction begins with 94 ° C for 2 minutes, then 94 ° C for 15 seconds of denaturation, and 60 ° C for 30 seconds of annealing and 68 ° C for 30 seconds for DNA extension. This was done by performing a total of 35 cycles.
  • Oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 26 to 28 were synthesized by the phosphoramidite method using a DNA synthesizer type 392 manufactured by PerkinElmer. The synthesis was performed according to the manual, and various oligonucleotides were deprotected with ammonia water at 55 ° C overnight. Oligonucleotide purification was carried out on a Perkin Elma OPC column. Or DNA synthesis contract company (Japan Bioservice Co., Ltd.) , Operon Co., Ltd., Proligo Co., Ltd., Sigmaji Anosys Co., Ltd.
  • Oligo 26 is a common sense primer (B) and oligo 27 is a common antisense primer (C), and both common primers are combined and used as an oligonucleotide for the amplification reaction.
  • Oligo 28 is a primer (A) for amplifying the Western type of the H. pylori CagA gene.
  • Example 16 DNA solution extracted from human gastric biopsy tissue with Dneasy Tissue Kit (Quiagen) and plasmid DNA solution cleaved with H.pylori East Asian gene and H.pylori Western European gene (Sample 2- 15) is used as a sample, the following reagents are added, and H.pylori
  • the type of CagA gene was analyzed.
  • the R reaction consists of 94 ° C for 2 minutes, then 94 ° C for 15 seconds of denaturation, and 60 ° C for 30 seconds for annealing and 68 ° C for 30 seconds for DNA extension. A total of 35 cycles were performed as a cycle.
  • the amplification reaction solution 51 was electrophoresed using a 3% agarose gel. The results are shown in Fig. 18. As shown electrophoresis results, CagA East Asian than 10 4 Copy, CagA western type was confirmed to be amplified from 1 0 3 Copy, sample force et extracted from gastric biopsy tissue also can be detected there were.
  • wild type nucleic acid sequence detection and mutant nucleic acid sequence detection which have been indispensable in the detection by electrophoresis in the detection of mutations, substitutions, single nucleotide polymorphisms, insertions or deletions, have been performed so far. Eliminating the need for separate work, it is possible to provide an effective means for reducing detection errors such as poor amplification due to samples as well as economic effects such as halving the amount of labor required for detection. It will be possible to make a significant contribution to the industry. Is expected.

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Abstract

L'invention concerne un procédé de détection d'une mutation dans une séquence d'acide nucléique, caractérisé par une détection sûre et une reproductibilité élevée, ainsi qu'un réactif associé. Le procédé de détermination de base permet de déterminer au moins un site de base dans une séquence d'acide nucléique contenue dans une solution de prélèvement. Ce procédé consiste à effectuer une réaction d'amplification des gènes en présence d'au moins une amorce oligonucléotidique (A) en vue de la détermination de base, laquelle amorce contient le site de base à déterminer, et d'amorces oligonucléotidiques (B) et (C).
PCT/JP2006/303265 2005-03-30 2006-02-23 Procede et kit de determination de base Ceased WO2006112141A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010279264A (ja) * 2009-06-03 2010-12-16 Tottori Univ Ｅｇｆｒ遺伝子変異検出プライマーセット、それを含むｅｇｆｒ遺伝子変異検出キット、およびそれを用いてｅｇｆｒ遺伝子変異検出を行うための核酸増幅装置
CN118755808A (zh) * 2024-09-05 2024-10-11 成都百思赛弗生物科技有限公司 一种多基因分型检测组合物的应用

Citations (2)

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Publication number Priority date Publication date Assignee Title
JPH02295496A (ja) * 1989-02-07 1990-12-06 Imperial Chem Ind Plc <Ici> 検定方法
JP2001137000A (ja) * 2000-09-19 2001-05-22 Nobuyuki Hamashima 遺伝子変異の判別方法

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JPH0889297A (ja) * 1994-09-26 1996-04-09 S R L:Kk Ｄｎａ点突然変異の検出方法及び試薬

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JPH02295496A (ja) * 1989-02-07 1990-12-06 Imperial Chem Ind Plc <Ici> 検定方法
JP2001137000A (ja) * 2000-09-19 2001-05-22 Nobuyuki Hamashima 遺伝子変異の判別方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010279264A (ja) * 2009-06-03 2010-12-16 Tottori Univ Ｅｇｆｒ遺伝子変異検出プライマーセット、それを含むｅｇｆｒ遺伝子変異検出キット、およびそれを用いてｅｇｆｒ遺伝子変異検出を行うための核酸増幅装置
CN118755808A (zh) * 2024-09-05 2024-10-11 成都百思赛弗生物科技有限公司 一种多基因分型检测组合物的应用

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