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WO2002046393A1 - Procede d'identification de polymorphisme nucleotidique - Google Patents

Procede d'identification de polymorphisme nucleotidique Download PDF

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Publication number
WO2002046393A1
WO2002046393A1 PCT/JP2001/010661 JP0110661W WO0246393A1 WO 2002046393 A1 WO2002046393 A1 WO 2002046393A1 JP 0110661 W JP0110661 W JP 0110661W WO 0246393 A1 WO0246393 A1 WO 0246393A1
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Prior art keywords
primer
nucleic acid
nucleotide
specific
wild
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Japanese (ja)
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Yutaka Takarada
Kohzo Hashimoto
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Toyobo Co Ltd
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Toyobo Co Ltd
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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
    • C12Q1/6858—Allele-specific amplification
    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to a method for identifying a mutation or polymorphism in a nucleic acid sequence and a kit for detecting a base polymorphism.
  • the present invention is particularly useful for diagnosis of genetic diseases, nucleotide polymorphism analysis, and the like.
  • the nucleotide polymorphism is a genotype having a sequence different from that of the wild-type, and the polymorphic gene plays an important role as a cause of inter-individual variation in the occurrence of side effects in drug metabolism and treatment failure. It is also known as a cause of individual differences such as basal metabolism known as constitution. In addition, they also serve as genetic markers for many diseases. Therefore, the elucidation of these mutations is clinically important, and phenotyping of rutin is particularly recommended for psychiatric patients and suicide volunteers in clinical research (Gram and Brsen, European Consensus Conference on Pharmacogenet i cs. Commi ss on of the European Communi ties, Luxembourg, 1990, pp.
  • nucleic acid sequencing can detect and identify nucleotide polymorphisms in nucleic acid sequences.However, preparation of type ⁇ nucleic acids, DNA polymerase reaction, polyacrylamide gel electrophoresis, analysis of nucleic acid sequences, etc. It requires a great deal of labor and time to perform. In addition, although labor saving can be achieved by using a recent automatic sequencer, there is a problem that an expensive device is required.
  • one of a pair of primers used in the gene amplification method includes a normal primer completely complementary to an end region of an amplification region of a normal gene and an end portion of an amplification region of an abnormal gene as one primer. Use an aberrant primer completely complementary to the region.
  • the abnormal primer has a nucleotide whose 3 'end is complementary to the nucleotide that caused the expected point mutation.
  • the sample gene is subjected to a gene amplification method by using such normal and abnormal primers separately. If the sample gene is normal, nucleic acid amplification will occur when the normal primer is used, but when the abnormal primer is used, the 3 'end of the primer will be complementary to the corresponding nucleotide in the sample gene. Therefore, no elongation reaction occurs and no amplification of nucleic acid occurs. On the other hand, if the sample gene is abnormal, amplification does not occur when the normal primer is used, and amplification occurs when the abnormal primer is used.
  • a nucleic acid sequence containing a point mutation may be amplified in advance, and a determination may be made as to whether an extension reaction occurs in each primer using the amplified nucleic acid.
  • This method can also be applied to base polymorphism analysis. That is, polymorphism analysis becomes possible by using a wild-type specific primer in place of a normal type primer and a polymorphism specific primer in place of an abnormal type primer.
  • nucleotide polymorphism can be clearly detected by the conventional method, but actually, there is a difference of only one nucleotide between the wild-type specific primer and the polymorphic specific primer.
  • a wild-type gene is extended or amplified using a polymorphism-specific primer and when a polymorphism gene is extended or amplified using a wild-type specific primer, a certain degree of reaction often occurs. In many cases, it is difficult to make a clear judgment. Whether or not extension or amplification occurs when mismatched primers are used depends on the type of equipment used and other delicate conditions, and the reproducibility is low. Therefore, in order to completely prevent the reaction from occurring when a mismatched primer is used, it is necessary to control the temperature conditions during the reaction extremely strictly. Can be a difficult task.
  • extension reaction may occur even though the extension of the mismatch is expected to occur by the polymerase proofreading function, despite the expectation that the extension reaction will not occur. There is a potential.
  • One embodiment is a method of hybridization using a labeled oligonucleotide probe. This method is accurate because a signal is obtained only when the probe is hybridized, but the detection step becomes complicated and the detection becomes complicated. If many polymorphisms are measured at one time, other types of labels are required.
  • An object of the present invention is to solve the above-mentioned problems and to provide a method capable of clearly and reproducibly detecting a polymorphism in a nucleic acid sequence and a reagent therefor.
  • Figures 1 to 6 show the fluorescence measurement data obtained with the Applied Biosystems ABI PRISM 310 Genetic Analyzer. Disclosure of the invention
  • the present inventors have conducted intensive studies and found that, in the above-described conventional method, when the wild-type specific primer hybridizes with the polymorphic nucleic acid, and when the polymorphic specific primer hybridizes with the wild-type nucleic acid, Measuring the length of the elongate allows easy and clear determination, and strict control of reaction conditions by associating a polymorphic nucleotide sequence with the second nucleotide from the 3 'end of the primer. The inventors have found that the primer extension reaction can be completely inhibited without performing this step, and have completed the present invention.
  • the present invention relates to a nucleic acid sequence containing a specific nucleotide polymorphism site contained in a sample, A wild-type specific primer and a polymorphic specific primer, each having a different length or label, are allowed to act simultaneously or separately, the primer is extended with DNA polymerase, and the extension product of the primer and / or its complementary strand are extended.
  • An object of the present invention is to provide a method for identifying a nucleotide polymorphism contained in a nucleic acid sample by detecting the length.
  • the present invention provides a nucleic acid sequence containing a specific nucleotide polymorphism site contained in a sample, together with a reverse primer, a wild-type specific primer and a polymorphism-specific primer having different lengths or labels, respectively, simultaneously or separately.
  • Another object of the present invention is to provide a method for identifying a nucleotide polymorphism contained in a nucleic acid sample by detecting whether or not a specific nucleic acid fragment has been amplified.
  • the present invention relates to a nucleic acid sequence comprising 2 or more specific nucleotide polymorphism site contained in a sample, each nucleotide polymorphism length corresponding to the type site or 2 or more wild-type with different labeled specific primers and 2 or more
  • the primers simultaneously or separately, extend the primers with DNA polymerase, and detect an extension product of the primers and / or a complementary strand thereof to include the primers in the nucleic acid sample. It is intended to provide a method for simultaneously identifying the above nucleotide polymorphisms.
  • the present invention relates to a nucleic acid sequence comprising 2 or more specific nucleotide polymorphism site contained in a sample, each nucleotide polymorphism length corresponding to the type site or 2 or more wild-type with different labeled specific primers and 2 or more
  • the polymorphism-specific primers together with two or more reverse primers corresponding to each base polymorphism site, simultaneously or separately, extend the primers with DNA polymerase, and detect amplification products of the primers
  • the present invention provides a method for simultaneously identifying two or more nucleotide polymorphisms contained in the nucleic acid sample.
  • the present invention provides a reagent kit for detecting a nucleotide polymorphism, wherein the second base from the 3 'end contains a primer, a DNA polymerase, a nucleotide and a size marker corresponding to a nucleotide expected to be polymorphic. Is provided.
  • the length of the wild-type specific primer and the length of the polymorphism-specific primer are different, whereby the length of the extension product is different.
  • the second base from the 3 ′ end of the primer is a base.
  • the wild-type specific primer and the polymorphism specific primer are not complementary to the specific nucleic acid sequence from the 3 'end to the 3' end from the 3 'end. Contains one nucleotide.
  • the method for detecting length is any one of electrophoresis, mass spectrometry, and liquid chromatography.
  • the DNA polymerase used in the primer extension reaction has 3 ′ exonuclease activity of double-stranded DNA.
  • the DNA polymerase is Pyrococcus sp. i 1 ic archaebacterium).
  • the nucleic acid sequence is amplified in advance.
  • the nucleic acid amplification method is any one selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA.
  • the wild-type-specific primer and the polymorphism-specific primer are pre-labeled.
  • the label is any one selected from the group consisting of an enzyme, a biotin, a fluorescent substance, a hapten, an antigen, an antibody, a radioactive substance, and a luminophore. is there.
  • a primer When a nucleic acid sequence is replicated, a primer, four types of deoxynucleoside triphosphates (dNTPs), and a DNA polymerase are allowed to act on a single-stranded denatured target nucleic acid, and the primer extension reaction is performed using the target nucleic acid as a ⁇ -type. The complementary strand of the resulting nucleic acid sequence is synthesized.
  • the polymorphism can be detected by performing an extension reaction using the wild-type specific primer and the polymorphism specific primer separately for the sample nucleic acid.
  • the target nucleic acid is not contained in an amount sufficient for detection, it is possible to amplify the nucleic acid fragment containing the polymorphic sequence in advance.
  • the nucleic acid amplification method in this case include PCR, NASBA (Nucleic acid sequence-based amplification method; Nature, Vol. 350, p. 91 (1991)), LCR (International Publication No. WO 89/12696, Kaihei 2_29344), SDA (Strand Displacement Amplification: Nucleic acid reserch Vol. 20, p. 1691 (1992)), RCR (International Publication No. 90/1069), TMA (Transcript ion mediated amplification method; J. Clin. Microbiol. Vol.
  • the PCR method involves repeating a cycle consisting of denaturation, annealing, and extension in the presence of a sample nucleic acid, four types of deoxynucleoside triphosphates, a pair of primers, and a thermostable DNA polymerase.
  • This is a method of exponentially amplifying a sample nucleic acid region sandwiched between the pair of primers. That is, the nucleic acid of the sample is denatured in the denaturation step, each primer is hybridized with a region on the single-stranded sample nucleic acid complementary to each other in the subsequent annealing step, and in the subsequent elongation step, the DNA is generated starting from each primer.
  • the DNA chain complementary to each single-stranded sample nucleic acid that becomes a type II by the action of the polymerase is extended to form a double-stranded DNA.
  • one double-stranded DNA is amplified to two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of primers is theoretically amplified 2 n times. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis. Therefore, using the gene amplification method, it is possible to detect a very small amount (even a single molecule) of sample nucleic acid, which was previously undetectable, and it is a very widely used technology in recent years. .
  • a wild type specific that can amplify a wild type nucleic acid The gene amplification method can be carried out by separately using polymorphism-specific primers capable of amplifying the polymorphic nucleic acid.
  • a polymorphic nucleic acid is a wild-type nucleic acid in which only one nucleotide of the wild-type nucleic acid is point-mutated and replaced with another nucleotide, or inserted or deleted in a part of the wild-type nucleic acid sequence. Nucleic acid sequence containing, and it is elucidated at which site the nucleotide is mutated. It has been elucidated that the constitutions and the like differ depending on such nucleotide polymorphisms.
  • the method of the present invention is a method for examining whether or not a sample nucleic acid has such expected polymorphism. is there.
  • the reaction occurs when the sample nucleic acid is wild-type, but does not occur when the polymorphism is used. Conversely, when a sample nucleic acid is extended or amplified using polymorphism-specific primers, the reaction occurs if the sample nucleic acid is polymorphic, but does not occur if the sample nucleic acid is wild-type. Therefore, one sample was divided into two, one was reacted with the wild-type specific primer, and the other was reacted with the polymorphic specific primer, to determine whether the reaction had occurred. It is possible to clearly determine whether the sample nucleic acid is wild-type or polymorphic.
  • higher organisms including humans, have one father-derived gene and one maternal-derived gene for one type of gene. You can also distinguish between homozygous and polymorphic or heterozygous. That is, in the case of heterozygous reaction, a wild-type gene and a polymorphic gene are both present, so that a reaction occurs both when a wild-type specific primer is used and when a polymorphic specific primer is used.
  • DNA polymerase derived from Thermus aquaticus which is often used in amplification reactions, does not have 3 'exonuclease activity.
  • the complementary strand of the sequence could not be synthesized Since the amplification reaction is continued as it is, the amplified nucleic acid fragment may contain a mutation. In other words, even if there is a mismatch at the 3 'end, the reaction proceeds, and it is thought that such a problem occurs.
  • DNA polymerase derived from Pyrococcus sp. K0D1 strain or Hyperthermophilic archaebacterium which has excellent extension reaction accuracy, If there is a mismatch at the 3 'end due to its 3' exonuclease activity, this problem may occur because the nuclease activity removes the mismatch and continues the extension reaction.
  • the arrangement of the primers is devised. That is, the primer used in the present invention is designed such that the second nucleotide from the 3 ′ end of the primer in the above combination corresponds to the nucleotide of the polymorphic sequence.
  • the primers are completely identical, so that the reaction occurs.
  • nucleotide polymorphism in a specific sequence can be identified.
  • a conventional nucleic acid sequence detection technique there is, for example, the Southern hybridization method (Masami Muramatsu, “Lab Manual Genetic Engineering Supplement”, published by Maruzen Co., Ltd., pp. 70-75).
  • a region of DNA having a base sequence complementary to the labeled DNA probe can be identified. That is, in the Southern hybridization method, nucleic acid fragments are electrophoresed on an agarose gel-polyacrylamide gel plate, separated according to the size (length) of the fragments, and denatured into single strands. Then, attach a membrane such as nitrocellulose or nylon to the plate, and transfer the nucleic acid fragments as they are in the electrophoresis pattern. After immobilization, a hybrid is formed with a nucleotide polymorphism-specific DNA probe labeled with RI (radioisotope) or the like, and the nucleic acid fragment on the membrane complementary to the probe is detected by autoradiography or the like.
  • RI radioisotope
  • the electrophoretic position and molecular weight of the target DNA fragment can be determined.
  • the presence or absence of amplification is used for the gene polymorphism because the above-described specific primers are used.
  • Electrophoresis is the simplest method. That is, after performing an extension reaction using a specific primer, it is possible to identify the polymorphism by confirming the length of the extension product or amplification product by electrophoresis. At this time, if the length of the extension or amplification product differs between the wild-type specific primer and the polymorphic specific primer, the polymorphism can be easily identified by electrophoresis, particularly by a genetic analyzer.
  • the length of the extension or amplification product differs depending on the type of polymorphism, it is possible to identify many types of polymorphisms at once. In addition, it is also possible to use a method for identifying the molecular weight, such as mass spectrometry or liquid chromatography.
  • a sequencer for determining a nucleic acid sequence can accurately detect a difference in the length of one base by electrophoresis.
  • the length of an extension product or an amplification product is adjusted using a wild-type specific primer and one or two polymorphism-specific primers corresponding to the number of polymorphisms. In this way, multiple types of polymorphisms can be accurately identified in one electrophoresis.
  • a primer for detecting a polymorphism a primer set consisting of a wild-type specific primer, at least one polymorphism-specific primer and a reverse primer can be preferably used.
  • the wild-type-specific primer and at least one polymorphism-specific primer In the first case, those having different numbers of bases are used.
  • the number of polymorphism-specific primers is the same as the number of polymorphism bases. For example, if the wild-type base is G and the polymorphism bases A and C are known, the type A polymorphism-specific primer If two types of primers and C-type primer are used, and if the polymorphic base is only A, use one (A-type) polymorphism-specific primer.
  • the chromosome containing a specific nucleotide polymorphism site or a fragment thereof contained in a sample is not particularly limited as long as it is a target nucleic acid containing a nucleotide polymorphism site that carries information on a target gene.
  • the target nucleic acid include an Alu sequence, exons and introns of a gene encoding a protein, and a promoter. More specifically, it includes genes related to various diseases including genetic diseases, drug metabolism, and lifestyle-related diseases (such as hypertension and diabetes). For example, hypertension includes the ACE gene.
  • a polymorphic nucleic acid of a chromosome or a nucleic acid fragment to be measured for a nucleotide polymorphism is a wild-type nucleic acid in which at least one nucleotide is point-mutated and replaced with another nucleotide, It is a nucleic acid containing an inserted or deleted sequence as part of a wild-type nucleic acid. It has been elucidated that the constitutions and the like differ depending on such nucleotide polymorphisms, and the method of the present invention is a method for examining whether or not a nucleic acid in a sample has such an expected polymorphism. It is.
  • each oligonucleotide primer in the present invention is 13 to 35 bases, preferably 16 to 30 bases.
  • the kit includes a primer (wild-type-specific primer and one or two or more polymorphism-specific primers) in which the second base from the 3 ′ end corresponds to a nucleotide predicted to be polymorphic, a reverse primer, DNA polymerase, containing nucleotides (dATP, dGTP, dTTP and dCTP).
  • nucleotide polymorphisms When two or more nucleotide polymorphisms are simultaneously detected, even if one kit contains the number of wild-type specific primers corresponding to the number of nucleotide polymorphisms and at least one polymorphism-specific primer Good. Two or more nucleotide polymorphisms are closely related to one disease, constitution, and the like, and a group of nucleotide polymorphisms that can determine the risk of a disease by detecting them at the same time is exemplified.
  • the same number of wild-type The total number of polymorphism-specific primers corresponding to the primers and each polymorphism site will be used. In this case, any combination of the wild-type specific primer and the polymorphic specific primer may be used.
  • a 19-base oligonucleotide having the base sequence of SEQ ID NO: 1 (hereinafter, referred to as primer 1), an oligonucleotide having the base sequence of SEQ ID NO: 2 (hereinafter, referred to as primer 1-2), and SEQ ID NO: 3
  • primer 1 an oligonucleotide having the nucleotide sequence shown in (1) (hereinafter, referred to as Primer 13) was obtained by requesting Japan Bioservices to synthesize.
  • Primer 1 has the nucleotide sequence at the second base from the 3 'end as a wild-type (C) nucleotide, has a 5' end labeled with 6 FAM, and primer 1-2 has the second base from the 3 'end. It has a nucleotide sequence of polymorphism (T), is labeled at the 5 'end with TAMRA, and primer 3 is a reverse primer paired with both primers 1 and 2.
  • the primer 1 has 19 bases and the primer 1 has 21 bases, and the primers 1 and 2 were designed so as to be able to detect the difference in the length of these 2 bases.
  • the difference in the number of base pairs between the two primers may be one or more, preferably one to three.
  • a 25 PL 1 solution containing the following reagents was prepared.
  • the obtained PCR product was diluted 10-fold, and the 11 was diluted with 19 1 Te immediate late Suppression Reagent (Applied Biosystems), followed by ice-cooling after 942 min of reaction. After centrifugation, the amplified product was detected using a Genetic Analyzer ABI PRISM310 from Applied Biosystems. As a result, the results shown in Figs. 1 to 3 below were obtained.
  • the genotype could be clearly determined by detecting the color and length of the label of the amplification product using a primer containing a polymorphic sequence at the second base from the 3 ′ end.
  • Example 2 Simultaneous Detection of Nucleotide Polymorphism of MTHFR (5, 10— Methylenetetrahydrofolate reductase) Gene and 2C19 Gene Polymorphism of Cytochrome P450
  • a 24-base oligonucleotide having the base sequence shown in SEQ ID NO: 4 (hereinafter referred to as primer 4), a 25-base oligonucleotide having the base sequence shown in SEQ ID NO: 5 (hereinafter referred to as primer 5), and SEQ ID NO:
  • An oligonucleotide having the nucleotide sequence shown in No. 6 (hereinafter, referred to as primer 6) was obtained by requesting Nippon Bioservice to synthesize it.
  • Primer 4 has a wild-type (G) nucleotide sequence at the second base from the 3 ′ end, has a 5 ′ end labeled with 6 FAM, and primer 5 has a base at the second base from the 3 ′ end. It has the nucleotide sequence of polymorphism (A), is labeled at the 5 'end with TAMRA, and primer 16 is a reverse primer paired with both primers 4 and 5.
  • G wild-type nucleotide sequence at the second base from the 3 ′ end
  • primer 5 has a base at the second base from the 3 ′ end. It has the nucleotide sequence of polymorphism (A), is labeled at the 5 'end with TAMRA, and primer 16 is a reverse primer paired with both primers 4 and 5.
  • the resulting PCR product was diluted 10-fold, and the 11 was diluted with 91 1 of Template Suppression Reagent (Applied Biosystems), and cooled with ice after 942 min of reaction. After centrifugation, the amplified products were detected with the Applied Biosystems Genetic Analyzer ABI PRISM310. As a result, the results shown in Figs. 4 to 6 below were obtained.
  • Amplification products were detected using a sample in which the polymorphism of the MTHFR gene was C / C and the genotype of 2C19 was GZG. As a result, 6 to 8 ⁇ 1 markers were assigned to 61 of the MTHFR gene. And a G-type peak labeled with 6 FAM at 77 bp of the 2C19 gene.
  • Amplification products were detected using a sample in which the polymorphism of the MTHFR gene was CZT and the genotype of 2C19 was GZA.
  • the 6FAM-labeled C-type peak at 61 bp of the MTHFR gene and the TAMRA-labeled T-type peak at 63 bp and the 6FAM-labeled G-type peak and 78 bp at 77 bp of the 2C19 gene at 63 bp.
  • Four of the type A peaks of TAMR A label were observed.
  • Amplification products were detected using a sample in which the polymorphism of the MTHFR gene was TZT and the genotype of 2C19 was AZA.
  • a T-type TAMRA-labeled peak at 63 bp of the MTHFR gene and a TAMRA-labeled A-type peak at 78 bp of the 2C19 gene were observed.
  • Example 2 is an example in which two types of gene polymorphisms are simultaneously detected. Similarly, by simultaneously using three or more types of primer sets (for example, primers 1, 2, and 3 constitute one primer set) Similarly, three or more nucleotide polymorphisms can be detected. However, in this case, if the number of bases of the amplification product detected by each primer set (for example, 61 bp and 63 bp when a primer set consisting of primers 1 to 3 is used) is different, the number of bases of the amplification product It is possible to detect nucleotide polymorphisms based on the difference in length.
  • primers 1, 2, and 3 constitute one primer set
  • three or more nucleotide polymorphisms can be detected. However, in this case, if the number of bases of the amplification product detected by each primer set (for example, 61 bp and 63 bp when a primer set consisting of primers 1 to 3 is used) is different, the number of bases of the a
  • the number of bases of the 61 bp (C type) amplification product is determined by primer 1 and primer-3, so the complementary position of reverse primer 3 may be shifted.
  • the number of bases of the amplification product can be easily adjusted.
  • polymorphism in a sample nucleic acid can be clearly and easily detected.
  • a method is provided.
  • the results can be obtained with good reproducibility even if the conditions of the gene amplification method are not so strict, and the judgment results do not differ depending on the type of the model.

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Abstract

L'invention concerne deux procédés. Un premier procédé permet d'identifier un polymorphisme nucléotidique contenu dans un échantillon d'acide nucléique, selon lequel il est prévu de traiter une séquence d'acide nucléique contenant un site de polymorphisme nucléotidique dans un échantillon simultanément avec ou séparément d'une amorce spécifique de type sauvage et d'une amorce spécifique de polymorphisme, de prolonger ces amorces par des polymérases d'ADN et de détecter les longueurs des produits ainsi prolongés de ces amorces ou de brins complémentaires desdites amorces. Un second procédé permet simultanément d'identifier au moins deux polymorphismes nucléotidiques contenus dans un échantillon d'acide nucléique, selon lequel il est prévu de traiter une séquence d'acide nucléique contenant au moins deux sites de polymorphismes nucléotidiques spécifiques dans un échantillon, simultanément avec ou séparément d'au moins deux amorces spécifiques de type sauvage et d'au moins deux amorces spécifiques de polymorphisme, correspondant respectivement aux sites de polymorphismes nucléotidiques, de prolonger ces amorces par des polymérases d'ADN, et de détecter les produits prolongés de ces amorces ou des brins complémentaires desdites amorces.
PCT/JP2001/010661 2000-12-07 2001-12-06 Procede d'identification de polymorphisme nucleotidique Ceased WO2002046393A1 (fr)

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Cited By (5)

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WO2004003199A1 (fr) * 2002-07-01 2004-01-08 Kankyo Engineering Co., Ltd. Methode d'obtention d'un acide nucleique ou d'un gene
WO2004061130A1 (fr) * 2002-12-27 2004-07-22 Toyo Boseki Kabushiki Kaisha Methode d'identification d'un polymorphisme nucleotidique
WO2017170101A1 (fr) * 2016-03-31 2017-10-05 積水メディカル株式会社 Procédé de détection d'une substitution de base unique en utilisant la chromatographie par échange d'ions
WO2019064483A1 (fr) * 2017-09-29 2019-04-04 積水メディカル株式会社 Procédé de détection d'une substitution de base unique à l'aide de la chromatographie par échange d'ions
CN116334256A (zh) * 2022-10-09 2023-06-27 中国食品发酵工业研究院有限公司 嗜热链球菌cicc 6038菌株的鉴定方法及其引物、试剂盒和应用

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JP5842811B2 (ja) * 2010-03-24 2016-01-13 凸版印刷株式会社 競合プライマーによる標的塩基配列の検出方法

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WO2004003199A1 (fr) * 2002-07-01 2004-01-08 Kankyo Engineering Co., Ltd. Methode d'obtention d'un acide nucleique ou d'un gene
WO2004061130A1 (fr) * 2002-12-27 2004-07-22 Toyo Boseki Kabushiki Kaisha Methode d'identification d'un polymorphisme nucleotidique
WO2017170101A1 (fr) * 2016-03-31 2017-10-05 積水メディカル株式会社 Procédé de détection d'une substitution de base unique en utilisant la chromatographie par échange d'ions
WO2019064483A1 (fr) * 2017-09-29 2019-04-04 積水メディカル株式会社 Procédé de détection d'une substitution de base unique à l'aide de la chromatographie par échange d'ions
CN109863245A (zh) * 2017-09-29 2019-06-07 积水医疗株式会社 使用离子交换层析的单碱基置换检测方法
US10626452B2 (en) 2017-09-29 2020-04-21 Sekisui Medical Co., Ltd. Method for detecting single base substitution using ion-exchange chromatography
KR20200057667A (ko) * 2017-09-29 2020-05-26 세키스이 메디칼 가부시키가이샤 이온 교환 크로마토그래피를 사용한 1염기 치환 검출 방법
JPWO2019064483A1 (ja) * 2017-09-29 2020-08-06 積水メディカル株式会社 イオン交換クロマトグラフィーを用いた一塩基置換検出方法
KR102389120B1 (ko) 2017-09-29 2022-04-20 세키스이 메디칼 가부시키가이샤 이온 교환 크로마토그래피를 사용한 1염기 치환 검출 방법
CN116334256A (zh) * 2022-10-09 2023-06-27 中国食品发酵工业研究院有限公司 嗜热链球菌cicc 6038菌株的鉴定方法及其引物、试剂盒和应用
CN116334256B (zh) * 2022-10-09 2023-11-10 中国食品发酵工业研究院有限公司 嗜热链球菌cicc 6038菌株的鉴定方法及其引物、试剂盒和应用

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