WO2005038026A1 - Procede de typage d'une mutation - Google Patents

Procede de typage d'une mutation Download PDF

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Publication number
WO2005038026A1
WO2005038026A1 PCT/JP2004/015214 JP2004015214W WO2005038026A1 WO 2005038026 A1 WO2005038026 A1 WO 2005038026A1 JP 2004015214 W JP2004015214 W JP 2004015214W WO 2005038026 A1 WO2005038026 A1 WO 2005038026A1
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WO
WIPO (PCT)
Prior art keywords
dna
sequence
target dna
mutation
identification code
Prior art date
Application number
PCT/JP2004/015214
Other languages
English (en)
Japanese (ja)
Inventor
Nobuto Koyama
Junichi Mineno
Shusaku Yamashita
Nao Yamanaka
Masahiro Sato
Hiroyuki Izu
Masanori Takayama
Kiyozo Asada
Ikunoshin Kato
Original Assignee
Takara Bio Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takara Bio Inc. filed Critical Takara Bio Inc.
Publication of WO2005038026A1 publication Critical patent/WO2005038026A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • RFLP is analyzed by performing Southern hybridization using a probe at that site. be able to.
  • polymorphisms that can be detected as RFLP are not so frequently present in the genome, so they are not suitable for comprehensive analysis of detailed polymorphisms.
  • SNP is the most frequently found polymorphism, and exists in human at about 1 kb. SNP analysis is useful for understanding the relationship between the efficacy and side effects of diseases and pharmaceuticals and individual differences, and conducting more effective and safe medical treatment.SNP screening and typing are performed on a large scale worldwide. ing.
  • Mutation detection methods utilize a method for determining a DNA base sequence, a method based on hybridization, a method based on primer extension, a method based on ligation, and an enzyme recognizing a special DNA structure. It is roughly divided into methods.
  • Patent Document 3 US Pat. No. 5,660,988
  • Patent Document 4 U.S. Pat.No. 5210015
  • step (g) ligating the encoding adapter complementary to the protruding end generated in step (f) with the target DNA on the microbead
  • step (i) A step of removing the decoder probe from the microbeads in step (h) and using the resulting microbeads in step (f) and subsequent steps.
  • the adapters in each group are a mixture of 4 N_1 B-terminal adapters, and the entire adapter is 4 XN.
  • NX 4 consists of N types of arrays.
  • Preferred examples of the type lis restriction enzyme used in the “step of determining the base sequence of the target DNA fragment” include Bbvl. As described below, this enzyme recognizes the GCAGC sequence and generates a 5'-terminal with a 4-base overhang.
  • mutant includes various forms of mutation such as “base substitution”, “deletion mutation”, “insertion mutation” and the like.
  • a target DNA is prepared.
  • the method for preparing the target DNA is not particularly limited, for example, genomic DNA, cDNA, DNA amplified by nucleic acid amplification methods such as these PCR, ICAN, etc., and these DNAs as vectors, for example, plasmid vectors or Batatelio phage vectors, DNA, a mixture of cloned DNA, cloned DNA from which the vector portion has been removed by restriction enzyme digestion, etc., nucleases such as restriction enzymes, chemical treatment such as acid treatment, or Those subjected to physical treatment, for example, ultrasonic treatment, may be mentioned.
  • the method for preparing the target DNA which is not particularly limited to the chain length of the target DNA, the method for determining the sample identification code and the nucleotide sequence of the region containing the mutation site, the nucleotide sequence around the mutation site, etc. Just select.
  • the molecular weight of the target DNA can be 10 to 10,000 bases, preferably 15 to 5000 bases, and more preferably 20 to 1000 bases.
  • the range of the target DNA is set so that the mutation site to be typed is located in the target DNA.
  • the method may be appropriately determined by a method for determining the base sequence of the region including the sample identification code and the mutation site. Just choose!
  • the nucleotide sequence length that can be determined by this method is about 20 bases, and thus the sample identification code and the mutation to be typed It must be included in the range of 20 bases or less.
  • SAGE method a polymerization method similar to the Serial Analysis of Gene Expression method (hereinafter, referred to as SAGE method) can be used.
  • the SAGE method is a method in which a large amount of gene expression information can be obtained by one sequence determination by polymerizing DNA fragmented with a type lis restriction enzyme and determining its base sequence (U.S. Pat. Book, Vel culescu VE, and 3 others, Science, Vol. 270, 484-487, 1995.
  • type lis restriction enzymes that cleave sites that are 20 bases or more apart from the recognition sequence are known, so the sample identification code and the mutation site are included in the region of 20 bases or less. Need to be. Therefore, the mutation site may be located within 19 bases, preferably within 15 bases, more preferably within 10 bases, from the end of the target DNA to which the sample identification code is added.
  • the relationship between the primer sequence and the mutation site is not particularly limited.
  • it is desirable to design the primer so that the primer sequence does not include the mutation site for the following reasons. That is, when the primer sequence contains a mutation site, it is necessary to prepare a primer having a plurality of sequences corresponding to the type of mutation, and when the mutation site is located other than near the 3 ′ end of the primer, Since there is a possibility that amplification will occur with primers containing bases that are not complementary to type II, there is a risk that DNA that does not accurately reflect the type II base sequence will be amplified.
  • the start adapter has a type lis restriction enzyme recognition sequence, and is designed such that digestion with this enzyme makes the end of the target DNA a protruding end.
  • a type lis restriction enzyme recognition sequence By flowing a type lis restriction enzyme through a flow cell filled with beads and reacting with the DNA on the beads, the four bases at the end of the target DNA are made to be protruding ends.
  • the type lis restriction enzyme is not particularly limited as long as it generates a protruding end, but an enzyme generating a 4-base protruding end, for example, Bbvl can be suitably used.
  • Preferable examples of the encoder adapter and decoder probe used in the present invention include those described in Non-patent Document 4.
  • An annealed oligonucleotide shown, adapter GAC is an annealed oligonucleotide shown by SEQ ID NO: 7 in the sequence list and an oligonucleotide shown by SEQ ID NO: 8 in the sequence list, and an adapter CCC Sequence listing
  • the oligonucleotides represented by SEQ ID NO: 9 and the oligonucleotides represented by SEQ ID NO: 10 in the Sequence Listing are annealed.
  • the oligonucleotides represented by SEQ ID NOs: 2, 4, 6, 8, and 10 are phosphorylated at the 5 'end.
  • the tag library pool was subjected to PCR using a type I, FAM-labeled PCR-R primer (SEQ ID NO: 12) and a biotinylated PCR-F primer (SEQ ID NO: 13).
  • the part containing the tag was amplified. Operations are performed for each pool from this PCR to the first sorting, and in this example, one pool is described. After digesting the PCR product with Pad (manufactured by NEB), T4 DNA polymerase (manufactured by Takara Bio Inc.) was allowed to act in the presence of dGTP to convert the tag portion to single-stranded.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de typage d'une mutation, caractérisé en ce qu'il consiste à préparer un ADN cible contenant un site de mutation devant être typé, à fixer un code d'identification d'échantillon sur cet ADN cible, puis à déterminer la séquence de base d'une région contenant ce code d'identification d'échantillon et le site de mutation dans l'ADN cible sur lequel est fixé ledit code.
PCT/JP2004/015214 2003-10-16 2004-10-15 Procede de typage d'une mutation WO2005038026A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2003-356850 2003-10-16
JP2003356850 2003-10-16

Publications (1)

Publication Number Publication Date
WO2005038026A1 true WO2005038026A1 (fr) 2005-04-28

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Application Number Title Priority Date Filing Date
PCT/JP2004/015214 WO2005038026A1 (fr) 2003-10-16 2004-10-15 Procede de typage d'une mutation

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WO (1) WO2005038026A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007097443A1 (fr) * 2006-02-20 2007-08-30 National University Corporation Hokkaido University Procede de determination de la sequence des bases d'un adn

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002500050A (ja) * 1998-01-09 2002-01-08 リンクス セラピューティクス， インコーポレイテッド 差次的に発現された遺伝子の固相選択
WO2003002709A2 (fr) * 2001-06-21 2003-01-09 K.U. Leuven Research & Development Nouvelle technologie de cartographie genetique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002500050A (ja) * 1998-01-09 2002-01-08 リンクス セラピューティクス， インコーポレイテッド 差次的に発現された遺伝子の固相選択
WO2003002709A2 (fr) * 2001-06-21 2003-01-09 K.U. Leuven Research & Development Nouvelle technologie de cartographie genetique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KADERALI L. ET AL.: "Primer-design for multiplexed genotyping", NUCLEIC ACID RESEARCH, vol. 31, no. 6, 15 March 2003 (2003-03-15), pages 1796 - 1802, XP002996256 *
OYAMA N.: "DNA micro beads array gijutsu o mochiita idenshi hatsugen kaiseki", NIPPON NOGEI KAGAKU KAISHI, vol. 76, no. 5, 2002, pages 474 - 479, XP002996255 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007097443A1 (fr) * 2006-02-20 2007-08-30 National University Corporation Hokkaido University Procede de determination de la sequence des bases d'un adn
US7951536B2 (en) 2006-02-20 2011-05-31 National University Corporation Hokkaido University Method for determining base sequence of DNA

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