JP2007190028A - 核酸分子の等温増幅方法 - Google Patents

核酸分子の等温増幅方法 Download PDF

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JP2007190028A
JP2007190028A JP2007061113A JP2007061113A JP2007190028A JP 2007190028 A JP2007190028 A JP 2007190028A JP 2007061113 A JP2007061113 A JP 2007061113A JP 2007061113 A JP2007061113 A JP 2007061113A JP 2007190028 A JP2007190028 A JP 2007190028A
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nucleic acid
double
primer
molecules
disease
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Jeffrey I Auerbach
アイ． オーバック ジェフリー
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Replicon Inc
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Replicon Inc
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Priority claimed from US08/383,327 external-priority patent/US5591609A/en
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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
    • C12Q1/6862—Ligase chain reaction [LCR]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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    • 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
    • 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
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

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Abstract

【課題】伝染病の因果作因の特定、個人が遺伝病を患い易い確立の予測、飲料水やミルクの純度の測定、組織サンプルの特定など法医学、医学、疫学および公衆衛生ならびに疾病の予測および診断に極めて重要な分析法を等温条件下で、単一のプライマを用いて核酸分子を増幅する方法の提供。
【解決手段】ポリメラーゼ、ヌクレオチド、クレ組換え酵素、プライマー及び制限エンドヌクレアーゼの存在下で、線状二本鎖核酸をインキュベートし、サンプル中に存在する二本鎖核酸分子の対象ポリヌクレオチド領域を増幅する。
【選択図】なし

Description
したがって、本発明は以下を提供する：
図１は、適当な５′アダプタ分子の例を示す。 図２Ａ及び２Ｂ（図面Ａ、Ｂ、Ｃ及びＤを含む）は、適当な３′アダプタ分子の例を示す。 図２Ａ及び２Ｂ（図面Ａ、Ｂ、Ｃ及びＤを含む）は、適当な３′アダプタ分子の例を示す。 図３Ａ及び３Ｂは、プライマ伸長生成物の３′末端の適合を示す。図３Ａの線Ａ、Ｂ、Ｃは別のプライマ伸長を介したプライマ伸長生成物の３′末端を修飾するための異なるアダプタ分子の使用を示す。図３Ｂの線Ｄは、３′末端を修飾するための連結の使用を示す。 図３Ａ及び３Ｂは、プライマ伸長生成物の３′末端の適合を示す。図３Ａの線Ａ、Ｂ、Ｃは別のプライマ伸長を介したプライマ伸長生成物の３′末端を修飾するための異なるアダプタ分子の使用を示す。図３Ｂの線Ｄは、３′末端を修飾するための連結の使用を示す。 図４Ａ、４Ｂ、４Ｃ及び４Ｄは、組換え部位を含むアダプタ分子を用いて適合させた直鎖状分子からの二本鎖環状分子の形成を示す。 図４Ａ、４Ｂ、４Ｃ及び４Ｄは、組換え部位を含むアダプタ分子を用いて適合させた直鎖状分子からの二本鎖環状分子の形成を示す。 図４Ａ、４Ｂ、４Ｃ及び４Ｄは、組換え部位を含むアダプタ分子を用いて適合させた直鎖状分子からの二本鎖環状分子の形成を示す。 図４Ａ、４Ｂ、４Ｃ及び４Ｄは、組換え部位を含むアダプタ分子を用いて適合させた直鎖状分子からの二本鎖環状分子の形成を示す。 図５は、逆方向反復塩基配列を有する３′アダプタ分子とのプライマ伸長生成物の適合から形成したヘヤピンループ分子を示す。 図６は、一対の並置逆方向反復塩基配列を有する３′アダプタ分子とのプライマ伸長生成物の適合から形成した「ボータイ」分子を示す。 図７は、直接反復組換え部位を有するヘヤピンループ及び「ボータイ」分子の一本鎖環状分子への変換を示す。 図８Ａ及び８Ｂは、本発明の増幅レプリコンを示す。図８Ａは、一本鎖環状分子の増幅時に二つのプライマの伸長から生じる双起点「ローリングサークル」を示す。図８Ｂは、二本鎖環状分子の増幅から生じる「シータ」及び「ローリングサークル」レプリコンを示す。 図８Ａ及び８Ｂは、本発明の増幅レプリコンを示す。図８Ａは、一本鎖環状分子の増幅時に二つのプライマの伸長から生じる双起点「ローリングサークル」を示す。図８Ｂは、二本鎖環状分子の増幅から生じる「シータ」及び「ローリングサークル」レプリコンを示す。 図９は、例１に記載した例示的等温増幅反応の概略図である。 図１０、例１に記載した別の例示的等温増幅反応の概略図である。図は、プロト−ロックス部位の部分に相補的なプライマＩの５′第四領域の使用を示す。 図１１は、例２に示したような二本鎖環状分子を形成するための連結の使用の概略図である。図１１において、プロト−ロックス部位の部分に相補的なプライマＩの５′第四領域は、必要に応じて省略される。 図１２は、例２に示したように、二本鎖環状分子を形成するための連結の別の使用の概略図である。図１２において、プロト−ロックス部位の部分に相補的なプライマ１の５′第四領域は、必要に応じて省略される。 図１３は、非修飾プライマを使用してＤＮＡリガーゼを採用した、例４に記載の別の例示的等温増幅反応の概略図である。
（配列表）

Claims (1)

  1. 明細書中に記載されるサンプル中に存在する二本鎖核酸分子の対象ポリヌクレオチド領域を増幅する方法。
JP2007061113A 1995-02-03 2007-03-09 核酸分子の等温増幅方法 Pending JP2007190028A (ja)

Applications Claiming Priority (2)

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US08/383,327 US5591609A (en) 1992-08-04 1995-02-03 Methods for the isothermal amplification of nucleic acid molecules
US08/533,852 US5614389A (en) 1992-08-04 1995-09-26 Methods for the isothermal amplification of nucleic acid molecules

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US (1) US5614389A (ja)
EP (1) EP0807186B1 (ja)
JP (2) JPH10513058A (ja)
AT (1) ATE191934T1 (ja)
AU (1) AU4969996A (ja)
CA (1) CA2212185C (ja)
DE (1) DE69607830T2 (ja)
WO (1) WO1996023904A1 (ja)

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ATE191934T1 (de) 2000-05-15
EP0807186B1 (en) 2000-04-19
WO1996023904A1 (en) 1996-08-08
CA2212185C (en) 2008-01-22
AU4969996A (en) 1996-08-21
DE69607830T2 (de) 2000-11-30
DE69607830D1 (de) 2000-05-25
CA2212185A1 (en) 1996-08-08
US5614389A (en) 1997-03-25
JPH10513058A (ja) 1998-12-15
EP0807186A1 (en) 1997-11-19

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