JPH08505535A - 一本鎖dna分子の生成方法 - Google Patents
一本鎖dna分子の生成方法Info
- Publication number
- JPH08505535A JPH08505535A JP6516386A JP51638694A JPH08505535A JP H08505535 A JPH08505535 A JP H08505535A JP 6516386 A JP6516386 A JP 6516386A JP 51638694 A JP51638694 A JP 51638694A JP H08505535 A JPH08505535 A JP H08505535A
- Authority
- JP
- Japan
- Prior art keywords
- primer
- molecule
- nucleic acid
- exonuclease
- nucleotide
- Prior art date
- Legal status (The legal status 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 status listed.)
- Granted
Links
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- 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
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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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- 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
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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- 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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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
Description
Claims (1)
- 【特許請求の範囲】 1.相補的な配列の核酸分子を実質的に含まない、所望の一本鎖核酸分子の生 成方法であって、下記工程: (A)前以て選択した核酸分子をプライマー分子の存在下でインキュベートし 、その際、該プライマー分子は該前以て選択した分子にハイブリダイズすること ができ、該プライマー分子は5'→3’エキソヌクレアーゼに対して耐性の領域 を含み; (B)鋳型に依存した該プライマーの伸長を行って該所望の核酸分子を生成さ せ;ついで (C)該前以て選択した分子を除去するに充分な条件下で該インキュベーショ ン液に5'→3’エキソヌクレアーゼを加え、それにより相補的な配列の核酸分 子を実質的に含まない該所望の一本鎖分子を得る からなることを特徴とする方法。 2.工程(B)において、所望の核酸分子を生成した後に、該分子にハイブリ ダイズすることができ鋳型に依存した仕方で伸長しうる第二のプライマー分子の 存在下で該分子をインキュベートし、それにより該所望の分子の配列に実質的に 相補的な配列を有する核酸分子を生成させる、請求項1に記載の方法。 3.工程(B)において、所望の核酸分子を生成した後に、該分子にハイブリ ダイズすることができる第二のプライマー分子の存在下、少なくとも一つのジデ オキシヌクレオチド誘導体とともに、非停止性のデオキシヌクレオチド誘導体の 不在下で該分子をインキュベートし、その際、該インキュベーションを鋳型に依 存した仕方での該プライマーの伸長を可能とするに充分な条件下で行う、請求項 1に記載の方法。 4.該プライマーが約10〜約30ヌクレオチドの長さを有する、請求項1に 記載の方法。 5.該プライマーが約20〜約25ヌクレオチドの長さを有する、請求項4に 記載の方法。 6.5'→3’エキソヌクレアーゼに対して耐性である該領域のエキソヌクレ アーゼ耐性が複数のホスホロチオエートヌクレオチド誘導体によってもたらされ る、請求項1に記載の方法。 7.該領域が約4つのホスホロチオエートヌクレオチド誘導体を含む、請求項 6に記載の方法。 8.該プライマーを検出可能なように標識してある、請求項1に記載の方法。 9.該プライマー分子に5'→3’エキソヌクレアーゼ耐性を付与する該ヌク レオチドが検出可能なように標識してある、請求項8に記載の方法。 10.該検出可能な標識が、酵素標識、蛍光標識、放射性同位体標識、および 化学ルミネセンス標識よりなる群から選ばれたものである、請求項7に記載の方 法。 11.該5'→3’エキソヌクレアーゼがT7遺伝子6エキソヌクレアーゼで ある、請求項1に記載の方法。 12.該5'→3’エキソヌクレアーゼがλエキソヌクレアーゼである、請求 項1に記載の方法。 13.該所望の一本鎖核酸分子が、鋳型に依存したプライマー伸長の間に標識 ヌクレオチドを導入することによって検出可能なように標識される、請求項1に 記載の方法。 14.該検出可能な標識が、酵素標識、蛍光標識、放射性同位体標識、および 化学ルミネセンス標識よりなる群から選ばれたものである、請求項13に記載の 方法。 15.約10〜約30ヌクレオチドの長さを有し5'→3’エキソヌクレアー ゼ耐性を付与する領域を含有するプライマー分子にハイブリダイズした標的核酸 分子を含む組成物。 16.5'→3’エキソヌクレアーゼ耐性を付与する該領域が約4つのホスホ ロチオエート結合を含む、請求項15に記載の組成物。 17.該プライマー分子が固相支持体に結合している、請求項15に記載の組 成物。 18.目的核酸中の特定の位置におけるヌクレオチド塩基を同定する方法であ っ て、 (A)目的核酸が二本鎖である場合には、該特定の位置を包含する不対ヌクレオ チド塩基を得るために該核酸を含有する試料を処理し、または該目的核酸が一本 鎖である場合は直接工程(B)を用い、その際、該目的核酸は該目的核酸の所定 の領域に5'→3’エキソヌクレアーゼ耐性を付与するに充分な数の5'→3’エ キソヌクレアーゼ耐性ヌクレオチド誘導体を含有し、 (B)工程(A)からの試料を、ハイブリダイズ条件下、同定しようとするヌク レオチド塩基の直ぐ隣に目的核酸中に存在するヌクレオチド塩基の連なりとハイ ブリダイズしうるオリゴヌクレオチドプライマーと接触させて該プライマーと該 目的核酸との間に二本鎖を生成させることにより、同定しょうとするヌクレオチ ド塩基が該二本鎖中のプライマーの3’末端の直ぐ下流の鋳型中の最初の不対塩 基となるようにし、 (C)工程(B)からの二本鎖を、dATP、dCTP、dGTPまたはdTT Pの実質的不在下、少なくとも2つの異なるヌクレオチド三リン酸誘導体と接触 させ、該誘導体は該最初の不対塩基に相補的な誘導体を含み、核酸鋳型に依存し たプライマー伸長反応のターミネーターであり、その際、該ターミネーターの少 なくとも一つは検出可能なマーカーで標識されており、該接触を該相補的ターミ ネーター誘導体と該最初の不対塩基との塩基対形成を可能とするに充分な条件下 で行い、 (D)該相補的ターミネーター誘導体をプライマーの3’末端中に導入するに充 分な鋳型依存プライマー伸長反応を起こさせ、 (E)該導入された誘導体を同定し、それにより該目的核酸中の特定の位置にお けるヌクレオチド塩基を同定する ことを特徴とする方法。 19.工程(C)において、工程(B)からの二本鎖を4つのターミネーター と接触させ、その際、これらターミネーターのうちの一つのみが検出可能なマー カーを有し、工程(C)を4回行い、これらターミネーターの異なる一つか各回 において標識されている、請求項18に記載の方法。 20.工程(C)において、工程(B)からの二本鎖を4つの標識したターミ ネーターと接触させ、これらターミネーターがそれぞれ異なる検出可能な標識を 有する、請求項18に記載の方法。 21.複製連鎖反応の所望のエキソヌクレアーゼ耐性増幅生成物の検出方法で あって、 (A)2つのプライマー分子を用いて複製連鎖反応を行い、その際、該プライマ ー分子の一つは該プライマーの5’末端に充分な数(最も好ましくは約4)のホ スホロチオエートヌクレオチド誘導体を含むことによって該末端に5'→3’エ キソヌクレアーゼに対する耐性が付与され、該反応は二本鎖増幅生成物を生成す るに充分なものであり、 (B)ついで、オリゴヌクレオチドにエキソヌクレアーゼに対する耐性を付与す る充分な数のホスホロチオエート結合を欠くオリゴヌクレオチドを分解する条件 下、増幅生成物を5'→3’エキソヌクレアーゼで処理し、 (C)固体支持体に結合した相補的オリゴヌクレオチドに複製連鎖反応の所望の 増幅生成物をハイブリダイズさせることにより、複製連鎖反応の所望の増幅生成 物を検出する ことを特徴とする方法。 22.該充分な数のホスホロチオエートヌクレオチド誘導体が約4である、請 求項21に記載の方法。 23.複製連鎖反応間での交差汚染を最小にする方法であって、該反応のプラ イマー分子の少なくとも一つは該プライマーの3’末端に充分な数(すなわち、 約4)のホスホロチオエートヌクレオチド誘導体を含むことによって5'→3’ エキソヌクレアーゼに対する耐性が付与され、該複製連鎖反応を行った後、該反 応の増幅生成物を該5'→3’エキソヌクレアーゼの存在下、充分な数のホスホ ロチオエート結合を欠く未使用のプライマーのオリゴヌクレオチド領域および増 幅生成物の分解を起こさせるに充分な条件下でインキュベートし、該分解により 該未使用のプライマーおよび該増幅生成物をさらなる複製連鎖反応において基質 として働き得ないようにさせ、それにより複製連鎖反応間での交差汚染を最小に することを特徴とする方法。 24.該充分な数のホスホロチオエートヌクレオチド誘導体が約4である、請 求項23に記載の方法。 25.密接な区画において、第一のプライマーを入れた第一の容器(該第一の プライマーはホスホロチオエートヌクレオチド誘導体を含む)と、ホスホロチオ エートヌクレオチド誘導体を欠く第二のプライマーを入れた第二の容器とを含み 、これら2つのプライマーを前以て決定した遺伝子配列を増幅するのに用いるこ とができるように特別に適合させたキット。 26.さらに5'→3’エキソヌクレアーゼを含む、請求項25に記載のキッ ト。
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US506193A | 1993-01-15 | 1993-01-15 | |
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US08/155,746 US5518900A (en) | 1993-01-15 | 1993-11-23 | Method for generating single-stranded DNA molecules |
US08/155,746 | 1993-11-23 | ||
PCT/US1994/000771 WO1994016090A1 (en) | 1993-01-15 | 1994-01-18 | Method for generating single-stranded dna molecules |
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- 1994-01-18 DE DE69432586T patent/DE69432586T2/de not_active Expired - Lifetime
- 1994-01-18 AT AT94907855T patent/ATE239090T1/de not_active IP Right Cessation
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JP2011512153A (ja) * | 2008-02-20 | 2011-04-21 | ジーン ブリッジズ ゲーエムベーハー | 核酸組換え法 |
JP2017518048A (ja) * | 2014-06-05 | 2017-07-06 | キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング | Dna増幅反応の最適化 |
Also Published As
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EP0679190A1 (en) | 1995-11-02 |
ATE239090T1 (de) | 2003-05-15 |
EP0679190B1 (en) | 2003-05-02 |
EP0679190A4 (en) | 1998-07-01 |
JP3330946B2 (ja) | 2002-10-07 |
WO1994016090A1 (en) | 1994-07-21 |
JP3421664B2 (ja) | 2003-06-30 |
JP2002209594A (ja) | 2002-07-30 |
AU674211B2 (en) | 1996-12-12 |
DE69432586T2 (de) | 2004-03-25 |
AU6126294A (en) | 1994-08-15 |
CA2153898A1 (en) | 1994-07-21 |
DE69432586D1 (de) | 2003-06-05 |
US5518900A (en) | 1996-05-21 |
CA2153898C (en) | 2003-10-28 |
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