JP2014501529A - マルチプレックス配列決定反応における核酸鋳型の完全性および同定を維持するための方法 - Google Patents
マルチプレックス配列決定反応における核酸鋳型の完全性および同定を維持するための方法 Download PDFInfo
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Abstract
Description
本願は、2011年4月7日に出願された米国仮特許出願第13/081,660号(この米国仮特許出願は、2010年12月23日に出願された米国特許出願第61/426,817号の利益および優先権を主張する)の利益および優先権を主張し、これらの出願の各々の全体の内容は本明細書中に参考として援用される。
本発明は一般に、マルチプレックス配列決定反応における核酸鋳型の完全性および同定を維持するための方法に関する。
合成による配列決定は、ヌクレオチドの、鋳型/プライマー二重鎖への、鋳型に依存する付加を伴う。ヌクレオチドの付加はポリメラーゼ酵素により媒介され、付加されたヌクレオチドは、それらの検出を容易とするために標識されうる。個別のDNAまたはRNAについてのハイスループットの配列情報を得るために、単一分子の配列決定が用いられている。試料をマルチプレックス処理する、すなわち、異なる患者試料をプールする能力は、費用を軽減し、合成による配列決定プラットフォームのスループットを増大させるために重要である。
本発明は一般に、分子検出アッセイの結果を検証し、試料調製でもたらされる誤差の検出を可能とするための方法を提供する。本発明は、核酸の配列決定、タンパク質の検出、ならびに被検体の存在および/または量の正確な測定を必要とする他の方法に適用可能である。本発明は、対象の被検体が検出される場合に限り識別子が存在するように、対象の被検体と独自に(uniquely)結合する2つ以上の識別子を使用する。このようにして、検出される被検体と結合する2つ以上の独立マーカーの同時検出を必要とすることにより、偽陽性および/または偽陰性の結果を回避する。
本発明の方法は、マルチプレックス配列決定反応において核酸鋳型の完全性を検証し、維持することに関する。本発明の方法は、鋳型核酸を得るステップと、配列識別子の対を鋳型へと組み込むステップと、鋳型を配列決定するステップとを伴う。
核酸鋳型は、デオキシリボ核酸(DNA)および/またはリボ核酸(RNA)を包含する。核酸鋳型は、合成の場合もあり、天然に存在する供給源に由来する場合もあり、合成配列および天然配列の両方を包含する場合もあり、PCR産物を包含する場合もある。一実施形態では、核酸鋳型分子を、タンパク質、脂質、および鋳型以外の核酸など、他の多様な成分を含有する生物学的試料から単離する。核酸鋳型分子は、動物、植物、細菌、真菌、または他の任意の細胞生物から得られる任意の細胞材料から得ることができる。本発明において用いるための生物学的試料には、ウイルス粒子またはウイルス調製物が含まれる。核酸鋳型分子は、生物から直接得ることもでき、生物から得られる生物学的試料、例えば、血液、尿、脳脊髄液、精液、唾液、痰、糞便、および組織から得ることもできる。任意の組織標本または体液標本は、本発明において用いるための核酸の供給源として用いることができる。核酸鋳型分子はまた、初代細胞培養物または細胞系などの培養細胞からも単離することができる。そこから鋳型核酸を得る細胞または組織は、ウイルスまたは他の細胞内病原体に感染している可能性がある。試料はまた、生物学的標本から抽出される全RNAの場合もあり、cDNAライブラリーの場合もあり、ウイルスDNAの場合もあり、ゲノムDNAの場合もある。
特定の実施形態では、配列識別子は、核酸鋳型に付着しているかまたはこれに組み込まれているバーコード配列である。バーコード配列は、第1のバーコード配列を鋳型の5’端に付着させ、第2のバーコード配列を鋳型の3’端に付着させるように、鋳型に付着させることができる。第1および第2のバーコード配列は、同じ場合もあり、異なる場合もある。バーコード配列は、配列決定される標的を包含する鋳型の隣接領域へと組み込むことができる。
本発明の方法は、バーコード処理された核酸鋳型を、固体支持体に付着または固定化するステップを包含することができる。このような方法は、例えば、それらの各々の内容が参照によりその全体において本明細書に組み込まれる、Sabotら(米国特許出願第2009/0226975号)、Adessiら(米国特許第7,115,400号)、およびKawashimaら(米国特許出願第2005/0100900号)において記載されている。
特定の実施形態では、本発明の方法は、鋳型を配列決定するステップの前に、バーコード処理された核酸鋳型を増幅するステップを包含する。このような方法は、例えば、それらの各々の内容が参照によりその全体において本明細書に組み込まれる、Sabotら(米国特許出願第2009/0226975号)、Adessiら(米国特許第7,115,400号)、およびKawashimaら(米国特許出願第2005/0100900号)において記載されている。
5’[公知の配列I]−[バーコード配列]−[公知の配列II]−[標的のポリヌクレオチド配列]−[公知の配列III]−[バーコード配列]−[公知の配列IV]−3’
という構造をとるように、公知の配列の領域を有する。
本発明はまた、固相増幅により生成させた、増幅された核酸を配列決定する方法も包含する。したがって、本発明は、核酸を配列決定する方法であって、上記のとおり固相増幅を用いて核酸鋳型のプールを増幅するステップと、核酸の配列決定反応を実行して、固相増幅反応により生成させた、少なくとも1つの増幅された核酸鎖の全体または一部の配列を決定するステップとを含む方法を提供する。
特許、特許出願、特許公開、雑誌、書籍、論文、ウェブコンテンツなど、他の文献に対する参照および引用は、本開示の全体において行った。このような文献の全ては、全ての目的のために、参照によりそれらの全体において本明細書に組み込まれる。
本発明は、その精神またはその本質的な特徴から逸脱しない限りにおいて、他の特定の形態においても実施することができる。したがって、前出の実施形態は、全ての点において、本明細書で記載される本発明について、限定的であるものではなく、例示的であると考えられるものとする。
Claims (46)
- マルチプレックス配列決定反応において、核酸鋳型の完全性を検証するための方法であって、
鋳型核酸を得るステップと、
少なくとも2つの配列識別子を該鋳型へと組み込むステップと、
該鋳型を配列決定するステップと
を含む方法。 - 第1の識別子を、前記鋳型の5’部分へと組み込み、第2の識別子を、該鋳型の3’部分へと組み込む、請求項1に記載の方法。
- 前記識別子を組み込んだ後で、前記鋳型が、5’から3’の順で以下の構成:第1の公知の配列、第1のバーコード配列、第2の公知の配列、標的ポリヌクレオチド配列、第3の公知の配列、第2のバーコード配列、および第4の公知の配列、を有する、請求項2に記載の方法。
- 第1の識別子を、前記鋳型の5’端へと組み込み、第2の識別子を、該鋳型の3’端へと組み込む、請求項1に記載の方法。
- 前記第1および第2の識別子が同じである、請求項2に記載の方法。
- 前記第1および第2の識別子が異なる、請求項2に記載の方法。
- 配列決定するステップの前に、前記鋳型を基材に付着させる、請求項1に記載の方法。
- 前記鋳型を前記基材に直接的に付着させる、請求項2に記載の方法。
- 前記鋳型を前記基材に間接的に付着させる、請求項2に記載の方法。
- 前記鋳型を増幅するステップをさらに含む、請求項7に記載の方法。
- 配列決定するステップが、合成によって配列決定するステップである、請求項1に記載の方法。
- 前記合成によって配列決定するステップが、合成によって単一分子を配列決定するステップである、請求項11に記載の方法。
- 配列決定するステップが、
プライマーを前記鋳型とハイブリダイズさせて、鋳型/プライマーの二重鎖を形成するステップと、
少なくとも1つの検出可能に標識されたヌクレオチドの存在下、ポリメラーゼ酵素が鋳型依存的な様式で該プライマーにヌクレオチドを付加することを可能とする条件下で、該二重鎖を該ポリメラーゼと接触させるステップと、
組み込まれた標識ヌクレオチドからのシグナルを検出するステップと、
該接触させるステップおよび該検出するステップを少なくとも1回逐次的に反復するステップと
を含み、ここで、組み込まれた標識ヌクレオチドの逐次的な検出により、前記核酸の配列を決定する、請求項1に記載の方法。 - 前記検出可能に標識されたヌクレオチドが、光学的に標識されたヌクレオチドである、請求項13に記載の方法。
- 前記光学的に標識されたヌクレオチドが、蛍光標識されたヌクレオチドである、請求項14に記載の方法。
- 前記識別子の対が、前記鋳型に付着した第1のバーコード配列および第2のバーコード配列である、請求項1に記載の方法。
- 第1のバーコード配列を、前記鋳型の5’端に付着させ、第2のバーコード配列を、該鋳型の3’端に付着させる、請求項16に記載の方法。
- 前記第1のバーコード配列および前記第2のバーコード配列が同じである、請求項17に記載の方法。
- 前記第1のバーコード配列および前記第2のバーコード配列が異なる、請求項17に記載の方法。
- 核酸を解析するための方法であって、
核酸鋳型を得るステップと、
バーコード配列の対を該鋳型に付着させるステップと、
該鋳型を配列決定するステップと
を含む方法。 - 第1の識別子を、前記鋳型の5’部分へと組み込み、第2の識別子を、該鋳型の3’部分へと組み込む、請求項20に記載の方法。
- 前記バーコード配列の対を付着させた後で、前記鋳型が、5’から3’の順で以下の構成:第1の公知の配列、第1のバーコード配列、第2の公知の配列、標的ポリヌクレオチド配列、第3の公知の配列、第2のバーコード配列、および第4の公知の配列、を有する、請求項21に記載の方法。
- 第1の識別子を、前記鋳型の5’端へと組み込み、第2の識別子を、該鋳型の3’端へと組み込む、請求項20に記載の方法。
- 配列決定するステップの前に、前記鋳型を基材に付着させる、請求項20に記載の方法。
- 前記鋳型を前記基材に直接的に付着させる、請求項24に記載の方法。
- 前記鋳型を前記基材に間接的に付着させる、請求項24に記載の方法。
- 前記鋳型を増幅するステップをさらに含む、請求項24に記載の方法。
- 配列決定するステップが、合成によって配列決定するステップである、請求項20に記載の方法。
- 前記合成によって配列決定するステップが、合成によって単一分子を配列決定するステップである、請求項28に記載の方法。
- 配列決定するステップが、
プライマーを前記鋳型とハイブリダイズさせて、鋳型/プライマーの二重鎖を形成するステップと、
少なくとも1つの検出可能に標識されたヌクレオチドの存在下、ポリメラーゼ酵素が鋳型依存的な様式で該プライマーにヌクレオチドを付加することを可能とする条件下で、該二重鎖を該ポリメラーゼと接触させるステップと、
組み込まれた標識ヌクレオチドからのシグナルを検出するステップと、
該接触させるステップおよび該検出するステップを少なくとも1回逐次的に反復するステップと
を含み、ここで、組み込まれた標識ヌクレオチドの逐次的な検出により、前記核酸の配列を決定する、請求項20に記載の方法。 - 前記検出可能に標識されたヌクレオチドが、光学的に標識されたヌクレオチドである、請求項29に記載の方法。
- 前記光学的に標識されたヌクレオチドが、蛍光標識されたヌクレオチドである、請求項31に記載の方法。
- 前記第1のバーコード配列および前記第2のバーコード配列が同じである、請求項23に記載の方法。
- 前記第1のバーコード配列および前記第2のバーコード配列が異なる、請求項23に記載の方法。
- 核酸を解析するための方法であって、
核酸鋳型を得るステップと、
第1のバーコード配列を該鋳型の5’部分に付着させ、第2のバーコード配列を該鋳型の3’部分に付着させるステップと、
該鋳型を増幅するステップと、
該鋳型を配列決定するステップと
を含む方法。 - 前記第1のバーコード配列および前記第2のバーコード配列を付着させた後で、前記鋳型が、5’から3’の順で以下の構成:第1の公知の配列、第1のバーコード配列、第2の公知の配列、標的ポリヌクレオチド配列、第3の公知の配列、第2のバーコード配列、および第4の公知の配列、を有する、請求項35に記載の方法。
- 第1の識別子を、前記鋳型の5’端へと組み込み、第2の識別子を、該鋳型の3’端へと組み込む、請求項35に記載の方法。
- 増幅するステップの前に、前記鋳型を固体支持体に固定化する、請求項35に記載の方法。
- 前記第1のバーコード配列および前記第2のバーコード配列が同じである、請求項35に記載の方法。
- 前記第1のバーコード配列および前記第2のバーコード配列が異なる、請求項35に記載の方法。
- 配列決定するステップが、
プライマーを前記鋳型とハイブリダイズさせて、鋳型/プライマーの二重鎖を形成するステップと、
少なくとも1つの検出可能に標識されたヌクレオチドの存在下、ポリメラーゼ酵素が鋳型依存的な様式で該プライマーにヌクレオチドを付加することを可能とする条件下で、該二重鎖を該ポリメラーゼと接触させるステップと、
組み込まれた標識ヌクレオチドからのシグナルを検出するステップと、
該接触させるステップおよび該検出するステップを少なくとも1回逐次的に反復するステップと
を含み、ここで、組み込まれた標識ヌクレオチドの逐次的な検出により、前記核酸の配列を決定する、請求項35に記載の方法。 - 被検体の検出を検証するための方法であって、該被検体と独自に結合する2つ以上の識別子の存在を検出するステップを含む方法。
- 前記識別子が核酸配列タグである、請求項42に記載の方法。
- 前記配列タグが、前記被検体に結合する部分と結合する、請求項43に記載の方法。
- 前記被検体がタンパク質である、請求項42に記載の方法。
- 前記識別子がペプチドである、請求項45に記載の方法。
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201061426817P | 2010-12-23 | 2010-12-23 | |
US61/426,817 | 2010-12-23 | ||
US13/081,660 US9163281B2 (en) | 2010-12-23 | 2011-04-07 | Methods for maintaining the integrity and identification of a nucleic acid template in a multiplex sequencing reaction |
US13/081,660 | 2011-04-07 | ||
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