JP2014504153A - 核酸合成のための方法およびデバイス - Google Patents
核酸合成のための方法およびデバイス Download PDFInfo
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- JP2014504153A JP2014504153A JP2013538904A JP2013538904A JP2014504153A JP 2014504153 A JP2014504153 A JP 2014504153A JP 2013538904 A JP2013538904 A JP 2013538904A JP 2013538904 A JP2013538904 A JP 2013538904A JP 2014504153 A JP2014504153 A JP 2014504153A
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Abstract
Description
本願は、2010年11月12日に出願された米国仮出願第61/412,937号、2010年11月30日に出願された米国仮出願第61/418,095号、2011年3月23日に出願された米国仮出願第61/466,814号、および2011年7月1日に出願された米国仮出願第61/503,722号の優先権およびそれらからの利益を主張し、それらの各々は出典明示によりその全文が本明細書に組み込まれる。
本明細書において提供される方法および装置は、予め定義された配列を有する核酸および核酸ライブラリーの合成およびアセンブリーに関する。より詳しくは、固体支持体上での標的ポリヌクレオチドの合成のため、および標的ポリヌクレオチドの選択のための方法および装置が提供される。
自然界から複製および増幅され、次いで、成分部分に分解されるDNA配列に、組換えDNA化学の技術を使用することは、今では一般的なことである。次いで、配列は、成分部分として、新規DNA配列に組み換えられるか、または再アセンブルされる。しかし、天然に入手可能な配列に対する確実性のために、研究者によって探索され得る可能性は大きく制限される。今では、短いDNA配列が、個々のヌクレオシドから直接合成されることは可能であるが、一般には、大きなセグメントまたはポリヌクレオチドのアセンブリー、すなわち、約400塩基対より長いポリヌクレオチド配列を直接構築することは非実用的なことであった。
本発明の態様は、高忠実度ポリマーを調製および/またはアセンブルするための方法および装置に関する。また、核酸アセンブリー反応を処理し、核酸をアセンブルするためのデバイスおよび方法も本明細書において提供される。カスタム核酸を合成する実用的な、経済的な方法を提供することが本発明の目的である。
X〜d x(C[スペーサー]/C[アンカー1+アンカー2])
[式中、dは、2種の核酸分子間の距離であり、C[スペーサー]は、スペーサーオリゴヌクレオチドの濃度であり、C[アンカー1+アンカー2]は、アンカーオリゴヌクレオチド1およびアンカーオリゴヌクレオチド2の混合物の濃度である]
を使用して設定される。
本発明は、中でも、高忠実度遺伝子アセンブリーのための新規方法およびデバイスを提供する。対象発明の特定の実施形態が論じられているが、上記の明細書は例示であって、制限ではない。本発明の多数の等価物は、本明細書を再検討すると当業者には明らかとなろう。本発明の全範囲は、特許請求の範囲を等価物のその全範囲とともに、本明細書をこのような変法と一緒に参照することによって決定されなければならない。
「Methods and Devices for Nucleic Acids Synthesis」と題された、2010年11月12日に出願された、米国仮出願第61/412,937号、「Methods and Devices for Nucleic Acids Synthesis」と題された、2010年11月30日に出願された米国仮出願第61/418,095号および「Methods and Devices for Nucleic Acids Synthesis」と題された、2011年3月23日に出願された米国仮出願第61/466,814号、2009年8月27日に出願されたPCT出願PCT/US2009/55267 、2010年11月3日に出願されたPCT出願PCT/US2010/055298 および2010年11月19日に出願されたPCT出願PCT/US2010/057405が参照される。本明細書において言及されたすべての刊行物、特許および配列データベースの項目は、個々の刊行物または特許が各々、具体的に、また参照により組み込まれるよう個々に指示されるように、出典明示によりその全文が本明細書に組み込まれる。
Claims (65)
- 予め定義された配列を有する少なくとも1種のポリヌクレオチドを製造する方法であって、
a.少なくとも第1および第2の複数の支持体結合一本鎖オリゴヌクレオチドを提供すること、ここで、第1および第2の複数のオリゴヌクレオチドの各々が、予め定義された配列を有し、支持体の別個の機構と結合しており、第1の複数のオリゴヌクレオチドの各々が、その3’末端に、第2の複数のオリゴヌクレオチドの3’末端の配列領域と相補的である配列領域を含む;
b.鎖伸長反応において、第1および第2の複数の支持体結合オリゴヌクレオチドと相補的である少なくとも第1および第2の複数の構築オリゴヌクレオチドを作製すること;
c.選択された機構で、複数の支持体結合アンカー一本鎖オリゴヌクレオチドを提供すること、ここで、複数の第1のアンカーオリゴヌクレオチドの5’末端が、第1の複数の支持体結合オリゴヌクレオチドの配列領域と同一である;
d.少なくとも第1および第2の複数の構築オリゴヌクレオチドを、複数のアンカーオリゴヌクレオチドとハイブリダイズすること;および
e.少なくとも第1および第2の複数の構築オリゴヌクレオチドをライゲーションし、それによって少なくとも1種のポリヌクレオチドを作製すること、
を含む、方法。 - 少なくとも第1および第2の複数の構築オリゴヌクレオチドが、少なくとも第1および第2の複数の支持体結合オリゴヌクレオチドから解離される、請求項1に記載の方法。
- 第1の複数の構築オリゴヌクレオチドを、第1の機構から選択された機構へ移すことと、第2の複数の構築オリゴヌクレオチドを、第2の機構から選択された機構へ移すこととをさらに含み、ここで、選択された機構が、複数の支持体結合アンカー一本鎖オリゴヌクレオチドを含む、請求項2に記載の方法。
- 選択された機構が、第1および第2の機構と同一支持体上にある、請求項3に記載の方法。
- 選択された機構が、第1および第2の機構と異なる支持体上にある、請求項4に記載の方法。
- さらに、
第3の複数の支持体結合一本鎖オリゴヌクレオチドを提供すること、ここで、第3の複数のオリゴヌクレオチドの各々が、予め定義された配列を有し、支持体の第3の別個の機構と結合しており、第3の複数のオリゴヌクレオチドの各々が、その3’末端に、第2の複数のオリゴヌクレオチドの3’末端の配列領域と相補的である配列領域を含む;
鎖伸長反応において、第3の複数の支持体結合オリゴヌクレオチドと相補的である第3の複数の構築オリゴヌクレオチドを作製すること;
選択された機構で、少なくとも第1、第2および第3の複数の構築オリゴヌクレオチドを、複数のアンカーオリゴヌクレオチドとハイブリダイズすること;および
少なくとも第1、第2および第3の複数の構築オリゴヌクレオチドをライゲーションし、それによって、少なくとも1種のポリヌクレオチドを製造すること、
を含む、請求項1に記載の方法。 - 少なくとも第3の複数のオリゴヌクレオチドを使用して、ステップb)からe)をさらに逐次反復し、複数のオリゴヌクレオチドの各々が、その3’末端に、次の複数のオリゴヌクレオチドの3’末端の配列領域と相補的である配列領域を含み、それによって、少なくとも1種のポリヌクレオチドを製造する、請求項1に記載の方法。
- 複数の支持体結合オリゴヌクレオチドの各々が、その3’末端にプライマー結合部位を有する、請求項1に記載の方法。
- プライマー結合部位が、ユニバーサルプライマー結合部位である、請求項8に記載の方法。
- プライマーの伸長を促進する条件下で、プライマーを、少なくとも第1および第2の複数の支持体結合オリゴヌクレオチドとアニーリングし、それによって伸長生成物二本鎖を形成することをさらに含む、請求項8に記載の方法。
- プライマー配列が、少なくとも1個のウラシルを含む、請求項10に記載の方法。
- 複数の支持体結合オリゴヌクレオチドが、固体支持体上で合成され、スポットされる、請求項1に記載の方法。
- 複数の支持体結合オリゴヌクレオチドが、その3’末端によって支持体上に固定化されている、請求項1に記載の方法。
- 支持体が、マイクロアレイデバイスである、請求項1に記載の方法。
- 少なくとも1つの機構が、プライマー伸長を促進する条件に付される、請求項11に記載の方法。
- N個の複数の予め定義された支持体結合一本鎖オリゴヌクレオチドを提供すること、ここで、第1の複数のオリゴヌクレオチドが、その3’末端に、第2のオリゴヌクレオチドの3’末端の配列領域と相補的である配列領域を含み、複数のオリゴヌクレオチドNが、その3’末端に、複数のオリゴヌクレオチド(N−1)の配列領域と相補的である配列領域を含む;および
その5’末端に、第1の複数の支持体結合オリゴヌクレオチドの配列領域と同一である配列を含む複数のアンカーオリゴヌクレオチドを提供すること、
を含む、請求項1に記載の方法。 - 支持体結合一本鎖オリゴヌクレオチドと相補的であるN個の複数の構築オリゴヌクレオチドが作製され、N個の複数の構築オリゴヌクレオチドが、ポリヌクレオチドの全配列にまたがり、ギャップを含まない、請求項16に記載の方法。
- 第1の複数の支持体結合オリゴヌクレオチドの3’末端配列領域が、アンカーオリゴヌクレオチドの5’末端領域と同一である、請求項15に記載の方法。
- 伸長二本鎖をさらに解離し、それによって、少なくとも第1および第2の複数の構築オリゴヌクレオチドを放出する、請求項10に記載の方法。
- ウラシルDNAグリコシラーゼ(UDG)およびDNAグリコシラーゼ−リアーゼエンドヌクレアーゼVIIIの混合物を使用して、プライマーをさらに除去する、請求項11に記載の方法。
- 予め定義された配列を有するポリヌクレオチドを作製する方法であって、
a.遊離一本鎖オーバーハングを含む複数の支持体結合二本鎖ポリヌクレオチドを合成し、複数のポリヌクレオチド配列が予め定義されたポリヌクレオチド配列を含むこと、ここで、一本鎖オーバーハングが、末端構築オリゴヌクレオチドの配列Nを含む;
b.一本鎖オーバーハングを含むステム−ループオリゴヌクレオチドを提供すること、ここで、一本鎖オーバーハングが、末端構築オリゴヌクレオチド配列Nと相補的である;
c.ステム−ループオリゴヌクレオチドを、予め定義された配列を有するポリヌクレオチドの遊離オーバーハングとハイブリダイズすること;
d.ステム−ループオリゴヌクレオチドを、予め定義された配列を有するポリヌクレオチドの遊離オーバーハングとライゲーションすること;および
e.末端構築オリゴヌクレオチド配列Nを含まないポリヌクレオチド配列を分解すること、
を含む、方法。 - N個の複数のオリゴヌクレオチドを含むオリゴヌクレオチドのプールを、アンカー支持体結合一本鎖オリゴヌクレオチドとハイブリダイズすることを含む請求項21に記載の方法であって、プール中の第1の複数のオリゴヌクレオチドが、その5’末端に、アンカーオリゴヌクレオチドの5’末端の配列領域と相補的である配列領域を含み、かつ複数のオリゴヌクレオチドNが、その3’末端に、複数のオリゴヌクレオチド(N−1)の配列領域と相補的である配列を含む、方法。
- 一本鎖特異的エキソヌクレアーゼを使用して、末端オリゴヌクレオチド配列を含まないポリヌクレオチド配列をさらに分解する、請求項21に記載の方法。
- エキソヌクレアーゼが、一本鎖特異的3’エキソヌクレアーゼ、一本鎖特異的エンドヌクレアーゼおよび一本鎖特異的5’エキソヌクレアーゼからなる群から選択される、請求項23に記載の方法。
- ステム−ループオリゴヌクレオチドが、II型制限部位を含み、II型制限エンドヌクレアーゼを使用してステム−ループオリゴヌクレオチドをさらに除去する、請求項22に記載の方法。
- ステム−ループオリゴヌクレオチドが、少なくとも1個のウラシルヌクレオチドを含み、ウラシルDNAグリコシラーゼ(UDG)およびDNAグリコシラーゼ−リアーゼエンドヌクレアーゼVIIIの混合物ステム−ループオリゴヌクレオチドをさらに除去する、請求項22に記載の方法。
- 予め定義されたポリヌクレオチド配列をさらに増幅する、請求項21に記載の方法。
- 複数の支持体結合二本鎖ポリヌクレオチドが、ポリメラーゼ鎖伸長によって支持体上で合成される、請求項21に記載の方法。
- 支持体からポリヌクレオチドをさらに放出する、請求項1に記載の方法。
- ポリヌクレオチドが、II型制限酵素を使用して放出される、請求項29に記載の方法。
- ポリヌクレオチドが、ウラシルDNAグリコシラーゼ(UDG)およびDNAグリコシラーゼ−リアーゼエンドヌクレアーゼVIIIの混合物を使用して放出される、請求項29に記載の方法。
- 予め定義された配列を有するポリヌクレオチドを合成する方法であって、
a.(i)5’末端が第1の複数のオリゴヌクレオチドの5’末端と相補的である、第1の複数の支持体結合アンカーオリゴヌクレオチドを有し、かつ(ii)5’末端が末端構築オリゴヌクレオチドNと相補的である、第2の複数の支持体結合アンカーオリゴヌクレオチドを有する、支持体を提供すること;
b.5’一本鎖オーバーハングを含む複数の支持体結合二本鎖ポリヌクレオチドを合成すること、ここで、複数のポリヌクレオチド配列が予め定義されたポリヌクレオチド配列を含み、予め定義されたポリヌクレオチド配列の一本鎖5’オーバーハングが、末端構築オリゴヌクレオチドN配列を含み、ポリヌクレオチド配列の一本鎖3’末端が、第1のオリゴヌクレオチド配列を含む;
c.複数の合成されたポリヌクレオチドを、第1のアンカーオリゴヌクレオチドとハイブリダイズすること;および
d.合成されたポリヌクレオチドをハイブリダイゼーション条件に付し、それによって、末端構築オリゴヌクレオチドNを有するポリヌクレオチドの5’オーバーハングを、第2の複数のアンカーオリゴヌクレオチドの5’末端とハイブリダイズし、それによって、予め定義された配列を有するポリヌクレオチドを選択すること、
を含む、方法。 - 遊離3’または5’末端を有するポリヌクレオチド配列をさらに分解する、請求項32に記載の方法。
- ポリヌクレオチド配列が、エキソヌクレアーゼを使用して分解される、請求項33に記載の方法。
- 支持体から予め定義された配列を有するポリヌクレオチドをさらに放出する、請求項32に記載の方法。
- ポリヌクレオチドが、II型エンドヌクレアーゼを使用して放出される、請求項35に記載の方法。
- ポリヌクレオチドが、ウラシルDNAグリコシラーゼ(UDG)およびDNAグリコシラーゼ−リアーゼエンドヌクレアーゼVIIIの混合物を使用して放出される、請求項35に記載の方法。
- 第1の複数のアンカーオリゴヌクレオチドが、第2の複数のアンカーオリゴヌクレオチドから、予め定義されたポリヌクレオチドの長さに対応する距離だけ離れている、請求項32に記載の方法。
- 支持体が、第1および第2のアンカーオリゴヌクレオチド間の距離を設定する支持体結合スペーサー一本鎖オリゴヌクレオチドをさらに含む、請求項38に記載の方法。
- 第1および第2のアンカーオリゴヌクレオチド間の距離が、第1および第2のアンカーオリゴヌクレオチドの濃度およびスペーサーオリゴヌクレオチドの濃度の関数である、請求項39に記載の方法。
- 複数の支持体結合二本鎖ポリヌクレオチドが、複数の支持体結合一本鎖オリゴヌクレオチドを鋳型として使用するポリメラーゼ鎖伸長によって合成される、請求項32に記載の方法。
- a.固体支持体;
b.固体支持体と関連している複数の別個の機構であって、各機構が、予め定義された配列を有する複数の支持体結合オリゴヌクレオチドを含み、第1の複数のオリゴヌクレオチドが、その5’末端に、第2のオリゴヌクレオチドの5’末端の配列領域と相補的である配列領域を含み、複数のオリゴヌクレオチドNが、その5’末端に、複数のオリゴヌクレオチド(N−1)の5’末端配列領域と相補的である配列を含む機構;および
c.その5’末端に、第1の複数の支持体結合オリゴヌクレオチドの配列領域と同一である配列を含む、少なくとも第1の複数のアンカーオリゴヌクレオチド、
を含む、核酸アレイ。 - 第2の複数の支持体結合アンカーオリゴヌクレオチドをさらに含み、第2のアンカーオリゴヌクレオチドの5’末端が、複数のオリゴヌクレオチドNの5’末端と同一である、請求項42に記載の核酸アレイ。
- 複数の、複数の支持体結合オリゴヌクレオチドと同一ではない配列を有する、複数の支持体結合オリゴヌクレオチドをさらに含む、請求項42に記載の核酸。
- 予め定義された配列を有する複数のポリヌクレオチドを製造する方法であって、
a.複数の機構を有する第1および第2の支持体を提供すること、ここで、各機構は、異なる予め定義された配列を有する複数の異なる支持体結合オリゴヌクレオチドを含む;
b.複数の支持体結合オリゴヌクレオチドを鋳型として使用して、異なる予め定義された配列を有する第1および第2の複数の異なる構築オリゴヌクレオチドを作製すること、ここで、第1および第2の複数の構築オリゴヌクレオチドは、3’末端相補配列を有する;
c.複数の機構を含む支持体を提供すること、ここで、各機構が、複数の支持体結合アンカー一本鎖オリゴヌクレオチドを含み、複数のアンカーオリゴヌクレオチド各々の5’末端が、第1の複数の構築オリゴヌクレオチドの5’末端と相補的である;
d.第1の複数の構築オリゴヌクレオチドを、アンカーオリゴヌクレオチドとハイブリダイズすること;
e.第2の複数の構築オリゴヌクレオチドを、第1の複数の構築オリゴヌクレオチドとハイブリダイズすること;
f.任意に、第3の複数の構築オリゴヌクレオチドを、第2の複数の構築オリゴヌクレオチドと逐次ハイブリダイズすること;
g.複数の構築オリゴヌクレオチドをライゲーションすること、
を含む、方法。 - 予め定義されたポリヌクレオチドの各々を定義する複数の構築オリゴヌクレオチドの各々が、異なる支持体上で合成される、請求項45に記載の方法。
- 複数の異なるポリヌクレオチドが、支持体結合アンカーオリゴヌクレオチドを含む支持体の異なる機構でアセンブルされる、請求項45に記載の方法。
- 複数の第1の構築オリゴヌクレオチドを作製するステップが、プライマーの伸長を促進する条件下で、少なくとも1個のウラシルを有するプライマー配列を、第1の複数の支持体結合オリゴヌクレオチドとアニーリングすること、およびウラシルDNAグリコシラーゼ(UDG)およびDNAグリコシラーゼ−リアーゼエンドヌクレアーゼVIIIの混合物を使用して、プライマーを除去すること、を含む、請求項45に記載の方法。
- 予め定義された配列を有する複数のポリヌクレオチドを合成する方法であって、
a.複数の機構を含む第1の支持体を提供すること、ここで、各機構は、複数の支持体結合アンカー一本鎖オリゴヌクレオチドを含み、複数のアンカーオリゴヌクレオチド各々の5’末端が、第1の複数の構築オリゴヌクレオチドの5’末端と相補的である;
b.複数の機構を有する第2の支持体を提供すること、ここで、各機構は、複数の一本鎖支持体結合鋳型オリゴヌクレオチドを含み、複数の支持体結合オリゴヌクレオチドは各々、異なる予め定義された配列を有する;
c.複数の支持体結合オリゴヌクレオチドを鋳型として使用するポリメラーゼ伸長によって、第1の複数の構築オリゴヌクレオチドを作製すること;
d.第2の支持体の各機構が、第1の支持体の対応する機構に対してアラインされるように、第1および第2の支持体を配置すること;
e.第1の複数のオリゴヌクレオチドの複数の対応するアンカーオリゴヌクレオチドとのハイブリダイゼーションを促進する条件下で、第1の複数の構築オリゴヌクレオチドを溶液中に放出すること;および
f.任意に、第2の複数の構築オリゴヌクレオチドを含む第3の支持体を用いてステップb〜eを反復すること、ここで、第2および第3の複数の構築オリゴヌクレオチドは3’末端相補配列を有する、
を含む方法。 - 複数の第1の構築オリゴヌクレオチドを作製するステップが、プライマーの伸長を促進する条件下で、少なくとも1個のウラシルを有するプライマー配列を、第1の複数の支持体結合オリゴヌクレオチドとアニーリングすること、ウラシルDNAグリコシラーゼ(UDG)およびDNAグリコシラーゼ−リアーゼエンドヌクレアーゼVIIIの混合物を使用してプライマーを除去すること、を含む、請求項49に記載の方法。
- アンカーオリゴヌクレオチドおよび第2の複数の構築オリゴヌクレオチドをさらにライゲーションする、請求項49に記載の方法。
- 第2の支持体が、第1の支持体の上部に向かい合って配置される、請求項49に記載の方法。
- 第1の複数の構築オリゴヌクレオチドを溶液中に放出するステップによって、アンカーオリゴヌクレオチドに向けた第1の複数のオリゴヌクレオチドの拡散が可能になる、請求項52に記載の方法。
- 第1の複数の構築オリゴヌクレオチドを溶液中に放出するステップが、アンカーオリゴヌクレオチドに向けた構築オリゴヌクレオチドの実質的な垂直拡散を可能にする多孔性膜の存在下にある、請求項53に記載の方法。
- 多孔性膜が、構築オリゴヌクレオチドの側方拡散を低減する、請求項54に記載の方法。
- 第2の支持体の各機構が、複数のオリゴヌクレオチドを含み、複数のオリゴヌクレオチドが、異なる予め定義された配列を有するオリゴヌクレオチドの少なくとも2つの集団を含み、オリゴヌクレオチドの少なくとも2つの集団が相補配列を有する、請求項49に記載の方法。
- オリゴヌクレオチドの2つの集団が、3’末端相補配列を含む、請求項56に記載の方法。
- オリゴヌクレオチドの2つの集団を溶液中に放出するステップによって、構築オリゴヌクレオチドの第1の集団の構築オリゴヌクレオチドの第2の集団とのハイブリダイゼーションおよびオリゴヌクレオチドの第1の集団のアンカーオリゴヌクレオチドとのハイブリダイゼーションが可能となる、請求項56に記載の方法。
- 溶液が、リガーゼを含む、請求項58に記載の方法。
- 第1の複数の構築オリゴヌクレオチドの化学量論が、アンカーオリゴヌクレオチドの化学量論よりも高い、請求項49に記載の方法。
- 複数のポリヌクレオチドを、ミスマッチヌクレオチドを含有する二本鎖ポリヌクレオチドの切断に適した条件下で、ミスマッチを認識し、切断する成分に曝露することをさらに含む、請求項49に記載の方法。
- 複数のポリヌクレオチドが、支持体上に固定化されているか、または溶液中にある、請求項61に記載の方法。
- ミスマッチを認識し、切断する成分が、ミスマッチエンドヌクレアーゼを含む、請求項62に記載の方法。
- ミスマッチ特異的エンドヌクレアーゼが、CEL I酵素である、請求項63に記載の方法。
- 第3の支持体が、複数のアンカーオリゴヌクレオチドとのハイブリダイゼーションによって固定化された複数のポリヌクレオチドを含む、請求項49に記載の方法。
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