JP2022550497A - バーコードに基づいた核酸配列アセンブリ - Google Patents
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Abstract
Description
本発明は、2019年6月21日に出願の米国仮特許出願第62/865,094号の利益を主張し、これは引用によって本明細書に組み込まれる。
本明細書で明記されるすべての公開物、特許、および特許出願は、個々の公開物、特許、または特許出願がそれぞれ参照により組み込まれるべく特別かつ個別に示されるかのような同じ程度で、参照により本明細書に組み込まれる。
いくつかの例では、遺伝子断片の数は、約1~約20、約2~約18、約3~約17、約4~約16、約6~約14、または約8~約12である。
98℃、30秒
98℃、10秒;63℃、10秒;72℃、10秒;12サイクルを繰り返す
72℃、2分
5’ベクター-ドメイン1(15)-ドメイン2(20)-ドメイン3(20)-ドメイン4(20)-3’ベクター
5’ベクター-ドメイン1(X)-定常ドメイン-ドメイン3(X)-3’ベクター
Claims (76)
- 核酸アセンブリのための方法であって、該方法は、
(a)第1の複数のポリヌクレオチドを提供する工程であって、ここで、前記第1の複数のポリヌクレオチドの各々のポリヌクレオチドは、配列相同性の第1の末端領域を含む、工程と、
(b)第2の複数のポリヌクレオチドを提供する工程であって、ここで、前記第2の複数のポリヌクレオチドの各々のポリヌクレオチドは、配列相同性の前記第1の末端領域に対する配列相同性の第2の末端領域を含む、工程と、
(c)核酸のライブラリーをアセンブルするために、前記第1の複数のポリヌクレオチドおよび前記第2の複数のポリヌクレオチドを、エキソヌクレアーゼ、エンドヌクレアーゼ、ポリメラーゼ、およびリガーゼを含む反応混合物と接触させる工程であって、ここで、核酸の少なくとも80%は、各々が、ライブラリーの核酸の各々の平均頻度の2倍(2x)以内の量でライブラリーにおいて存在する、工程と、を含む、方法。 - 前記第1の複数のポリヌクレオチドは、最大で100の異なる配列を含む、請求項1に記載の方法。
- 前記 第2の複数のポリヌクレオチドは、最大で100の異なる配列を含む、請求項1に記載の方法。
- 少なくとも10,000の核酸がアセンブルされる、請求項1に記載の方法。
- 少なくとも100,000の核酸がアセンブリされる、請求項1に記載の方法。
- 前記第1の複数のポリヌクレオチドの各々のポリヌクレオチドは、最大で2500塩基長を含む、請求項1に記載の方法。
- 前記第2の複数のポリヌクレオチドの各ポリヌクレオチドは、最大で2500の塩基長を含む、請求項1に記載の方法。
- 前記エキソヌクレアーゼは、エキソヌクレアーゼIIIである、請求項1に記載の方法。
- 前記エンドヌクレアーゼは、フラップエンドヌクレアーゼである、請求項1に記載の方法。
- 前記フラップエンドヌクレアーゼは、フラップエンドヌクレアーゼ1、エキソヌクレアーゼ1、XPG、Dna2、またはGEN1である、請求項9に記載の方法。
- 前記ポリメラーゼは、5’~3’のポリメラーゼ活性を含む、請求項1に記載の方法。
- 前記ポリメラーゼはDNAポリメラーゼである、請求項1に記載の方法。
- 前記リガーゼは、少なくとも2つの核酸の結合を触媒する、請求項1に記載の方法。
- 核酸アセンブリのための方法であって、該方法は、
(a)5’~3’の順番でバーコード配列、第1の制限エンドヌクレアーゼ部位、第2の制限エンドヌクレアーゼ部位、および第1の超可変領域配列を含む第1の核酸をデノボ合成する工程と、
(b)5’~3’の順番で任意の定義された配列長さの第1の領域、自己切断型ペプチド配列、第1の可変領域配列に隣接する第1の相補的領域、および第1の可変領域配列を含む第2の核酸をデノボ合成する工程と、
(c)第3の核酸を生成するために、前記第1の核酸と前記第2の核酸を接触させる工程と、
(d)5’~3’の順番でベクター配列、第2の可変領域配列に隣接する第2の相補的領域、第2の可変領域配列、第2の超可変領域配列、第1の制限エンドヌクレアーゼ部位、およびバーコード配列を含む第4の核酸を提供する工程と、
(e)前記第3の核酸および前記第4の核酸を制限エンドヌクレアーゼと接触させる工程と、
(f)1つ以上の酵素を含む反応混合物を使用して前記第3の核酸および前記第4の核酸をアセンブルする工程と、を含む、方法。 - 前記第1の制限エンドヌクレアーゼ部位、または前記第2の制限エンドヌクレアーゼ部位は、IIS型制限エンドヌクレアーゼ(TIIS-RE)部位である、請求項14に記載の方法。
- 前記制限エンドヌクレアーゼは、IIS型制限エンドヌクレアーゼである、請求項14に記載の方法。
- 反応混合物はリガーゼを含む、請求項14に記載の方法。
- 前記第1の超可変領域配列および前記第2の超可変領域配列は、各々が相補性決定領域(CDR)を含む、請求項14に記載の方法。
- 前記CDRはCDR3である、請求項18に記載の方法。
- 前記自己切断型ペプチドはP2Aである、請求項14に記載の方法。
- 前記第1の可変領域配列の約100の変異体が合成される、請求項14に記載の方法。
- 前記第2の可変領域配列の約130の変異体が合成される、請求項14に記載の方法。
- 第1のバーコード配列に相補的な第1のプライマー、およびアンプリコンの少なくとも99%が欠失がない第2のプライマーを用いて、核酸を増幅する工程と、をさらに含む、請求項14に記載の方法。
- 核酸アセンブリのための方法であって、該方法は、
(a)第1の可変領域配列を含む第1の核酸をデノボ合成する工程と、
(b)第2の可変領域配列を含む第2の核酸をデノボ合成する工程と、
(c)5’~3’の順番で固定された可変性配列の第1の領域、任意の定義された配列長さの第1の領域、自己切断型ペプチド配列、第1の可変領域配列に隣接する第1の相補的領域、および固定された可変性配列の第2の領域を含む第3の核酸をデノボ合成する工程と、
(d)前記第1の核酸、前記第2の核酸、前記第3の核酸を、エキソヌクレアーゼ、エンドヌクレアーゼ、ポリメラーゼ、およびリガーゼを含む反応混合物と接触させる工程と、を含む、方法。 - 前記第1の可変領域配列、または前記第2の可変領域配列は、超可変領域配列を用いて増幅される、請求項24に記載の方法。
- 前記超可変領域配列は、CDRを含む、請求項25に記載の方法。
- 前記CDRはCDR3である、請求項26に記載の方法。
- 任意の定義された長さの1つ以上の領域を含む配列と接触させる工程をさらに含む、請求項24に記載の方法。
- 前記第1の可変領域配列の約100の変異体が合成される、請求項24に記載の方法。
- 前記第2の可変領域配列の約130の変異体が合成される、請求項24に記載の方法。
- 前記自己切断型ペプチドはP2Aである、請求項24に記載の方法。
- 前記エキソヌクレアーゼは、エキソヌクレアーゼIIIである、請求項24に記載の方法。
- 前記エンドヌクレアーゼは、フラップエンドヌクレアーゼである、請求項24に記載の方法。
- 前記フラップエンドヌクレアーゼは、フラップエンドヌクレアーゼ1、エキソヌクレアーゼ1、XPG、Dna2、またはGEN1である、請求項33に記載の方法。
- 前記ポリメラーゼは、5’~3’のポリメラーゼ活性を含む、請求項24に記載の方法。
- 前記ポリメラーゼはDNAポリメラーゼである、請求項24に記載の方法。
- 前記リガーゼは、少なくとも2つの核酸の結合を触媒する、請求項24に記載の方法。
- 前記固定された可変性配列の第1の領域、および前記固定された可変性配列の第2の領域は、各々が約10~約100の塩基対である、請求項24に記載の方法。
- 前記固定された可変性配列の第1の領域、および前記固定された可変性配列の第2の領域は、各々が約40の塩基対である、請求項24に記載の方法。
- 核酸アセンブリのための方法であって、該方法は、
(a)任意の定義された長さの配列の第1の領域を含む第1の核酸を提供する工程と、
(b)任意の定義された長さの配列の第2の領域を含む第2の核酸を提供する工程と、
(c)5’~3’の順番で第1の可変領域配列に隣接する第1の相補的領域、第1の可変領域配列、および第1の超可変領域配列を含む第3の核酸をアセンブルする工程と、
(d)5’~3’の順番で第2の可変領域配列に隣接する第2の相補的領域、第2の可変領域配列、および第2の超可変領域配列を含む第4の核酸をアセンブルする工程と、
(e)前記第1の核酸と、前記第2の核酸と、前記第3の核酸と第4の核酸とを接触させる工程と、
(f)工程(e)から産物を増幅する工程と、を含む、方法。 - エラー修正工程をさらに含む、請求項40に記載の方法。
- 工程(e)の間に、エキソヌクレアーゼ、エンドヌクレアーゼ、ポリメラーゼ、およびリガーゼを含む反応混合物を接触させる工程をさらに含む、請求項40に記載の方法。
- 前記第1の超可変領域配列および前記第2の超可変領域配列は、各々が相補性決定領域(CDR)を含む、請求項40に記載の方法。
- 前記CDRはCDR3である、請求項43に記載の方法。
- 前記第1の核酸は、約300~約700の塩基対を含む、請求項40に記載の方法。
- 前記第2の核酸は、約200~約600の塩基対を含む、請求項40に記載の方法。
- 前記第3の核酸は、約200~約600の塩基対を含む、請求項40に記載の方法。
- 前記第4の核酸は、約200~約600の塩基対を含む、請求項40に記載の方法。
- 核酸アセンブリのための方法であって、該方法は、
(a)
i.5’~3’の順番で第1の可変領域配列に隣接する第1の相補的領域、および第1の可変領域配列を含む第1の核酸と、
ii.5’~3’の順番で固定された可変性配列の第1の領域、および第1の超可変領域配列を含む第2の核酸と、
iii.第2の可変領域配列を含む第3の核酸と、
iv.5’~3’の順番で制限エンドヌクレアーゼ部位、および固定された可変性配列の第2の領域を含む第4の核酸と、
v.5’~3’の順番で固定された可変性配列の第2の領域、第2の超可変領域配列、および可変定常領域配列を含む第5の核酸と、をデノボ合成する工程と、
(b)前記第1の核酸、前記第2の核酸、前記第3の核酸、前記第4の核酸、および前記第5の核酸を、エキソヌクレアーゼ、エンドヌクレアーゼ、ポリメラーゼ、およびリガーゼを含む反応混合物と接触させる工程と、
(c)工程(b)の構築物を、ベクター配列へとクローン化する工程と、を含む、方法。 - 前記第1の超可変領域配列および前記第2の超可変領域配列は、各々が相補性決定領域(CDR)を含む、請求項49に記載の方法。
- 前記CDRはCDR3である、請求項49に記載の方法。
- 1つ以上の可変定常領域を接触させる工程をさらに含む、請求項49に記載の方法。
- 前記エキソヌクレアーゼは、エキソヌクレアーゼIIIである、請求項49に記載の方法。
- 前記エンドヌクレアーゼは、フラップエンドヌクレアーゼである、請求項49に記載の方法。
- 前記フラップエンドヌクレアーゼは、フラップエンドヌクレアーゼ1、エキソヌクレアーゼ1、XPG、Dna2、またはGEN1である、請求項54に記載の方法。
- 前記ポリメラーゼは、5’~3’のポリメラーゼ活性を含む、請求項49に記載の方法。
- 核酸アセンブリのための方法であって、該方法は、
(a)5’~3’の順番で第1の可変領域配列に隣接する第1の相補的領域、および第1の可変領域配列を含む第1の核酸を提供する工程と、
(b)5’~3’の順番で固定された可変性配列の第1の領域、第1の超可変領域配列、制限エンドヌクレアーゼ部位、第2の超可変領域配列、およびユニバーサルプライマーを含む第2の核酸配列を提供する工程と、
(c)第3の核酸を生成するために、前記第1の核酸と前記第2の核酸を増幅させる工程と、
(d)第1の可変領域配列に隣接する第1の相補的領域、および任意の定義された長さの配列の第1の領域を含むベクター配列を提供する工程と、
(e)前記第3の核酸と前記ベクター配列とを接触させる工程と、
(f)5’~3’の順番で自己切断型ペプチド配列、第2の可変領域配列に隣接する第2の相補的領域、および第2の可変領域配列を含む第4の核酸と接触させる工程と、を含む、方法。 - 第1の超可変領域配列および第2の超可変領域配列は、各々が相補性決定領域(CDR)を含む、請求項57に記載の方法。
- 前記CDRはCDR3である、請求項58に記載の方法。
- 前記自己切断型ペプチドはP2Aである、請求項57に記載の方法。
- 核酸アセンブリのための方法であって、該方法は、
(a)
i.第1の可変領域配列に隣接する第1の相補的領域、および第1の可変領域配列を含む第1の核酸と、
ii.第1の超可変領域配列を含む第2の核酸と、
iii.第2の可変領域配列を含む第3の核酸と、
iv.5’~3’の順番で第1の超可変領域配列、固定された可変性の第1の領域、およびバーコードを含む第4の核酸と、をデノボ合成する行程と、
(b)第5の核酸を生成するために、前記第1の核酸および前記第2の核酸を増幅させる工程と、
(c)第5の核酸を生成するために、前記第3の核酸および前記第4の核酸を増幅させる行程と、
(d)第7の核酸を生成するために、前記第5の核酸および第6の核酸を、エキソヌクレアーゼ、エンドヌクレアーゼ、ポリメラーゼおよびリガーゼを含む反応混合物と接触させる行程と、
(e)前記第7の核酸を環状化させる行程と、
(f)前記第7の核酸をバーコードを使用して配列決定および同定する行程と、
(g)前記第7の核酸を増幅する行程と、
(h)前記第7の核酸を、エキソヌクレアーゼ、エンドヌクレアーゼ、ポリメラーゼおよびリガーゼを含む反応混合物を使用してベクターにアセンブルする行程と、を含む、方法。 - 前記第1の可変領域配列、または前記第2の可変領域配列は、超可変領域配列を用いて増幅される、請求項61に記載の方法。
- 前記超可変領域配列はCDRを含む、請求項62に記載の方法。
- CDRはCDR3である、請求項63に記載の方法。
- 任意の定義された長さの1つ以上の領域を含む配列と接触する行程をさらに含む、請求項61に記載の方法。
- 前記第1の可変領域配列の約100の変異体が合成される、請求項61に記載の方法。
- 前記第2の可変領域配列の約130の変異体が合成される、請求項61に記載の方法。
- 前記自己切断型ペプチドはP2Aである、請求項61に記載の方法。
- 前記エキソヌクレアーゼは、エキソヌクレアーゼIIIである、請求項61に記載の方法。
- 前記エンドヌクレアーゼは、フラップエンドヌクレアーゼである、請求項61に記載の方法。
- 前記フラップエンドヌクレアーゼは、フラップエンドヌクレアーゼ1、エキソヌクレアーゼ1、XPG、Dna2、またはGEN1である、請求項70に記載の方法。
- 前記ポリメラーゼは、5’~3’のポリメラーゼ活性を含む、請求項61に記載の方法。
- 前記ポリメラーゼはDNAポリメラーゼである、請求項61に記載の方法。
- 前記リガーゼは、少なくとも2つの核酸の結合を触媒する、請求項61に記載の方法。
- 前記固定された可変性配列の第1の領域および前記固定された可変性配列の第2の領域は、各々が約10~約100の塩基対である、請求項61に記載の方法。
- 前記固定された可変性配列の第1の領域および前記固定された可変性配列の第2の領域は、各々が約40の塩基対である、請求項61に記載の方法。
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