JP6335918B2 - 制限酵素を用いない標的富化 - Google Patents
制限酵素を用いない標的富化 Download PDFInfo
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- JP6335918B2 JP6335918B2 JP2015546460A JP2015546460A JP6335918B2 JP 6335918 B2 JP6335918 B2 JP 6335918B2 JP 2015546460 A JP2015546460 A JP 2015546460A JP 2015546460 A JP2015546460 A JP 2015546460A JP 6335918 B2 JP6335918 B2 JP 6335918B2
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Description
例示的な実施形態をより詳細に記載する前に、本明細書に用いられる用語の意味及び範囲を説明及び規定する以下の定義を示す。
本開示は、侵襲的切断によってアダプタをゲノム配列に付加する方法、及び該方法を行うためのキットを提供する。ある特定の実施形態では、該方法を用いて、各々がそれにライゲートしたアダプタ配列を含有する無作為に生成されたゲノム断片のライブラリを作製することができる。これらの実施形態は、全ゲノムシークエンシングにおいて特定の用途を有する。他の実施形態では、該方法を用いて、各々がそれにライゲートしたアダプタ配列を含有する標的ゲノム断片のライブラリを作製することができる。これらの実施形態は、例えば標的化再シークエンシング用途及びSNPのマッピングにおいて特定の用途を有する。
上記のような主題の方法を行うためのキットも本開示によって提供される。主題のキットは少なくとも記載のハロプローブ、及び上記方法を行うのに好適な反応試薬(例えば緩衝剤等)を含有する。キットの様々な構成要素は別個の容器に存在していてもよく、又はある特定の適合する構成要素が必要に応じて単一容器に予め合わせられていてもよい。
Claims (20)
- (a)無作為に剪断されたゲノムDNAを、ハロプローブにハイブリダイズするステップであって、第1の環状複合体を作製するステップであって、前記ハロプローブが、
(i)前記無作為に剪断されたゲノムDNAの断片中の種々の領域にハイブリダイズするフランキング配列及び中央配列を含む第1のオリゴヌクレオチドと、
(ii)前記第1のオリゴヌクレオチドの前記中央配列に相補的な1つ又は複数の第2のオリゴヌクレオチドと
を含む、ステップと、
(b)前記第1の環状複合体中の前記ゲノム断片のオーバーハング末端を酵素的に消化するステップであって、前記1つ又は複数の第2のオリゴヌクレオチドの5’末端及び3’末端が、消化されたゲノム断片の3’末端及び5’末端にライゲーション可能に隣接した第2の環状複合体を得る、ステップと、
(c)(b)の前記消化されたゲノム断片の末端を、前記1つ又は複数の第2のオリゴヌクレオチドの末端にライゲートするステップであって、環状DNA分子を作製する、ステップと、
を含む方法。 - (d)前記1つ又は複数の第2のオリゴヌクレオチドによって与えられる部位に結合する1つ又は複数のプライマを使用して、前記環状DNA分子から前記消化されたゲノム断片を増幅するステップを含む、請求項1に記載の方法。
- (e)(d)の増幅生成物をシークエンシングするステップであって、前記消化されたゲノム断片の少なくとも一部のヌクレオチド配列を得る、ステップ、
を更に含む、請求項2に記載の方法。 - 前記第1のオリゴヌクレオチドが捕捉部分を含み、前記方法がステップ(a)とステップ(b)との間に、該捕捉部分を使用して前記第1の環状複合体を単離するステップを含む、請求項1〜3のいずれか一項に記載の方法。
- 前記シークエンシングするステップが、前記1つ又は複数の第2のオリゴヌクレオチド中のシークエンシングプライマ部位にハイブリダイズするプライマを使用して行われる、請求項3に記載の方法。
- 前記酵素的に消化するステップが、ポリメラーゼの存在下で一本鎖特異的両方向性エキソヌクレアーゼを使用して前記第1の環状複合体を消化することを含む、請求項1〜5のいずれか一項に記載の方法。
- 前記一本鎖特異的両方向性エキソヌクレアーゼがエキソヌクレアーゼVIIである、請求項1〜6のいずれか一項に記載の方法。
- 前記酵素的に消化するステップが、Pfu DNAポリメラーゼ/Taq DNAポリメラーゼカクテル、T4 DNAポリメラーゼ/エキソヌクレアーゼVIIカクテルによる処理、マングビーンヌクレアーゼによる処理、別の3’エンドヌクレアーゼと組み合わせたフラップエンドヌクレアーゼによる処理を含む、請求項1〜7のいずれか一項に記載の方法。
- 前記無作為に剪断されたゲノムDNAが化学的な、物理的な又はトランスポゼースにより触媒される断片化方法を用いてゲノムDNAから作製される、請求項1〜8のいずれか一項に記載の方法。
- 前記物理的な断片化方法が超音波処理、ネブライゼーション又は剪断を含む、請求項8に記載の方法。
- 前記1つ又は複数の第2のオリゴヌクレオチドが、前記第1のオリゴヌクレオチドの中央配列に相補的な単一オリゴヌクレオチドである、請求項1〜10のいずれか一項に記載の方法。
- 前記1つ又は複数の第2のオリゴヌクレオチドが、各々が前記第1のオリゴヌクレオチドにハイブリダイズする第1の領域と、1つ又は複数の増幅プライマの結合部位をもたらす第2の領域とを含む2つのオリゴヌクレオチドである、請求項1〜11のいずれか一項に記載の方法。
- (a)無作為に剪断されたゲノムDNAを、該無作為に剪断されたゲノムDNAの断片にハイブリダイズする領域を含むRNAオリゴヌクレオチドにハイブリダイズするステップであって、RNA/DNA二重鎖を作製する、ステップと、
(b)前記RNA/DNA二重鎖中のゲノム断片のオーバーハング末端を酵素的に消化するステップであって、規定の末端を有する消化されたゲノム断片を含む二重鎖を得る、ステップと、
(c)(b)の前記二重鎖のRNAオリゴヌクレオチドを消化するステップであって、前記消化されたゲノム断片を放出する、ステップと、
(d)(c)の前記消化されたゲノム断片を、
(i)前記消化されたゲノム断片の末端にハイブリダイズするフランキング配列及び中央配列を含む第1のオリゴヌクレオチドと、
(ii)前記第1のオリゴヌクレオチドの中央配列に相補的な1つ又は複数の第2のオリゴヌクレオチドと、
を含むハロプローブとハイブリダイズするステップであって、前記第2のオリゴヌクレオチドの5’末端及び3’末端が、前記消化されたゲノム断片の3’末端及び5’末端にライゲーション可能に隣接した第2の複合体を得る、ステップと、
(e)前記消化されたゲノム断片の末端を前記第2のオリゴヌクレオチドにライゲートするステップであって、環状DNA分子を作製する、ステップと、
を含む方法。 - 前記RNAオリゴヌクレオチドが捕捉部分を含み、前記方法が該捕捉部分を使用して(b)の前記酵素的に消化された第1の複合体を単離するステップを含む、請求項13に記載の方法。
- 前記消化するステップ(c)を、NaOH又はRNaseH処理を使用して行う、請求項13又は14に記載の方法。
- (f)前記第1のオリゴヌクレオチドの中央配列中の部位に結合する1つ又は複数のプライマを使用して、前記環状DNA分子から前記消化されたゲノム断片を増幅するステップを含む、請求項13〜15のいずれか一項に記載の方法。
- (g)(f)の増幅生成物をシークエンシングするステップを更に含む、請求項16に記載の方法。
- 前記シークエンシングするステップを、前記1つ又は複数の第2のオリゴヌクレオチドによって与えられるシークエンシングプライマ部位にハイブリダイズするプライマを使用して行う、請求項17に記載の方法。
- 前記酵素的に消化するステップが、エキソヌクレアーゼVIIを使用して前記第1の複合体を消化することを含む、請求項13〜18のいずれか一項に記載の方法。
- 前記第1のオリゴヌクレオチドが捕捉部分を含み、前記方法がライゲートするステップ(e)の前に(d)の前記第2の複合体を単離するステップを含む、請求項13〜18のいずれか一項に記載の方法。
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