JP2019506172A - プライマー伸長中のプライマー−プライマー相互作用の排除 - Google Patents
プライマー伸長中のプライマー−プライマー相互作用の排除 Download PDFInfo
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Abstract
Description
用語「ポリメラーゼ」は、天然(その天然種から精製される)であってもまたは組換え体(形質転換宿主で産生され、そしてそこから精製される)であってもよい核酸ポリメラーゼを指す。本発明の背景において、ポリメラーゼは、元来のアミノ酸配列、または本発明の方法にしたがって修飾塩基で複製を失速させる能力をポリメラーゼが維持する限り、修飾アミノ酸配列を有することも可能である。
分子反転プローブ(MIP)は、次世代配列決定適用において、ターゲット配列を濃縮させるために用いられてきている。用いるプローブプールは、数百そしてさらに数千のターゲットアンプリコンを生成しうる。しかし、数百または数千の他のプローブの背景においてよく働くプローブを設計することは容易ではない。これは、最終ライブラリーにおいて、望ましくない産物を生成する、プローブ間の相互作用が起こりうるためである。この状況は、これらのプローブにおいてユニークな識別子配列を用いた際に悪化する可能性もあり、これはこれらが配列決定を通じた分子同定を可能にするために、プローブ内に操作されるランダム配列であるためである。分子反転プローブ内へのウラシル塩基の取り込みは、2つのプローブが相互作用した際に、ポリメラーゼ伸長をブロッキングすることにより、望ましくない産物の生成を減少させるはずである。
Claims (15)
- プライマー−プライマー相互作用を減少させつつ、プライマー伸長によって核酸鎖を合成する方法であって:ターゲット核酸鎖を、核酸ポリメラーゼ、およびポリメラーゼによるヌクレオチド取り込みを失速させる少なくとも1つの修飾ヌクレオチドを含むプライマーと接触させる工程を含む、前記方法。
- 前記プライマーが、ターゲット配列に非相補的なリンカー配列によって分離されている、ターゲット配列に相補的な2つのアームからなる一本鎖オリゴヌクレオチドである、請求項1の方法。
- 前記修飾ヌクレオチドが、ウラシル、脱塩基修飾ヌクレオチドおよびピリミジン二量体からなる群より選択される、請求項1〜2の方法。
- プライマー−プライマー相互作用を減少させつつ、試料中のターゲット核酸を増幅する方法であって:
a. 試料を、核酸ポリメラーゼ、およびポリメラーゼによるヌクレオチド取り込みを失速させる少なくとも1つの修飾ヌクレオチドを含む、ターゲット核酸に相補的な第一のプライマーと接触させて、プライマー伸長産物を生成する、プライマー伸長工程;および
b. 該試料を、プライマー伸長産物に相補的な第二のプライマーと接触させる、指数関数的増幅工程
を含む、前記方法。 - 前記プライマー伸長産物を、工程bの前に二本鎖アダプターに連結する、請求項4の方法。
- 前記アダプターおよび第一のプライマーが、普遍的増幅プライマーの結合部位を含み、そして前記第二のプライマーが普遍的プライマーである、請求項4〜5の方法。
- 前記プライマーが、ターゲット配列に非相補的なリンカー配列によって分離されている、ターゲット配列に相補的な2つのアームからなる一本鎖オリゴヌクレオチドである、請求項4〜6の方法。
- 前記指数関数的増幅工程が、修飾ヌクレオチドに耐性であるポリメラーゼを利用する、請求項4〜7の方法。
- 工程bの前に精製工程をさらに含み、ここで第一のプライマーおよびテンプレート核酸が、プライマー伸長産物から分離される、請求項4〜8の方法。
- 前記修飾ヌクレオチドがウラシルである、請求項4〜9の方法。
- 工程bの前にキャリーオーバー防止工程をさらに含み、ここで試料をウラシルDNAグリコシラーゼと接触させる、請求項10の方法。
- 請求項1〜3にしたがって、核酸鎖を合成するためのキット。
- 請求項4〜11にしたがって、試料におけるターゲット核酸を増幅するためのキット。
- 請求項1〜3にしたがって、核酸鎖を合成するための反応混合物。
- 請求項4〜11にしたがって、試料におけるターゲット核酸を増幅するための反応混合物。
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EP4269582A3 (en) | 2017-12-21 | 2024-01-24 | F. Hoffmann-La Roche AG | Target enrichment by unidirectional dual probe primer extension |
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