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Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry
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ナノポールは,DNAのC5-サイトシン5つの変異種を区別する
Zachary L Wescoe1, Jacob Schreiber, Mark Akeson
1Department of Biomolecular Engineering, Baskin School of Engineering, MS SOE2, University of California , Santa Cruz, California 95064, United States.
Journal of the American Chemical Society
|October 28, 2014
まとめ
この研究は,5-メチルサイトシン (mC) や5-カルボキシルサイトシン (caC) のような改変塩基を含む5つのサイトシン変種を正確に識別することにより,DNAシーケンシングを前進させ,エピジェネティクスを理解するために不可欠です.
科学分野:
- バイオケミストリー バイオケミストリー
- ゲノミクスゲノミクスとは
- エピジェネティクス エピジェネティクス
背景:
- 酵素によるナノ孔配列化は,単一核酸DNAの読み取りを可能にします.
- カノニカル塩基の区別は,5-メチルサイトシン (mC) と5-ヒドロキシメチルサイトシン (hmC) が確立されています.
- 哺乳類の細胞は,5-ヒドロキシメチルシトシン (hmC) の変換中に,5-カルボキシルシトシン (caC) と5-ホルミルシトシン (fC) を生成する.
研究 の 目的:
- DNA内のサイトシンとその主要C5変種 (mC, hmC, caC, fC) の正確な識別のための方法を開発する.
- ナノ孔配列解析を用いてCG二核酸でこれらの塩基を区別する精度を評価する.
主な方法:
- M2MspAナノポールを介して制御されたDNA転位のためにphi29DNAポリメラーゼを使用した.
- イオン電流の痕跡を記録するために,パッチクランプ増幅器を使用しました.
- ベース呼び出しのための3つの連続したイオン電流状態に基づく決定境界を実装しました.
主要な成果:
- サイトシン,mC,hmC,caC,fCをCG二核酸で呼び出す正確な塩基を達成しました.
- シングルパス読み取りの精度は91.6%から98.3%でした.
- 精度は,CGダイヌクレオチドを取り巻く最も近い隣接ベースによって影響を受けた.
結論:
- 酵素制御と組み合わせたナノ孔配列化は,複数のサイトシン変種を確実に区別することができます.
- この方法は,caCとfCを含むことで,表遺伝的景観のより包括的な理解を提供します.
- 最も近い隣の塩基同一性は,改変されたサイトシンのシーケンシング精度に影響を与える重要な要因です.
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