JP5535415B2 - 核酸分子の配列決定の方法 - Google Patents
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Description
本発明は、核酸分子の配列を決定する方法に関する。
ヒトの全ゲノムを明らかにするという目標は、大小いずれのスケールの適用についても迅速なDNA配列決定をする技術に対する関心を喚起した。重要なパラメータは配列決定の速さ、一度の配列解析において読み取ることの可能な配列長、および必要とされる鋳型の核酸の量である。これらの研究上の挑戦は、事前に増幅せずに、および事前に遺伝材料を配列決定用ベクターへクローニングする必要なく、単一の細胞の遺伝情報の配列決定を目指すことを提案する。現状では、大きなスケールのゲノムプロジェクトは、多数の生物または患者について現実的に実施するには費用がかかりすぎる。さらに、ヒトの疾患について遺伝学的基礎の知識が増加するにつれ、臨床適用を可能にする、正確な、高処理量DNA配列決定の必要性は常に増加し続けると考えられる。核酸の単分子の塩基対配列の決定の実用的な方法、好ましくは高速かつ読み取り長の長いものは、必要な測定能を提供する。
本発明は、複数のヌクレオチド塩基をもつ標的核酸分子の配列決定の方法に関する。本方法は、標的核酸に相補的な活性部位において、ヌクレオチド類似体を付加するために適した位置において、互いに配向された、核酸重合酵素と標的核酸分子の複合体の提供に関連する。多種のヌクレオチド類似体が活性部位の近傍に、各種のヌクレオチド類似体が標的核酸配列の異なるヌクレオチドに対し相補的になるように提供される。ヌクレオチド類似体は活性部位において、付加したヌクレオチド類似体が次なるヌクレオチド類似体の付加を受けられるように、付加されるヌクレオチド類似体が標的核酸ヌクレオチドに対し相補的になるように重合される。重合段階の結果、活性部位に付加されたヌクレオチド類似体が同定される。標的核酸配列を決定するため、複数のヌクレオチド類似体の提供段階、重合段階、および同定段階が繰り返される。
本発明は、複数のヌクレオチド塩基をもつ標的核酸分子の配列決定の方法に関するものである。本方法は標的核酸に相補的な活性部位にヌクレオチド類似体を付加するために適切な位置において、互いに関して配向する核酸重合酵素と標的核酸分子の複合体の提供に関連する。多種のヌクレオチド類似体が、各種のヌクレオチド類似体が標的核酸配列において異なるヌクレオチドに対し相補的になるように、活性部位の近傍に提供される。ヌクレオチド類似体は活性部位において、付加したヌクレオチド類似体が、次なるヌクレオチド類似体の付加を受けられるように、付加されるヌクレオチド類似体が標的核酸ヌクレオチドに対し相補的になるように重合される。重合段階の結果、活性部位に付加されたヌクレオチド類似体が同定される。標的核酸配列を決定するため、複数のヌクレオチド類似体の提供段階、重合段階、および同定段階が繰り返される。
Claims (60)
- 複数のヌクレオチド塩基を有する標的核酸分子を配列決定する方法であって、
核酸重合酵素の活性部位においてかつ標的核酸に対し相補的である位置において伸長する核酸鎖にヌクレオチド類似体を付加するために適切な位置において互いに関して配向された、核酸重合酵素およびプライマーが結合した標的核酸分子の複合体を提供する段階;
複数の型の標識されたヌクレオチド類似体を提供する段階であって、各々の型の標識されたヌクレオチド類似体が前記標的核酸配列における異なるヌクレオチドに対し相補的である段階;
前記活性部位において標識されたヌクレオチド類似体を重合する段階であって、付加される標識されたヌクレオチド類似体が、標的核酸のヌクレオチドに対し相補的であり、かつ前記活性部位に付加された後に次の標識されたヌクレオチド類似体と重合することができる段階;
該重合する段階の結果として前記活性部位において付加されたヌクレオチド類似体を同定する段階であって、該同定が前記活性部位の近傍の標識されたヌクレオチドの滞留時間に基づく段階;および
前記重合する段階および前記同定する段階を継続して標的核酸配列を決定する段階、
を含む方法。
- 核酸重合酵素が、DNAポリメラーゼ、RNAポリメラーゼ、逆転写酵素、およびその混合物からなる群より選択される、請求項1記載の方法。
- 核酸重合酵素が耐熱性ポリメラーゼである、請求項1記載の方法。
- 核酸重合酵素が熱失活性ポリメラーゼである、請求項1記載の方法。
- 標的核酸分子が、二本鎖DNA、単鎖DNA、単鎖DNAヘアピン、DNA/RNAハイブリッド、ポリメラーゼの結合のための認識部位をもつRNA、およびRNAヘアピンからなる群より選択される、請求項1記載の方法。
- 核酸重合酵素が、複製起点、二本鎖標的核酸のニックもしくはギャップ、単鎖標的核酸の二次構造、アクセサリータンパク質により作成される結合部位、またはプライマーが結合した単鎖核酸において、標的核酸分子複合体に結合される、請求項1記載の方法。
- 核酸重合酵素に、その活性を改変するために1つまたはそれ以上のアクセサリータンパク質を提供する、請求項1記載の方法。
- アクセサリータンパク質が、単鎖結合タンパク質、プライマーゼ、およびヘリカーゼからなる群より選択される、請求項7記載の方法。
- 核酸重合酵素がプロセシブ(processive)である、請求項1記載の方法。
- 核酸重合酵素が非プロセシブ(non-processive)である、請求項1記載の方法。
- ヌクレオチド類似体が、リボヌクレオチド、デオキシリボヌクレオチド、修飾リボヌクレオチド、修飾デオキシリボヌクレオチド、ペプチドヌクレオチド、修飾ペプチドヌクレオチド、および修飾リン酸‐糖骨格をもつヌクレオチドからなる群より選択される、請求項1記載の方法。
- 複数のヌクレオチド類似体を提供する段階の前または該提供する段階の間に、オリゴヌクレオチドプライマーを標的核酸分子にハイブリダイズする段階をさらに含む、請求項1記載の方法。
- オリゴヌクレオチドプライマーが、リボヌクレオチド、デオキシリボヌクレオチド、修飾リボヌクレオチド、修飾デオキシリボヌクレオチド、ペプチド核酸、修飾ペプチド核酸、および修飾リン酸‐糖骨格をもつヌクレオチドからなる群より選択されるヌクレオチドを含む、請求項12記載の方法。
- ヌクレオチド類似体に標識を提供する、請求項1記載の方法。
- 標識が、発色団、蛍光部分、酵素、抗原、重金属、磁気プローブ、色素、りん光群、放射性物質、化学発光部分、散乱または蛍光ナノ粒子、ラマンシグナル発生部分、および電気化学的検出部分からなる群より選択される、請求項14記載の方法。
- 標識がヌクレオチド類似体に、その塩基、糖部分、αリン酸、βリン酸、またはγリン酸で付着する、請求項14記載の方法。
- 標識がリンカーによってヌクレオチド類似体に付着する、請求項14記載の方法。
- 標識がリンカーを用いずにヌクレオチド類似体に付着する、請求項14記載の方法。
- 前記活性部位において多くのさらなるヌクレオチド類似体を同定する段階の間または該同定する段階の後で、且つ重合する段階の前に、標識をヌクレオチド類似体から除去する段階をさらに含む、請求項14記載の方法。
- 除去段階が標識の退色により実施される、請求項19記載の方法。
- 標識の除去を誘導および調節するために調整された放射光を用いた光退色により退色が実施される、請求項20記載の方法。
- 除去段階がヌクレオチド類似体からの標識の切断により実施される、請求項19記載の方法。
- βまたはγリン酸標識されたヌクレオチド類似体が酵素的に切断される、請求項22記載の方法。
- 複数の型のヌクレオチド類似体の各々が、同定段階の間に互いに識別される異なる標識をもつ、請求項14記載の方法。
- 3つまたはそれ以下の、複数の型のヌクレオチド類似体が異なる標識をもつ、請求項14記載の方法。
- 異なる型のヌクレオチド類似体が、同じ標識であるが、塩基蛍光団、クエンチングされた蛍光団、または蛍光性ヌクレオチド類似体の存在による異なる特性により識別される標識をもつ、請求項14記載の方法。
- 核酸重合酵素が標識をもち、同定段階が該標識とヌクレオチド類似体の間の相互作用に基づくシグナルの検出により実施される、請求項1記載の方法。
- 標識が蛍光共鳴エネルギー移動ドナーまたはアクセプターである、請求項27記載の方法。
- 同定段階が、遠隔場顕微分光、近接場顕微分光、エバネッセント波または導波管照射、ナノ構造増強、およびそれらの組合せからなる群より選択される光学的手順により実施される、請求項1記載の方法。
- 同定段階が、単光子および/または多光子励起、蛍光共鳴エネルギー移動、または光変換の使用により実施される、請求項1記載の方法。
- 同定段階が、スペクトル波長識別、蛍光寿命の測定および分離、蛍光団同定および/またはバックグラウンド抑制により達成される、請求項1記載の方法。
- 蛍光団同定および/またはバックグラウンド抑制において、多重励起源の迅速な切替を使用する、請求項31記載の方法。
- 前記複合体を提供する段階が
(1)オリゴヌクレオチドプライマーまたは(2)標的核酸分子のいずれかを支持体上に配置する段階;
プライマーが結合した標的核酸分子複合体を形成するために、(1) 配置されたオリゴヌクレオチドプライマーに標的核酸分子をハイブリダイズする、または(2) 配置された標的核酸分子にオリゴヌクレオチドプライマーをハイブリダイズする段階;および
標的核酸分子に沿った移動および活性部位におけるオリゴヌクレオチドプライマーの伸長のために適切な位置において、プライマーが結合した標的核酸分子複合体上の核酸重合酵素を提供する段階
を含む、請求項1記載の方法。
- ハイブリダイズの段階が、支持体上に配置されたオリゴヌクレオチドプライマーに、標的核酸分子の末端を結合することにより実施される、請求項33記載の方法。
- 支持体およびオリゴヌクレオチドプライマーまたは標的核酸分子のいずれかが、抗原‐抗体結合対、ストレプトアビジン‐ビオチン結合対、光活性化した結合分子、および相補的核酸対からなる群より選択される、共有結合対または非共有結合対の対応する成分と、可逆的または不可逆的に結合する、請求項33記載の方法。
- オリゴヌクレオチドプライマーが支持体上に配置され、配置されたオリゴヌクレオチドプライマーに標的核酸分子がハイブリダイズする、請求項33記載の方法。
- 標的核酸分子が支持体上に配置され、配置された標的核酸分子にオリゴヌクレオチドプライマーがハイブリダイズする、請求項33記載の方法。
- 前記複合体を提供する段階が
標的核酸を含み、且つ核酸重合酵素のための認識部位をもつ二本鎖核酸分子を、支持体上に配置する段階、および
核酸重合酵素を、標的核酸分子上の標的核酸分子に沿った移動のために適切な位置において提供する段階
を含む、請求項1記載の方法。
- 前記複合体を提供する段階が、核酸重合酵素を、標的核酸複合体が核酸重合酵素に対して相対的に移動するために適切な位置で支持体上に配置する段階を含む、請求項1記載の方法。
- 支持体および核酸重合酵素が、抗原‐抗体結合対、ストレプトアビジン‐ビオチン結合対、光活性化結合分子、および相補的核酸対からなる群より選択される、共有結合対または非共有結合対の対応する成分により、可逆的または不可逆的に結合される、請求項39記載の方法。
- 核酸重合酵素または標的核酸を固体支持体上に配置する、請求項1記載の方法。
- 核酸重合酵素または標的核酸を小孔をもつゲル内に配置する、請求項1記載の方法。
- 標的核酸および核酸重合酵素を固体支持体上に互いに近傍に配置する、請求項1記載の方法。
- 同定段階が、遊離ヌクレオチド類似体から生じるバックグラウンドノイズを減少することにより実施される、請求項1記載の方法。
- 前記同定する段階が、
光放射を活性部位に指向させる段階、および
活性部位において重合されたヌクレオチド類似体を検出する段階
を含む、請求項44記載の方法。
- 同定段階により活性部位において重合されたヌクレオチド類似体が遊離ヌクレオチド類似体から識別される、請求項44記載の方法。
- 同定段階が、支持体および前記核酸重合酵素を含む制限領域において実施される、請求項44記載の方法。
- 同定段階がナノ構造において実施される、請求項47記載の方法。
- ナノ構造が、検出段階を増強する、パンクチュエート(punctuate)構造、針状(acicular)構造、または共鳴ナノ構造である、請求項48記載の方法。
- 活性部位において重合されていないヌクレオチド類似体が、ミクロ構造を通って、制限領域までおよび制限領域から迅速に移動する、請求項47記載の方法。
- ミクロ構造が、
異なるヌクレオチド類似体を制限領域へ指向させるための複数のチャネル、および
制限領域から材料を除去させるための排出チャネル、
を含む、請求項50記載の方法。
- 同定段階が、活性部位近傍にて小さな曲率半径を有する対象物の近傍において増強された電磁放射を用いた電磁場増強により実施される、請求項44記載の方法。
- 同定段階が、プライマーが結合した標的核酸分子が位置する空洞の近接場照射をさらに含む、請求項44記載の方法。
- 同定段階が、複合体近傍の光ファイバーを用いて実施される、請求項44記載の方法。
- 同定段階が、ゲート時間光子検出を含む、請求項44記載の方法。
- アレイ上の複数の異なる位置における異なる核酸分子の配列決定により方法が実施される、請求項1記載の方法。
- 同時にまたは連続して同じ標的核酸を配列決定する段階、およびそのような配列決定からのアウトプットを組合せる段階により実施される、請求項1記載の方法。
- 標的核酸分子を配列決定するために適切な装置であって、
支持体と、
プライマーが結合した標的核酸分子に結合した核酸重合酵素であって、該核酸重合酵素または標的核酸分子が前記支持体上に配置される核酸重合酵素と、
不透明層中に孔を含むミクロ構造であって、該孔が、該支持体、該核酸重合酵素、および前記標的核酸分子を含む制限領域を規定し、該制限領域が、前記核酸重合酵素の活性部位でかつ前記標的核酸分子に相補的である位置での成長中の核酸の重合に基づき標識されたヌクレオチド類似体から標識を検出するよう構成された、ミクロ構造と、
前記重合の結果として活性部位において付加されたヌクレオチド類似体を同定することができる検出器であって、該同定が、前記活性部位の近傍の標識されたヌクレオチドの滞留時間に基づく、検出器と、
を含む装置。
- ミクロ構造が、
異なる型のヌクレオチド類似体を制限領域へ指向させるための複数のチャネルと、
制限領域から材料を除去させるための排出チャネルと
を含む、請求項58記載の装置。
- 光放射を制限領域に集中させ、制限領域から放射を収集するための、制限領域近傍の光学導波管
をさらに含む請求項58記載の装置。
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