JP5256316B2 - 核酸分子の配列決定の方法 - Google Patents
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- C12Q1/6869—Methods for sequencing
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- Y10S436/00—Chemistry: analytical and immunological testing
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
- Y10T436/143333—Saccharide [e.g., DNA, etc.]
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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 (24)
- 近接場励起のためのシステムであって、
透明な層の上にある、孔を有する不透明な層であって、該孔の直径が、励起光の波長の半分より小さい層と、
前記孔のうちの少なくとも1つの孔の中に近接場励起容量を形成するための前記透明な層を介して励起光を導くのに適合した照射源と、
該励起光への曝露に基づき前記少なくとも1つの孔の中の近接場励起容量から放射される光を検出する検出器であって、該放射される光が前記透明な層を介して通過する検出器と、
を含むシステム。 - 前記不透明な層の孔が、1または複数の蛍光標識を有する試薬溶液である、請求項1記載のシステム。
- 前記試薬溶液が、複数の型の標識されたヌクレオチド類似体を含む、請求項2記載のシステム。
- 前記孔が、一本鎖ポリメラーゼ酵素、核酸鋳型、および蛍光標識されたヌクレオチド類似体を含む試薬溶液、ならびに鋳型を標的とした核酸合成のために必要な別の試薬を含み、ここで、前記蛍光標識されたヌクレオチド類似体から放射された光を用いて前記鋳型の配列が決定される、請求項1記載のシステム。
- 前記ポリメラーゼ酵素が、DNAポリメラーゼ、RNAポリメラーゼ、逆転写酵素、およびそれらの混合物からなる群から選択される、請求項4記載のシステム。
- 前記核酸鋳型が、二本鎖もしくは一本鎖DNA、一本鎖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伴うRNA、またはRNAヘアピンである、請求項4記載のシステム。
- 前記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から選択される、請求項4記載のシステム。
- 前記ヌクレオチド類似体が、該ヌクレオチド類似体の塩基、糖成分、αリン酸、βリン酸、またはγリン酸において結合した蛍光標識を含む、請求項4記載のシステム。
- 前記ヌクレオチド類似体が、該ヌクレオチド類似体の末端のリン酸の位置に結合した標識を有する、請求項4記載のシステム。
- 前記核酸鋳型が、近接場励起容量内に固定化される、請求項4記載のシステム。
- 前記ポリメラーゼ酵素が、近接場励起容量内に固定化される、請求項4記載のシステム。
- 標識されたヌクレオチド類似体からの放射に対応するヌクレオチド塩基を同定し、その同定結果を保存するコンピュータをさらに含む、請求項4記載のシステム。
- 核酸を配列決定するための方法であって、
請求項4〜12のいずれか一項記載のシステムを供するステップと、
前記透明な層を介して孔を照射して、該孔の中に近接場励起容量を形成するステップと、
前記透明な層を介して通過し、前記孔から放射された光を、検出器を用いて検出するステップと、
を含む方法。 - 前記ポリメラーゼ酵素が、DNAポリメラーゼ、RNAポリメラーゼ、逆転写酵素、およびそれらの混合物からなる群から選択される、請求項13記載の方法。
- 前記核酸鋳型が、二本鎖もしくは一本鎖DNA、一本鎖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伴うRNA、またはRNAヘアピンである、請求項13記載の方法。
- 前記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から選択される、請求項13記載の方法。
- 前記標識されたヌクレオチド類似体が、該ヌクレオチド類似体の塩基、糖成分、αリン酸、βリン酸、またはγリン酸に結合した標識を含む、請求項13記載の方法。
- 前記標識されたヌクレオチド類似体が、該ヌクレオチド類似体の末端のリン酸の位置に結合した標識を有する、請求項13記載の方法。
- 前記核酸鋳型が、前記近接場励起容量内に固定化される、請求項13記載の方法。
- 前記ポリメラーゼ酵素が、前記近接場励起容量内に固定化される、請求項13記載の方法。
- 前記試薬溶液が、全部で4つの型のヌクレオチド類似体を含み、ここで該ヌクレオチド類似体のそれぞれの型が、アデニン、グアニン、シトシン、およびチミンからなる群から選択される、請求項13記載の方法。
- 前記4つの型のヌクレオチド類似体のそれぞれが識別標識を含み、前記照射により、該ヌクレオチド類似体が光を放射して、ヌクレオチド類似体が鋳型に依存した様式で重合化酵素により組み込まれていることを示す、請求項21記載の方法。
- 前記近接場励起容量から放射される光が、前記励起光の方向と逆平行である方向に沿って前記検出器に向けられる、請求項1記載のシステム。
- 前記励起光が、前記透明な層の下の位置から透明な層に向けられ、前記近接場励起容量から放射される光が、前記透明な層の下の位置で前記検出器に向けられる、請求項1記載のシステム。
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CA (1) | CA2373385C (ja) |
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