JP6396299B2 - 配列決定方法 - Google Patents
配列決定方法 Download PDFInfo
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- JP6396299B2 JP6396299B2 JP2015535114A JP2015535114A JP6396299B2 JP 6396299 B2 JP6396299 B2 JP 6396299B2 JP 2015535114 A JP2015535114 A JP 2015535114A JP 2015535114 A JP2015535114 A JP 2015535114A JP 6396299 B2 JP6396299 B2 JP 6396299B2
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- C—CHEMISTRY; METALLURGY
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- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Description
a.その少なくともいくつかが単一ヌクレオチドを含有する液滴のストリームを生成するステップであって、前記液滴のストリーム中の単一ヌクレオチドの順序が検体中のヌクレオチドの配列に対応する、ステップ;
b.各液滴中に複数の生体プローブタイプを導入するステップであって、各タイプが(i)異なる検出可能要素を検出不能な状態で備え、(ii)検体を構成する異なる相補的な単一ヌクレオチドを捕捉するように構成された、ステップ;
c.前記液滴中に含有される単一ヌクレオチドをその相補的なプローブに結合させ、使用済プローブをもたらすステップ;および
d.前記検出可能要素を使用済プローブから検出可能な状態に解放させるステップ
により特徴づけられる、ポリヌクレオチド検体中のヌクレオチド塩基の配列を決定する方法が提供される。
本発明の好適な実施形態において、各生体プローブは、末端が2つの異なる二本鎖オリゴヌクレオチド領域に結合した一本鎖ヌクレオチド領域を備えることを特徴とし、オリゴヌクレオチド領域の少なくとも1つは、特徴的な検出特性を有する検出可能要素を備え、検出可能要素は、オリゴヌクレオチド領域上において、同じ数の検出可能要素が対応する数の単一ヌクレオチドに結合した場合よりも検出可能特性が検出できないように配置される。
(5’)GGCACGATGGXXAXXGCCCGCACTTCAGCGGGCAAYAACCATCGTGCCTGCAGGCTCGACCTTTATTCGCGGCACTTCAGCCGCGAATAAAGGTCGAGCCTGC(3’)
[XはQuasar 570(フルオロフォア)で標識されたT塩基であり、YはBHQ−2クエンチャで標識されたT塩基である]を有する103ヌクレオチド一本鎖オリゴヌクレオチド前駆体(例えばATDBio)を、その水溶液を95℃まで加熱した後、1℃当たり10分の割合で室温までゆっくり冷却することにより、第49ヌクレオチド塩基辺りで折り畳む。この時間の終わりに、第49ヌクレオチド塩基(ここではT)が一本鎖ヌクレオチド領域ならびにその両側にそれぞれ24および27ヌクレオチド塩基対長の2つの二本鎖オリゴヌクレオチドを備えた、本発明によるクローズドループプローブが形成される。
Claims (20)
- a.ポリヌクレオチド検体の分解により、その少なくともいくつかが単一ヌクレオチドを含有する液滴のストリームを生成するステップであって、前記液滴のストリーム中の単一ヌクレオチドの順序が前記検体中のヌクレオチドの配列に対応する、ステップ;
b.各液滴中に複数の生体プローブタイプを導入するステップであって、各タイプが(i)末端が2つの異なる二本鎖オリゴヌクレオチド領域に結合した一本鎖ヌクレオチド領域であって、前記オリゴヌクレオチド領域の少なくとも1つが、特徴的な検出特性を有する検出可能要素を備え、前記検出可能要素が、前記オリゴヌクレオチド領域上において、同じ数の検出可能要素が対応する数の単一ヌクレオチドに結合した場合よりも検出可能特性が検出できないように配置される、一本鎖ヌクレオチド領域を備え、(ii)検体を構成する異なる相補的な単一ヌクレオチドを捕捉するように構成された、ステップ;
c.ポリメラーゼ及び/又はリガーゼにより、前記液滴中に含有される単一ヌクレオチドをその相補的なプローブに結合させ、使用済プローブをもたらすステップ;および
d.前記検出可能要素を使用済プローブから検出可能な状態に解放させるステップ
により特徴づけられ、
前記検出可能要素がフルオロフォアであり、
各二本鎖オリゴヌクレオチド領域は長さが最大で50のヌクレオチド対であり、
前記一本鎖ヌクレオチド領域が、チミン、グアニン、シトシン、アデニン、及びウラシルの1つから選択される1つのヌクレオチドからなる、ポリヌクレオチド検体中のヌクレオチド塩基の配列を決定する方法。 - 前記プローブは、フルオロフォアが検出される波長または波長包絡線において本質的に非蛍光性であることを特徴とする、請求項1に記載の方法。
- 前記オリゴヌクレオチド領域が、前記一本鎖ヌクレオチド領域によって互いに接続された第1および第2の二本鎖オリゴヌクレオチドを備え、前記二本鎖オリゴヌクレオチドの少なくとも1つが、互いに近接した複数のフルオロフォアで標識されることを特徴とする、請求項2に記載の方法。
- 前記二本鎖オリゴヌクレオチドの少なくとも1つが、前記フルオロフォアに近接したクエンチャで標識されることを特徴とする、請求項1または請求項2に記載の方法。
- 前記単一ヌクレオチドが、チミン、グアニン、シトシン、アデニンおよびウラシルの1つから選択されるヌクレオチド塩基からなることを特徴とする、請求項1〜4のいずれか1項に記載の方法。
- 各二本鎖オリゴヌクレオチドが、10〜30のヌクレオチド対からなることを特徴とする、請求項1〜5のいずれか1項に記載の方法。
- 二本鎖オリゴヌクレオチドにおける最大で10のヌクレオチド対が、フルオロフォアで標識されることを特徴とする、請求項1〜6のいずれか1項に記載の方法。
- 二本鎖オリゴヌクレオチドにおける最大で10のヌクレオチド対が、クエンチャで標識されることを特徴とする、請求項1〜7のいずれか1項に記載の方法。
- クローズドループである前記一本鎖ヌクレオチド領域から離れた末端をそれぞれ備える2つの別々の二本鎖オリゴヌクレオチドが用いられることを特徴とする、請求項1〜8のいずれか1項に記載の方法。
- 前記二本鎖オリゴヌクレオチドが、末端を折り畳み、前記一本鎖ヌクレオチド領域を備えるギャップを残すことにより、一本鎖オリゴヌクレオチド前駆体から誘導されることを特徴とする、請求項9に記載の方法。
- 前記生体プローブが、少なくとも1つの制限酵素認識部位を備えることを特徴とする、請求項1〜10のいずれか1項に記載の方法。
- 前記制限酵素認識部位が、単一ヌクレオチドを生体プローブの一本鎖ヌクレオチド領域に結合させることによりもたらされることを特徴とする、請求項11に記載の方法。
- 前記使用済プローブが、一本鎖ヌクレオチド領域への単一ヌクレオチドの結合により形成された制限酵素認識部位を備えることを特徴とする、請求項1〜12のいずれか1項に記載の方法。
- 前記生体プローブのそれぞれタイプが、異なる検出可能要素を有することを特徴とする、請求項1〜13のいずれか1項に記載の方法。
- ステップcが、ポリメラーゼおよびリガーゼの存在下で行われることを特徴とする、請求項1〜14のいずれか1項に記載の方法。
- ステップdが、制限酵素およびエキソヌクレアーゼの存在下で行われることを特徴とする、請求項1〜15のいずれか1項に記載の方法。
- 前記検出可能要素が、光学または分光手段により検出されることを特徴とする、請求項1〜16のいずれか1項に記載の方法。
- 前記液滴のストリームが、油担体中の水性液滴のストリームを備えることを特徴とする、請求項1〜17のいずれか1項に記載の方法。
- 前記液滴が直径50ミクロン未満であることを特徴とする、請求項18に記載の方法。
- 前記ポリヌクレオチド検体が、自然発生のDNAおよびRNAに由来することを特徴とする、請求項1〜19のいずれか1項に記載の方法。
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