JP2008104463A - ポリヌクレオチド配列決定方法 - Google Patents
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Abstract
【解決手段】以下の工程を含む、ポリヌクレオチドの配列を決定する方法を提供する:
(i)定められた位置に固定化されたポリヌクレオチド進行型酵素と、標的ポリヌクレオチドとを、 酵素活性の誘導に十分な条件下で接触させる工程;
(ii)酵素とポリヌクレオチドとの相互作用の結果である効果を検出する工程、ここで、該効果は、非線形光シグナルまたは非線形シグナルに結合した線形シグナルの測定により検出する。
【選択図】なし
Description
本発明は、ポリヌクレオチドの配列を決定する方法に関する。
ヒトゲノムにコードされる三十億塩基のDNAのマッピングにおいてヒトゲノムプロジェクトによって示されるように、ポリヌクレオチドの配列決定を可能にすることは、科学的に非常に重要である。
本発明は、酵素が標的ポリヌクレオチドと結合し、それに沿って移動する際に起こる、ポリヌクレオチド進行型酵素における立体構造および/または質量および/またはエネルギー分布の変化が、二次または三次高調波発生に基づくものを含む、非線形光学的イメージングを用いて検出できるということの認識に基づくものである。
(i)定められた位置に固定化されたポリヌクレオチド進行型酵素と標的ポリヌクレオチドとを酵素活性に十分な条件下で接触させる工程;および、
(ii)酵素とポリヌクレオチドとの相互作用の結果である効果を検出する工程、
ここで、該効果は、非線形光シグナル、または非線形シグナルと結合した線形シグナルの測定によって検出される。
本発明を添付の図面を参照して説明する、ここで:
図 1は、二次高調波発生を利用するイメージングシステムの模式図である; そして、
図 2は、特定のポリヌクレオチドの取り込みの際にポリメラーゼによって生じた二次高調波シグナルを示す。
本発明は、ポリヌクレオチド進行型酵素が、標的ポリヌクレオチド上の個々の塩基と相互作用するか、または、合成中のポリヌクレオチド分子にヌクレオチドを取り込む際に起こる立体構造および/または質量および/またはエネルギー分布の変化を同定するために通常の非線形光学的測定を利用する。
P = X(1)E1 + X(2)E2 + X(3)E3 + .....
ここで、Pは、誘導分極、X(n)は、n次の非線形感受率(susceptibility)、およびEは、電場ベクトルである。第一項は通常の光の吸収および反射を表現する; 第二項は二次高調波発生 (SHG)、和および差周波数発生を表現する;そして第三項は、光散乱、誘導ラマン過程、三次高調波発生 (TGH)、および2および3光子吸収の両方を表現する。
この実験において、緑色蛍光タンパク質 (GFP)とポリメラーゼとの融合タンパク質を、当該技術分野において周知の組換え技術によって作成した。
CAAGGAGAGGACGCTGCTTGTCGAAGGTAAGGAACGGACGAGAGAAGGGAGAG
配列番号2
CTCTCCCTTCTCTCGTC
Claims (25)
- 以下の工程を含む、ポリヌクレオチドの配列を決定する方法:
(i)定められた位置に固定化されたポリヌクレオチド進行型酵素と、標的ポリヌクレオチドとを、酵素活性の誘導に十分な条件下で接触させる工程;
(ii)酵素とポリヌクレオチドとの相互作用の結果である効果を検出する工程、
ここで効果は、非線形光シグナルまたは非線形シグナルと結合した線形シグナルの測定によって検出する。 - 効果が、非線形シグナルの測定によって検出される請求項1の方法。
- 非線形光学的検出が、二次または三次高調波発生イメージングである、請求項1または請求項2の方法。
- 非線形光学的検出が、ラマン分光法または表面増強ラマン分光法である、請求項1または請求項2の方法。
- 双極子分子が酵素上または酵素の近くに位置する請求項1-4のいずれかの方法。
- 該分子がスチリル色素分子である請求項5の方法。
- 該分子が緑色蛍光タンパク質である請求項5の方法。
- 双極子分子が、ポリヌクレオチドの個々の塩基に結合している請求項5または請求項6の方法。
- 酵素がポリメラーゼである、請求項1-8のいずれかの方法。
- 酵素がヘリカーゼまたはプライマーゼ酵素である、請求項1-8のいずれかの方法。
- 工程(i)が、ヌクレオシド三リン酸、dATP、dTTP、dGTPおよびdCTPの添加を含む、請求項10の方法。
- ヌクレオシド三リン酸が、パルス単色光によって選択的に除去できる1以上の保護基を含む、請求項11の方法。
- 金属ナノ粒子が酵素上または酵素の近くに位置している請求項1-12のいずれかの方法。
- ナノ粒子が金または銀ナノ粒子である請求項13の方法。
- ナノ粒子がポリヌクレオチドの個々の塩基の1以上の上に取り込まれている請求項13または請求項14の方法。
- 酵素が固体支持体に固定化されている請求項1-15のいずれかの方法。
- 固体支持体に固定化された酵素が複数存在する請求項16の方法。
- 固体支持体が金属粗面を有する請求項16または請求項17の方法。
- 支持体が銀または金である請求項16-18のいずれかの方法。
- 検出を原子間力顕微鏡法または近接場走査型光学顕微鏡法とともに行う請求項1-19のいずれかの方法。
- 局在型表面プラズモン共鳴の適用をさらに含む、請求項1-20のいずれかの方法。
- 酵素が細胞中の定められた位置に固定化されている、請求項1-14のいずれかの方法。
- 少なくとも1つの固定化されたポリメラーゼ、およびポリメラーゼ上またはポリメラーゼの近くに位置する少なくとも1つの双極子分子を含む、固体支持体材料。
- 表面に細胞を固定化して含む固体支持体材料であって、該細胞が細胞中の固定された位置にポリメラーゼ酵素を維持して含む、固体支持体材料。
- その表面にポリヌクレオチドと相互作用する酵素を固定化して有する固体支持体、および、酵素上または酵素の近くに位置する双極子分子を含む、非線形光シグナルを検出するためのイメージングシステム設定。
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GBGB0112238.1A GB0112238D0 (en) | 2001-05-18 | 2001-05-18 | Sequencing method |
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EP (1) | EP1389241B1 (ja) |
JP (2) | JP4094961B2 (ja) |
KR (1) | KR20030094412A (ja) |
CN (1) | CN1537172B (ja) |
AT (1) | ATE383442T1 (ja) |
AU (1) | AU2002257933B2 (ja) |
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2001
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Patent Citations (2)
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WO2000053805A1 (en) * | 1999-03-10 | 2000-09-14 | Asm Scientific, Inc. | A method for direct nucleic acid sequencing |
WO2001025480A2 (en) * | 1999-10-06 | 2001-04-12 | Medical Biosystems Ltd. | Dna sequencing method |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2012137485A (ja) * | 2010-12-08 | 2012-07-19 | Osaka Prefecture Univ | 金属ナノ粒子集積構造体を利用した圧力検出装置、温度検出装置、圧力検出方法、および温度検出方法 |
JP2018527020A (ja) * | 2015-07-21 | 2018-09-20 | オムニオム インコーポレイテッドOmniome, Inc. | 核酸の配列決定方法及びシステム |
US11499185B2 (en) | 2015-07-21 | 2022-11-15 | Pacific Biosciences Of California, Inc. | Nucleic acid sequencing methods and systems |
Also Published As
Publication number | Publication date |
---|---|
WO2002095070A3 (en) | 2003-09-18 |
CA2447222A1 (en) | 2002-11-28 |
EP1389241B1 (en) | 2008-01-09 |
US20170022556A1 (en) | 2017-01-26 |
PT1389241E (pt) | 2008-04-17 |
KR20030094412A (ko) | 2003-12-11 |
HK1061047A1 (en) | 2004-09-03 |
CN1537172B (zh) | 2013-02-20 |
US20090029383A1 (en) | 2009-01-29 |
DE60224524T2 (de) | 2009-01-08 |
ATE383442T1 (de) | 2008-01-15 |
BR0209841A (pt) | 2006-02-07 |
JP4907498B2 (ja) | 2012-03-28 |
JP2004535800A (ja) | 2004-12-02 |
CN1537172A (zh) | 2004-10-13 |
AU2002257933B2 (en) | 2006-08-10 |
RU2003136429A (ru) | 2005-05-27 |
DK1389241T3 (da) | 2008-05-26 |
GB0112238D0 (en) | 2001-07-11 |
US20040241678A1 (en) | 2004-12-02 |
IS7039A (is) | 2003-11-17 |
CA2447222C (en) | 2012-09-18 |
NZ529349A (en) | 2006-10-27 |
US9476094B2 (en) | 2016-10-25 |
DE60224524D1 (de) | 2008-02-21 |
ES2299569T3 (es) | 2008-06-01 |
MXPA03010411A (es) | 2004-06-25 |
IL158743A0 (en) | 2004-05-12 |
WO2002095070A2 (en) | 2002-11-28 |
RU2291197C2 (ru) | 2007-01-10 |
JP4094961B2 (ja) | 2008-06-04 |
EP1389241A2 (en) | 2004-02-18 |
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