JP7013069B2 - 低塩条件下での等温増幅 - Google Patents
低塩条件下での等温増幅 Download PDFInfo
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- JP7013069B2 JP7013069B2 JP2016559274A JP2016559274A JP7013069B2 JP 7013069 B2 JP7013069 B2 JP 7013069B2 JP 2016559274 A JP2016559274 A JP 2016559274A JP 2016559274 A JP2016559274 A JP 2016559274A JP 7013069 B2 JP7013069 B2 JP 7013069B2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6844—Nucleic acid amplification reactions
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Description
Tm=(wA+xT)*2+(yG+zC)*4-16.6*log10(0.050)+16.6*log10([Na+])
(式中、w、x、yおよびzはそれぞれオリゴヌクレオチド配列中の塩基A、T、G、Cの数である)。16.6*log10([Na+])という用語は、塩濃度の変化のためにTmを調整し、log10(0.050)という用語は50mM Na+で塩調整をするために調整する。反応混合物が、オリゴヌクレオチドのTmに影響を及ぼし得る1種または複数の一価および二価塩を含有してもよい。ナトリウムイオンはDNA鎖との間で塩架橋を形成するのにはるかに有効であるので、二本鎖DNAを安定化するのに有意な効果を有する。融解温度(Tm)計算は、アニーリングが、0.01~1.0Mの間の濃度の一価カチオン(Na+またはK+のいずれか)の存在下、pH7.0で、50mMプライマーの標準条件下で起こり、非対称配列が少なくとも8塩基長であり、少なくとも1個のGまたはCを含有すると仮定する。(Nakano et al,(1999)Proc.Nucleic Acids Res.27:2957-65およびhttp://www.basic.northwestern.edu/biotools/oligocalc.html参照)。
PEGおよび低塩条件の存在下での単一細胞からのDNAのMDA反応の反応速度論および感度。
Claims (16)
- a)標的核酸鋳型を用意するステップと;
b)前記標的核酸鋳型を、鎖置換活性を有するDNAポリメラーゼと、デオキシリボヌクレオシド三リン酸(dNTP)混合物と、3’末端および5’末端を有するプライマーと、分子クラウディング試薬と、緩衝液とを含む反応混合物と接触させるステップであって、前記緩衝液は20mMの前記反応混合物の塩濃度を維持するステップと;
c)前記標的核酸鋳型を30℃における等温増幅条件下一定反応温度で増幅するステップと
を含む、多置換増幅(multiple displacement amplification)(MDA)によって核酸を増幅する方法であって、
前記標的核酸鋳型の投入量が少なくとも5フェムトグラムであり、
前記プライマーがランダムプライマーであり、および、
前記分子クラウディング試薬が、ポリエチレングリコールからなる群から選択される、方法。 - 前記プライマーが5ヌクレオチド~9ヌクレオチドの間の長さである、請求項1に記載の方法。
- 細菌由来の前記標的核酸鋳型の前記投入量が少なくとも5フェムトグラムである、請求項1に記載の方法。
- ヒト由来の前記標的核酸の前記投入量が少なくとも5ピコグラムである、請求項1に記載の方法。
- 前記分子クラウディング試薬が、PEG400、PEG2000、PEG6000、PEG8000またはこれらの組み合わせからなる群から選択される、請求項1に記載の方法。
- 前記核酸鋳型を増幅するステップが高ストリンジェンシー条件下で行われる、請求項1に記載の方法。
- 前記DNAポリメラーゼがphi29 DNAポリメラーゼである、請求項1に記載の方法。
- 前記プライマーがチオ化されている、請求項1に記載の方法。
- 前記プライマーがヌクレオチド類似体を含む、請求項1に記載の方法。
- 前記プライマーが3’末端ヌクレオチドと、前記3’末端ヌクレオチドに隣接するヌクレオチドとの間のホスホロチオエート結合を含む、請求項9に記載の方法。
- 前記プライマーがヌクレオチド塩基の前にあるロックド核酸(LNA)を含む、請求項9に記載の方法。
- 前記プライマーの前記ヌクレオチド類似体が2-アミノ-デオキシアデノシン(2-アミノ-dA)である、請求項9に記載の方法。
- 前記プライマーが、プライマーダイマー形成を防ぐためのヌクレオチド類似体2-チオ-デオキシチミジン(2-チオ-dT)をさらに含む、請求項12に記載の方法。
- 前記プライマーがヘキサマーである、請求項1に記載の方法。
- 前記プライマーがNNNN*N*Nの配列を含む、請求項14に記載の方法。
- 前記ヘキサマーが(atN)(atN)(atN)(atN)(atN)*N(式中、(atN)は前記ヘキサマーの5’末端ヌクレオチドであり、*Nは前記ヘキサマーの3’末端ヌクレオチドであり、「N」はデオキシアデノシン(dA)、デオキシシチジン(dC)、デオキシグアノシン(dG)またはデオキシチミジン(dT)を表し、(atN)は2-アミノ-dA、dC、dGおよび2-チオ-dTのランダム混合物を表し、「*」はホスホロチオエート結合を表す)の一般構造を有する、請求項14に記載の方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/225,887 US9587263B2 (en) | 2014-03-26 | 2014-03-26 | Isothermal amplification under low salt condition |
US14/225,887 | 2014-03-26 | ||
PCT/US2015/021272 WO2015148218A2 (en) | 2014-03-26 | 2015-03-18 | Isothermal amplification under low salt condition |
Publications (3)
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JP2017508474A JP2017508474A (ja) | 2017-03-30 |
JP2017508474A5 JP2017508474A5 (ja) | 2018-04-19 |
JP7013069B2 true JP7013069B2 (ja) | 2022-01-31 |
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JP2016559274A Active JP7013069B2 (ja) | 2014-03-26 | 2015-03-18 | 低塩条件下での等温増幅 |
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US (2) | US9587263B2 (ja) |
EP (1) | EP3122888B1 (ja) |
JP (1) | JP7013069B2 (ja) |
CN (1) | CN106460040B (ja) |
CA (1) | CA2939282C (ja) |
WO (1) | WO2015148218A2 (ja) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
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US10443094B2 (en) * | 2014-03-26 | 2019-10-15 | General Electric Company | Solid phase isothermal amplification |
WO2016011177A1 (en) * | 2014-07-18 | 2016-01-21 | Fluidigm Corporation | Reagents and kit compositions for single-cell whole genome amplification |
CN111621548A (zh) * | 2016-04-26 | 2020-09-04 | 序康医疗科技(苏州)有限公司 | 扩增dna的方法 |
EP3555314B1 (en) * | 2016-12-19 | 2023-08-16 | Quantum-Si Incorporated | Loading molecules into sample wells for analysis |
US11732254B2 (en) | 2017-06-30 | 2023-08-22 | Pacific Biosicences of California, Inc. | Size selection purification using a thermoplastic silica nanomaterial |
JP7348603B2 (ja) * | 2018-04-02 | 2023-09-21 | エニュメラ・モレキュラー・インコーポレイテッド | 核酸分子を計数するための方法、システム、および組成物 |
CN112899152B (zh) * | 2021-02-02 | 2023-11-17 | 中国科学院苏州纳米技术与纳米仿生研究所 | 核酸快速扩增和检测的微流控芯片、检测方法及系统 |
CN114277103A (zh) * | 2022-01-21 | 2022-04-05 | 杭州飞时达生物科技有限公司 | 一种基于六聚体随机引物高效滚环扩增方法 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6096548A (en) | 1996-03-25 | 2000-08-01 | Maxygen, Inc. | Method for directing evolution of a virus |
US6511803B1 (en) | 1997-10-10 | 2003-01-28 | President And Fellows Of Harvard College | Replica amplification of nucleic acid arrays |
DE19943374A1 (de) | 1999-09-10 | 2001-03-29 | Max Planck Gesellschaft | Verfahren zum Anbinden von Nukleinsäuren an eine Festphase |
US7993839B2 (en) * | 2001-01-19 | 2011-08-09 | General Electric Company | Methods and kits for reducing non-specific nucleic acid amplification |
US7662594B2 (en) * | 2002-09-20 | 2010-02-16 | New England Biolabs, Inc. | Helicase-dependent amplification of RNA |
US7955795B2 (en) * | 2003-06-06 | 2011-06-07 | Qiagen Gmbh | Method of whole genome amplification with reduced artifact production |
WO2004058987A2 (en) * | 2002-12-20 | 2004-07-15 | Qiagen Gmbh | Nucleic acid amplification |
KR101077603B1 (ko) | 2004-01-28 | 2011-10-27 | 삼성전자주식회사 | 카르복실기 또는 아미노기로 코팅된 고체상 물질을 이용한핵산의 증폭 방법 |
US8426126B2 (en) | 2004-03-18 | 2013-04-23 | Applied Biosystems, Llc | Modified surfaces as solid supports for nucleic acid purification |
KR100785016B1 (ko) | 2006-05-22 | 2007-12-12 | 삼성전자주식회사 | 단일 마이크로 챔버에서 핵산의 농축 및 증폭을 수행하는방법 및 장치 |
WO2008051928A2 (en) * | 2006-10-23 | 2008-05-02 | The Salk Institute For Biological Studies | Target-oriented whole genome amplification of nucliec acids |
US20110195457A1 (en) | 2010-02-09 | 2011-08-11 | General Electric Company | Isothermal amplification of nucleic acid using primers comprising a randomized sequence and specific primers and uses thereof |
EP2566985A4 (en) | 2010-05-06 | 2014-08-06 | Ibis Biosciences Inc | INTEGRATED SAMPLE PREPARATION SYSTEMS AND MIXTURES OF STABILIZED ENZYMES |
US9353393B2 (en) | 2012-02-15 | 2016-05-31 | General Electric Company | Methods and kits for reducing non-specific nucleic acid amplification |
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2014
- 2014-03-26 US US14/225,887 patent/US9587263B2/en active Active
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2015
- 2015-03-18 EP EP15768311.1A patent/EP3122888B1/en active Active
- 2015-03-18 CN CN201580015896.3A patent/CN106460040B/zh active Active
- 2015-03-18 CA CA2939282A patent/CA2939282C/en active Active
- 2015-03-18 WO PCT/US2015/021272 patent/WO2015148218A2/en active Application Filing
- 2015-03-18 JP JP2016559274A patent/JP7013069B2/ja active Active
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2017
- 2017-01-18 US US15/408,509 patent/US10597691B2/en active Active
Non-Patent Citations (1)
Title |
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Analytical biochemistry,2006年,Vol.355,p.298-303 |
Also Published As
Publication number | Publication date |
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CN106460040A (zh) | 2017-02-22 |
WO2015148218A3 (en) | 2015-11-19 |
CA2939282A1 (en) | 2015-10-01 |
EP3122888A4 (en) | 2017-11-22 |
CN106460040B (zh) | 2021-01-08 |
EP3122888A2 (en) | 2017-02-01 |
US20150275282A1 (en) | 2015-10-01 |
US9587263B2 (en) | 2017-03-07 |
US10597691B2 (en) | 2020-03-24 |
US20170121747A1 (en) | 2017-05-04 |
EP3122888B1 (en) | 2019-06-26 |
CA2939282C (en) | 2024-04-23 |
JP2017508474A (ja) | 2017-03-30 |
WO2015148218A2 (en) | 2015-10-01 |
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