JP2014512834A - 内部標識プライマーを含む核酸の配列決定、増幅および検出のための方法 - Google Patents
内部標識プライマーを含む核酸の配列決定、増幅および検出のための方法 Download PDFInfo
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- JP2014512834A JP2014512834A JP2014508823A JP2014508823A JP2014512834A JP 2014512834 A JP2014512834 A JP 2014512834A JP 2014508823 A JP2014508823 A JP 2014508823A JP 2014508823 A JP2014508823 A JP 2014508823A JP 2014512834 A JP2014512834 A JP 2014512834A
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Abstract
Description
i)少なくとも1つの標識ヌクレオチドを含む、少なくとも1つの核酸プライマーを提供するステップと、
ii)前記少なくとも1つの標識核酸プライマーを用いて、標的核酸を増幅するための酵素および試薬を提供するステップと、
iii)標的核酸プライマーの増幅に適する条件下で反応の成分をインキュベートするステップと、
iv)標識ヌクレオチドを介して、増幅された核酸を検出するステップと
を含み、ここで、
核酸プライマーが、以下の配列:
5’−NaXNb−3’
を含み、式中、
Nは、任意のヌクレオチドまたは修飾ヌクレオチドであることが可能であり、
aおよびbは、ヌクレオチドの数を表し、1〜150の範囲であることが可能であり、
Xは、前記少なくとも1つの標識ヌクレオチドである。
少なくとも1つの標識ヌクレオチドを含む核酸プライマー
を含み、ここで、
この核酸プライマーは、以下の配列:
5’−NaXNb−3’
を含み、式中、
Nは、任意のヌクレオチドまたは修飾ヌクレオチドであることが可能であり、
aおよびbは、ヌクレオチドの数を表し、1〜150の範囲であることが可能であり、そして
Xは、前記少なくとも1つの標識ヌクレオチドである。
これらの例におけるPCRは、以下の通りに実行した:
0.2μlのMulti Taq2 DNAポリメラーゼ(QIAGEN、Hilden、Germany)を、ホットスタート用DNAポリメラーゼとして用いた。2.5μlのReaction MixB(QIAGEN、Hilden、Germany)を、PCR緩衝液として用いた。全てBiomers.net(Ulm、Germany)製である、0.4μMの未標識D16_revリバースプライマー(5’−gtttgtgtgtgcatctgtaagcatgtatc−3’;配列番号3)と組み合わせた、0.4μMの内部標識フォワードプライマーであるD16−P−intern(5’−gggggtctaagagcXtgtaaaaag−3’;配列番号1;配列中、Xは、チミジンヌクレオチドの環外アミノ基へと連結されたATTO550を表す)または0.4μMの標識フォワードプライマーであるD16−P−ATTO550(5’−ATTO550−gggggtctaagagcttgtaaaaag−3’;配列番号2)を、PCRの実行に用いた。
任意の内部塩基において標識されたプライマーを用いると、バックグラウンドノイズの大幅な低減が明らかである。しかし、さらなる実験では、プライマー内の内部標識の位置を選択することにより、効果をさらに増強しうることが示される。
Claims (11)
- 核酸を配列決定するかまたは核酸を増幅し、そして検出するための方法であって、前記方法は:
i)少なくとも1つの標識ヌクレオチドを含む、少なくとも1つの核酸プライマーを提供するステップと、
ii)前記少なくとも1つの標識核酸プライマーを用いて、標的核酸を増幅するための酵素および試薬を提供するステップと、
iii)標的核酸プライマーの増幅に適する条件下で反応の成分をインキュベートするステップと、
iv)前記標識ヌクレオチドを介して、増幅された核酸を検出するステップと
を含み、ここで
前記核酸プライマーは、以下の配列:
5’−NaXNb−3’
を含み、式中、
Nは、任意のヌクレオチドであることが可能であり、
「a」および「b」は、ヌクレオチドの数を表し、1〜150の範囲であることが可能であり、
Xは、前記少なくとも1つの標識ヌクレオチドである、方法。 - ステップiv)が、前記増幅された核酸をそれらの長さに従い分離するステップを含む、請求項1に記載の方法。
- 標識を、前記ヌクレオチドの環外アミノ基または2’−アミノ基を介して前記ヌクレオチドへと連結する、請求項1または2に記載の方法。
- 「a」が3〜25である、請求項1から3のいずれか一項に記載の方法。
- 「b」が3〜25である、請求項1から4のいずれか一項に記載の方法。
- 前記核酸プライマーの長さが10〜300ntである、請求項1から5のいずれか一項に記載の方法。
- 前記標識が、フルオロフォア、発色団、放射性同位体、および化学発光物質からなる標識の群から選択され、そして5−カルボキシフルオレセインまたは6−カルボキシフルオレセイン(FAM(商標))、VIC(商標)、NED(商標)、フルオレセイン、フルオレセインイソチオシアネート(FITC)、IRD−700/800、シアニン色素、例えば、CY3(商標)、CY5(商標)、CY3.5(商標)、CY5.5(商標)、Cy7(商標)、キサンテン、6−カルボキシ−2’,4’,7’,4,7−ヘキサクロロフルオレセイン(HEX)、6−カルボキシ−1,4−ジクロロ−2’,7’−ジクロロ−フルオレセイン(TET(登録商標))、6−カルボキシ−4’,5’−ジクロロ−2’,7’−ジメトキシフルオレセイン(JOE(商標))、N,N,N’,N’−テトラメチル−6−カルボキシローダミン(TAMRA(商標))、6−カルボキシ−X−ローダミン(ROX)、5−カルボキシローダミン−6G(R6G5)、6−カルボキシローダミン−6G(RG6)、ローダミン、ローダミングリーン、ローダミンレッド、ローダミン110、Rhodamin 6G(登録商標)、BODIPY TMRなどのBODIPY色素、Oregon Green、ウンベリフェロンなどのクマリン、Hoechst33258などのベンズイミド;フェナントリジン、例えば、Texas Red(登録商標)、California Red(登録商標)、Yakima Yellow、Alexa Fluor(登録商標)350、Alexa Fluor(登録商標)405、Alexa Fluor(登録商標)430、Alexa Fluor(登録商標)488、Alexa Fluor(登録商標)500、Alexa Fluor(登録商標)514、Alexa Fluor(登録商標)532、Alexa Fluor(登録商標)546、Alexa Fluor(登録商標)555、Alexa Fluor(登録商標)568、Alexa Fluor(登録商標)594、Alexa Fluor(登録商標)610、Alexa Fluor(登録商標)633、Alexa Fluor(登録商標)647、Alexa Fluor(登録商標)660、Alexa Fluor(登録商標)680、Alexa Fluor(登録商標)700、Alexa Fluor(登録商標)750、PET(登録商標)、臭化エチジウム、アクリジニウム色素、カルバゾール色素、フェノキサジン色素、ポルフィリン色素、ポリメチン(polymethin)色素、Atto390、Atto425、Atto465、Atto488、Atto495、Atto520、Atto532、Atto550、Atto565、Atto590、Atto594、Atto620、Atto633、Atto 647N、Atto655、Atto RhoG6、Atto Rho11、Atto Rho12、Atto Rho101、BMN(商標)−5、BMN(商標)−6、CEQ8000 D2、CEQ8000 D3、CEQ8000 D4、DY−480XL、DY−485XL、DY−495、DY−505、DY−510XL、DY−521XL、DY−521XL、DY−530、DY−547、DY−550、DY−555、DY−610、DY−615、DY−630、DY−631、DY−633、DY−635、DY−647、DY−651、DY−675、DY−676、DY−680、DY−681、DY−700、DY−701、DY−730、DY−731、DY−732、DY−750、DY−751、DY−776、DY−780、DY−781、DY−782、6−カルボキシ−4’,5’−ジクロロ−2’,7’−ジメトキシフルオレセイン(JOE)、TET(商標)、CAL Fluor(登録商標)Gold540、CAL Fluor RED590、CAL Fluor Red610、CAL Fluor Red635、IRDye(登録商標)700Dx、IRDye(登録商標)800CW、Marina Blue(登録商標)、Pacific Blue(登録商標)、Yakima Yellow(登録商標)、6−(4,7−ジクロロ−2’,7’−ジフェニル−3’,6’−ジピバロイルフルオレセイン−6−カルボキサミド)−ヘキシル−1−O−(2−シアノエチル)−(N,N−ジイソプロピル)−ホスホルアミダイト(SIMA)、CAL Fluor(登録商標)Gold540、CAL Fluor(登録商標)Orange560、CAL Fluor Red635、Quasar570、Quasar670、LIZ、Sunnyvale Red、LC Red(登録商標)610、LC Red(登録商標)640、LC Red(登録商標)670、およびLC Red(登録商標)705を含むフルオロフォアの群から選択される、請求項1から6のいずれか一項に記載の方法。
- 前記増幅の方法が、ポリメラーゼ連鎖反応(PCR)、リガーゼ連鎖反応(LCR)、転写ベースの増幅系(TAS)、核酸配列ベースの増幅(NASBA)、ローリングサークル増幅(RCA)、転写媒介増幅(TMA)、自己持続配列複製(3SR)およびQβ増幅、鎖置換増幅(SDA)、多重置換増幅(MDA)、ループ媒介等温増幅(LAMP)、ヘリカーゼ依存性増幅(HDA)、スマート増幅法(SMAP)、定量的リアルタイムPCR(qPCR)、逆転写PCR(RT−PCR)、サンガー配列決定からなる群より選択される、請求項1から7のいずれか一項に記載の方法。
- 核酸を配列決定するかまたは核酸を増幅し、そして検出するためのキットであって、前記キットは:
少なくとも1つの標識ヌクレオチドを含む核酸プライマー
を含み、ここで、
前記核酸プライマーが、以下の配列:
5’−NaXNb−3’
を含み、式中、
Nは、任意のヌクレオチドまたは修飾ヌクレオチドであることが可能であり、
aおよびbは、ヌクレオチドの数を表し、1〜150の範囲であることが可能であり、
Xは、前記少なくとも1つの標識ヌクレオチドである、キット。 - 核酸を増幅するための酵素および試薬をさらに含む、請求項9に記載のキット。
- 核酸を配列決定するかまたは核酸を増幅し、そして検出するための方法における、請求項9もしくは10に記載のキットまたは少なくとも1つの標識ヌクレオチドを含む核酸プライマーの使用であって、前記核酸プライマーは、以下の配列:
5’−NaXNb−3’
を有し、式中、
Nは、任意のヌクレオチドであることが可能であり、
「a」および「b」は、ヌクレオチドの数を表し、1〜150の範囲であることが可能であり、
Xは、前記少なくとも1つの標識ヌクレオチドである、使用。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013140771A (ja) * | 2011-12-09 | 2013-07-18 | Gigaphoton Inc | ターゲット供給装置 |
JP2020504074A (ja) * | 2016-12-22 | 2020-02-06 | イルミナ ケンブリッジ リミテッド | クマリン化合物および蛍光標識としてのそれらの使用 |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104561248A (zh) * | 2013-10-22 | 2015-04-29 | 常州金麦格生物技术有限公司 | 用于检测靶核酸的引物和其应用 |
CA2939621C (en) * | 2014-02-13 | 2019-10-15 | Takara Bio Usa, Inc. | Methods of depleting a target molecule from an initial collection of nucleic acids, and compositions and kits for practicing the same |
KR101677127B1 (ko) * | 2014-11-28 | 2016-11-18 | (주)에스엘에스 | 말 유전자 타이핑용 프라이머 세트 |
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EP3559262A4 (en) * | 2016-12-22 | 2020-07-22 | Illumina, Inc. | NETWORKS PRESENTING QUALITY CONTROL TRACERS |
CN109232663B (zh) * | 2018-11-08 | 2020-09-25 | 云南大学 | 一种钌配合物的制备方法及其艾滋病毒逆转录酶抑制应用 |
CN111454926B (zh) * | 2020-05-11 | 2022-10-28 | 南京君华基因科技有限公司 | 一种优化的扩增目标核酸的聚合酶、复合体系及应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000079009A2 (en) * | 1999-06-22 | 2000-12-28 | Invitrogen Corporation | Improved primers and methods for the detection and discrimination of nucleic acids |
WO2002057479A2 (en) * | 2000-12-27 | 2002-07-25 | Invitrogen Corporation | Primers and methods for the detection and discrimination of nucleic acids |
WO2006053259A2 (en) * | 2004-11-12 | 2006-05-18 | Transgenomic, Inc. | Fluorescent mutation detection with mismatch cutting dna endonucleases |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3223104A1 (de) | 1982-06-21 | 1983-12-22 | Hoechst Ag, 6230 Frankfurt | Photopolymerisierbares gemisch und damit hergestelltes photopolymerisierbares kopiermaterial |
IL88923A (en) | 1988-01-12 | 1995-07-31 | Hoffmann La Roche | Gene encoding a thermostable dna polymerase from thermus aquaticus said dna polymerase and its purification |
US5654419A (en) * | 1994-02-01 | 1997-08-05 | The Regents Of The University Of California | Fluorescent labels and their use in separations |
US6361940B1 (en) | 1996-09-24 | 2002-03-26 | Qiagen Genomics, Inc. | Compositions and methods for enhancing hybridization and priming specificity |
US6579680B2 (en) | 2000-02-28 | 2003-06-17 | Corning Incorporated | Method for label-free detection of hybridized DNA targets |
CN1141399C (zh) * | 2000-04-30 | 2004-03-10 | 厦门大学 | 用于均相荧光pcr检测的孪生引物 |
US9261460B2 (en) * | 2002-03-12 | 2016-02-16 | Enzo Life Sciences, Inc. | Real-time nucleic acid detection processes and compositions |
KR101038137B1 (ko) * | 2002-06-28 | 2011-05-31 | 프리메라디엑스, 인크. | 서열 차이를 감지하는 방법 |
US20050147976A1 (en) * | 2003-12-29 | 2005-07-07 | Xing Su | Methods for determining nucleotide sequence information |
GB2413796B (en) * | 2004-03-25 | 2006-03-29 | Global Genomics Ab | Methods and means for nucleic acid sequencing |
CN101124337B (zh) * | 2004-08-24 | 2010-11-17 | 康乃尔研究基金会有限公司 | 使用内切核酸酶剪切/连接酶重新密封反应和毛细管电泳或微阵列检测核酸差异 |
US20060105348A1 (en) * | 2004-11-15 | 2006-05-18 | Lee Jun E | Compositions and methods for the detection and discrimination of nucleic acids |
US20060188902A1 (en) * | 2005-01-03 | 2006-08-24 | The Gov. of the USA as represented by the Secretary of the Dept. of Health and Human | Primer for nucleic acid detection |
US8153372B2 (en) * | 2006-12-19 | 2012-04-10 | The Board Of Regents For Oklahoma State University | Method for simultaneously determining in a single multiplex reaction gender of donors and quantities of genomic DNA and ratios thereof, presence and extent of DNA degradation, and PCR inhibition within a human DNA sample |
-
2012
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- 2017-07-05 JP JP2017132022A patent/JP2017200488A/ja not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000079009A2 (en) * | 1999-06-22 | 2000-12-28 | Invitrogen Corporation | Improved primers and methods for the detection and discrimination of nucleic acids |
WO2002057479A2 (en) * | 2000-12-27 | 2002-07-25 | Invitrogen Corporation | Primers and methods for the detection and discrimination of nucleic acids |
WO2006053259A2 (en) * | 2004-11-12 | 2006-05-18 | Transgenomic, Inc. | Fluorescent mutation detection with mismatch cutting dna endonucleases |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013140771A (ja) * | 2011-12-09 | 2013-07-18 | Gigaphoton Inc | ターゲット供給装置 |
JP2020504074A (ja) * | 2016-12-22 | 2020-02-06 | イルミナ ケンブリッジ リミテッド | クマリン化合物および蛍光標識としてのそれらの使用 |
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