[go: up one dir, main page]

CN1123328A - 高温、高传真脱氧核糖核酸(DNA)聚合酶(HiFi Bst) - Google Patents

高温、高传真脱氧核糖核酸(DNA)聚合酶(HiFi Bst) Download PDF

Info

Publication number
CN1123328A
CN1123328A CN94113990.5A CN94113990A CN1123328A CN 1123328 A CN1123328 A CN 1123328A CN 94113990 A CN94113990 A CN 94113990A CN 1123328 A CN1123328 A CN 1123328A
Authority
CN
China
Prior art keywords
dna
bst
hifi bst
polymerase
hifi
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN94113990.5A
Other languages
English (en)
Inventor
洪国藩
翟峰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Center for Excellence in Molecular Cell Science of CAS
Original Assignee
Center for Excellence in Molecular Cell Science of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Center for Excellence in Molecular Cell Science of CAS filed Critical Center for Excellence in Molecular Cell Science of CAS
Priority to CN94113990.5A priority Critical patent/CN1123328A/zh
Priority to US08/544,643 priority patent/US5747298A/en
Priority to EP95308132A priority patent/EP0712927A3/en
Priority to US08/642,684 priority patent/US5834253A/en
Publication of CN1123328A publication Critical patent/CN1123328A/zh
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/6869—Methods for sequencing

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明表明，一种将嗜热脂肪芽孢杆菌(Bacillusstearothermophilus)，经紫外线等处理制备所获得的高温、高传真DNA聚合酶(HiFi Bst)。具有高效地切除不配对的核苷酸的活力，使DNA顺序反应具有独特的专一性，从而保证了DNA顺序测定图谱的高质量。

Description

高温、高传真脱氧核糖核酸(DNA)聚合酶(HiFi Bst)
本发明涉及用于DNA顺序测定的聚合酶和它的分析系统。
我们从嗜热脂防芽孢杆菌(Bacillus stearothermophilus)中制备得到DNA聚合酶，进而又用Subtilisin将此DNA聚合酶水解成二个片段。其中一个大片段定名为Bst。经过反复研究，证明Bst具有如下九个重要的技术指标：
(1)Bst可在65—70℃测定DNA顺序，从而消除堆积现象；
(2)在此高温条件下，无非专一的引物与模块的结合，从而消除了杂带；
(3)包括胞嘧啶在内的所有四种核苷酸都能均匀地渗入，消除了“C”不清的现象；
(4)此高温DNA顺序测定系统既可用于标准的Sanger一步法，也可用于将同位素标记和聚合—终止反应分开的二步法；
(5)此高温体系同样适用于双链DNA顺序测定；
(6)此高温体系同样适用于36S标记和32P标记的核苷酸；
(7)二种去压缩剂—7-deaza-dGTP和dITP均可在此高温体系中使用；
(8)此高温体系在室温可放置二个星期而不失活力，充分满足了DNA顺序测定自动化的高稳定性要求；
(9)此高温体系既适用于荧光法自动化DNA顺序测定，也适用于放射性同位素法自动化DNA顺序测定。
经与其他DNA聚合酶比较，Bst的特点总结如下；
特性           T7  Klenow  Taq  Bst  Vent  AMVRT
3′→5′外切酶 无   低     无         无    无
5′→3′外切酶 无   无     有    无   无    无
延伸率         高   中     中    高   中    低
对核苷酸衍生物的利用dITP           能   能     不     能   能    ？
7-deaza-dGT    能   能     能     能   能
条带的均一性  极好  差     好    较好  好   一般
顺序测定温度(℃)＜55 ＜50  ＜70    ＜65 ＜75  ＜42
从上表可看出，Bst的特性使它成为测定DNA顺序的理想的酶。从上表也可看出，关于Bst的3′→5′外切酶的特性不知。而其余的酶均没有3′→5′活力(Klenow具有较低的3′→5′活力。由于Klenow的延伸率不高，条带均一性差，及顺序测定温度低，在DNA顺序测定中已不应用)。
我们用紫外线处理Bacillus Stearothermophilus获得高产菌Mo-1。从Mo-1中获得HiFi Bst。用双链DNA作模板，证明HiFi Bst有3′→5′外切酶活力，并具有如下两个特性；(1)它切除配对的核苷酸，也切除不配对的核苷酸。(2)切除不配对的核苷酸的速度大于切除配对核苷酸的速度。这两个特性是目前国际上所有用于DNA顺序测定的DNA聚合酶所没有的，从而使HiFi Bst具有特殊的高度校正活力。本发明提供了DNA顺序测定中校正活力最好的DNA聚合酶HiFi Bst，因为HiFi Bst可以有效切除不配对的核苷酸，使DNA顺序反应具有独特的专一性，从而保证了DNA顺序测定图谱的高质量。
本文中简写说明：
TE：    100mmol/L Tris(pH8.0)1mmol/L EDTA(pH8.0)
HiFi Bst反应缓冲液：15mM Tris-Cl，pH8.5，15mM MgCl2
DE-81：whatman产品
图1是活力测定结果
Figure A9411399000061
：HiFi Bst切除非配对核苷酸的活力反应
■：HiFi Bst切除配对核苷酸的活力反应
X轴：单位：小时
Y轴：计数/30秒(由FH-408自动定标器测定)
在下述的实施例中对本发明进行具体的描述：
实施例1：高温、高传真DNA聚合酶(HiFi Bst)的制备嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)，经紫外线处理获得高产菌Mo-1，经制备得到DNA聚合酶，进而用枯草杆菌蛋白酶(Subtilisin)将此DNA聚合酶水解成二个片段[Ye，S.and Hong，G.，Scientia Sinica，30，503-506(1987)]，其中大片段即是高温、高传真DNA聚合酶(HiFi Bst)。
实施例2：引物的设计
5′  C A T T T T G C T G C C G G T C A    3′
实施例3：DNA模板设计
(a)3′——G T A A A A C G A C G G C C A G T C T T——5′
(b)3′——G T A A A A C G A C G G C C A G T C G G——5′
实施例4：引物的同位素标记
5′ C A T T T T G C T G C C G G T C A    3′
——G T A A A A C G A C G G C C A G T C T T——
        ↓  α32pdATP，dGTP，Taq酶
C A T T T T G C T G C C G G T C A G  A* A*(*表示32P标记)
实施例5：将标记引物与DNA模板配对
                引物
    ＋                 ＋
DNA模板(a)          DNA模板(b)
        5′C A T T T T G C T G C C G G T C A G A*A* 3′
    3′——G T A A A A C G A C G G C C A G T C G G——5′
 5′C A T T T T G C T G C C G G T C A G A* A*  3′3′——G T A A A A C G A C G G C C A G T C T  T —5′
实施例6：HiFi Bst切除配对核苷酸的活力的测定：
将退火的模板DNA(a)和标记的引物混合物溶于10μl TE中，加入4μl HiFi Bst反应缓冲液，2单位HiFi Bst酶，加水至20μl，充分混合后，以3μl/管分装入多个0.5ml微型离心管中，再加3μl石蜡油封管，置65℃水浴中保温。每间隔一定时间取出一管，将管内反应混合物滴到DE-81纸上，在定标仪上进行初级计数。然后用0.3M磷酸钠缓冲液(pH6.8)洗涤三次，每次10分钟，然后进行第二次计数，二次计数之差即为被HiFi Bst切除的游离的同位素的量。外切酶活力以单位时间内产生游离同位素的量表示。
实施例7：HiFi Bst切除非配对核苷酸的活力的测定
将退火的模板DNA(b)和标记的引物混合物溶于10μl TE中，加入4μl Bst反应缓冲液，2单位HiFi Bst酶加水至20μl，充分混匀后，以3μl/管分装入多个0.5ml微型离心管中，再加入3μl石蜡油封管，置65℃水浴中保温。每间隔一定时间取出一管，将管内反应混合物滴到DE-81纸上，在定标仪上进行初级计数。然后用0.3M磷酸钠缓冲液(pH6.8)洗涤三次，每次10分钟，然后进行第二次计数，二次计数之差即为被HiFi Bst切除的游离的同位素的量。外切酶活力以单位时间内产生游离同位素的量表示。

Claims (2)

1.一种用于DNA顺序测定的高温、高传真DNA聚合酶(HiFi Bst)和它的分析系统，其特征在于：
(1)它切除配对的核苷酸，也切除不配对的核苷酸；
(2)切除不配对的核苷酸的速度大于切除配对核苷酸的速度；
(3)引物的设计为：
5′ C A T T T T G C T G C C G G T C A    3′
(4)DNA模板设计
(a)3′——G T A A A A C G A C G G C C A G T C T T——5′
(b)3′——G T A A A A C G A C G G C C A G T C G G——5′
(5)引物的同位素标记：
5′ C A T T T T G C T G C C G G T C A    3′
——G T A A A A C G A C G G C C A G T C T T——
       ↓   α32pdATP，dGTP，Taq酶
C A T T T T G C T G C C G G T C A G A* A*(*表示32P标记)
(6)将标记引物与DNA模板配对：
         引物
    ＋         ＋
DNA模板(a)   DNA模板(b)
      5′C A T T T T G C T G C C G G T C A G A* A* 3′
   3′——G T A A A A C G A C G G C C A G T C G G——5′
5′C A T T T T G C T G C C G G T C A G A* A*  3′3′——G T A A A A C G A C G G C C A G T C T  T —5′
2.根据权利要求1中所述的高温、高传真DNA聚合酶(HiFi Bst)其特征在于是用紫外线处理嗜热脂肪芽孢杆菌(Bacillus stearother-mophilus)，获得高产菌，从中制备的DNA聚合酶，再经枯草杆菌蛋白酶(Subtilisin)将此酶水解成二个片段，其中大片段即是HiFi Bst。
CN94113990.5A 1994-11-17 1994-11-17 高温、高传真脱氧核糖核酸(DNA)聚合酶(HiFi Bst) Pending CN1123328A (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN94113990.5A CN1123328A (zh) 1994-11-17 1994-11-17 高温、高传真脱氧核糖核酸(DNA)聚合酶(HiFi Bst)
US08/544,643 US5747298A (en) 1994-11-17 1995-10-18 DNA polymerase with proof-reading 3'-5' exonuclease activity Bacillus stearothermophilus
EP95308132A EP0712927A3 (en) 1994-11-17 1995-11-14 Bst DNA polymerase with proof-reading 3'-5' exonuclease activity
US08/642,684 US5834253A (en) 1994-11-17 1996-05-03 Bacillus stearothermophilus DNA polymerase with proof-reading 3'-5' exonuclease activity

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN94113990.5A CN1123328A (zh) 1994-11-17 1994-11-17 高温、高传真脱氧核糖核酸(DNA)聚合酶(HiFi Bst)

Publications (1)

Publication Number Publication Date
CN1123328A true CN1123328A (zh) 1996-05-29

Family

ID=5036946

Family Applications (1)

Application Number Title Priority Date Filing Date
CN94113990.5A Pending CN1123328A (zh) 1994-11-17 1994-11-17 高温、高传真脱氧核糖核酸(DNA)聚合酶(HiFi Bst)

Country Status (3)

Country Link
US (1) US5747298A (zh)
EP (1) EP0712927A3 (zh)
CN (1) CN1123328A (zh)

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834253A (en) * 1994-11-17 1998-11-10 Shanghai Institute Of Biochemistry, Chinese Academy Of Sciences Bacillus stearothermophilus DNA polymerase with proof-reading 3'-5' exonuclease activity
US5814506A (en) * 1995-08-02 1998-09-29 New England Biolabs, Inc. Over-expression and purification of a truncated thermostable DNA polymerase by protein fusion
US6818431B1 (en) * 1995-10-18 2004-11-16 Shanghai Mendel Dna Center Co. Ltd. DNA polymerase having ability to reduce innate selective discrimination against fluorescent dye-labeled dideoxynucleotides
US6165765A (en) * 1995-10-18 2000-12-26 Shanghai Institute Of Biochemistry, Chinese Academy Of Sciences DNA polymerase having ability to reduce innate selective discrimination against fluorescent dye-labeled dideoxynucleotides
US6291164B1 (en) 1996-11-22 2001-09-18 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
SG86311A1 (en) * 1997-04-10 2002-02-19 Univ Singapore Bacillus stearothermophilus dna polymerase i (klenow) clones with reduced 3'-to-5' exonuclease activity
DE60133992D1 (de) 2000-02-17 2008-06-26 Qiagen Gmbh Thermostabile chimäre nucleinsäurepolymerasen und ihre verwendungen
US6632645B1 (en) 2000-03-02 2003-10-14 Promega Corporation Thermophilic DNA polymerases from Thermoactinomyces vulgaris
US6436677B1 (en) * 2000-03-02 2002-08-20 Promega Corporation Method of reverse transcription
US6994963B1 (en) * 2000-07-10 2006-02-07 Ambion, Inc. Methods for recombinatorial nucleic acid synthesis
DE10049211A1 (de) 2000-10-05 2002-04-18 Qiagen Gmbh Thermostabile Polymerase aus Thermococcus pacificus
CN1384203A (zh) * 2001-04-30 2002-12-11 香港科技创业股份有限公司 具有高度延伸专一性的dna低温循环延伸反应方法
US20030134292A1 (en) * 2001-10-30 2003-07-17 Farchaus Joseph W. Thermostable DNA polymerases and methods of making same
US20030180741A1 (en) 2001-12-21 2003-09-25 Holly Hogrefe High fidelity DNA polymerase compositions and uses therefor
US7148049B2 (en) 2002-04-02 2006-12-12 Roche Molecular Systems, Inc. Thermostable or thermoactive DNA polymerase molecules with attenuated 3′-5′ exonuclease activity
WO2004050916A1 (en) * 2002-12-02 2004-06-17 Solexa Limited Recovery of original template
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
EP2152864A1 (en) * 2007-06-13 2010-02-17 Amersham Biosciences Corp. Polymerase stabilization
CA2872141C (en) 2012-05-31 2016-01-19 Board Of Regents, The University Of Texas System Method for accurate sequencing of dna
GB201217770D0 (en) * 2012-10-04 2012-11-14 Base4 Innovation Ltd Biological probes and the use thereof
WO2015031206A1 (en) 2013-08-30 2015-03-05 University Of Connecticut Modified epstein-barr virus dna polymerase and methods for isothermal dna amplification
EP4151751A1 (en) 2016-04-14 2023-03-22 T2 Biosystems, Inc. Methods and systems for amplification in complex samples
CN112041462B (zh) 2018-03-23 2024-06-07 德克萨斯州立大学董事会 具有极低的错误水平的dsDNA有效测序

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE547920T1 (de) * 1991-12-18 1994-02-03 New England Biolabs Inc Rekombinante thermostabile DNA-Polymerase von Archaebakterie.
DE547359T1 (de) * 1991-12-18 1993-11-25 New England Biolabs Inc Gereinigte thermostabile DNS-Polymerase aus Pyrococcus-Arten.

Also Published As

Publication number Publication date
US5747298A (en) 1998-05-05
EP0712927A3 (en) 1999-04-28
EP0712927A2 (en) 1996-05-22

Similar Documents

Publication Publication Date Title
CN1123328A (zh) 高温、高传真脱氧核糖核酸(DNA)聚合酶(HiFi Bst)
US11697843B2 (en) Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
EP0437459B1 (en) Methods for dna sequencing with thermus aquaticus dna polymerase
JP3010738B2 (ja) 核酸の交雑、増幅方法
US9487823B2 (en) Nucleic acid amplification
JP3421664B2 (ja) ヌクレオチド塩基の同定方法
EP1583843B1 (en) Single primer whole genome amplification
JP5809385B2 (ja) 環状ゲノムのローリングサークル増幅
CA3123151A1 (en) Method of nucleic acid enrichment using site-specific nucleases followed by capture
JP7389551B2 (ja) Ｍｅｔエキソン１４欠失の検出と、関連する治療法
EP3642400B1 (en) Methods and kits of constructing sequencing library for use in detecting chromosome copy number variation
US7838270B2 (en) Target-dependent transcription using deletion mutants of N4 RNA polymerase
EP3625356A1 (en) In vitro isolation and enrichment of nucleic acids using site-specific nucleases
CN1333827A (zh) 合成cDNA的方法
WO2020099675A1 (en) Optimization of in vitro isolation of nucleic acids using site-specific nucleases
KR20210112350A (ko) 다중 복제수 변이 검출 및 대립 유전자 비율 정량화를 위한 정량적 앰플리콘 서열분석
CA2050276C (en) Improved primer extension reactions
Kim et al. Probing the structure of a putative intermediate in homologous recombination: the third strand in the parallel DNA triplex is in contact with the major groove of the duplex
CN1900312A (zh) 实时pcr检测中抗高温聚合酶核酸外切酶活性的荧光探针
WO2020073748A1 (zh) 一种测序文库的构建方法
CN109957611B (zh) 一种特异性定量PCR反应混合液、miRNA定量检测试剂盒以及检测方法
WO2018073323A1 (en) Method for complete, uniform and specific amplification of ultra-low amounts of input dna
CN117701688A (zh) 一种动态监测rna和dna的探针自复位分析方法
HK40101410A (zh) Dna甲基化检测的标准品及其应用体系
Ballantyne et al. Document Title: Improved Detection of Male DNA in Post-Coital Samples

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C01 Deemed withdrawal of patent application (patent law 1993)
WD01 Invention patent application deemed withdrawn after publication