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 PDFInfo
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- 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
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12Q1/6869—Methods for sequencing
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
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
■: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。
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 |
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Application Number | Priority Date | Filing Date | Title |
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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) |
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CN1123328A true CN1123328A (zh) | 1996-05-29 |
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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 |
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US (1) | US5747298A (zh) |
EP (1) | EP0712927A3 (zh) |
CN (1) | CN1123328A (zh) |
Families Citing this family (23)
Publication number | Priority date | Publication date | Assignee | Title |
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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 |
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 |
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 |
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 |
EP1925669B1 (en) | 2000-02-17 | 2010-12-08 | Qiagen GmbH | Thermostable chimeric nucleic acid polymerases and uses thereof |
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 |
AU2003295085A1 (en) * | 2002-12-02 | 2004-06-23 | Solexa Limited | Recovery of original template |
US7666593B2 (en) * | 2005-08-26 | 2010-02-23 | Helicos Biosciences Corporation | Single molecule sequencing of captured nucleic acids |
CA2689068A1 (en) * | 2007-06-13 | 2008-12-18 | Ge Healthcare Bio-Sciences Corp. | Polymerase stabilization |
WO2013181170A1 (en) | 2012-05-31 | 2013-12-05 | 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 |
CA2921654A1 (en) | 2013-08-30 | 2015-03-05 | University Of Connecticut | Modified epstein-barr virus dna polymerase and methods for isothermal dna amplification |
JP2019517780A (ja) | 2016-04-14 | 2019-06-27 | ティー2 バイオシステムズ,インコーポレーテッド | 複合試料における増幅のための方法及びシステム |
WO2019183640A1 (en) | 2018-03-23 | 2019-09-26 | Board Of Regents, The University Of Texas System | Efficient sequencing of dsdna with extremely low level of errors |
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DE547359T1 (de) * | 1991-12-18 | 1993-11-25 | New England Biolabs Inc | Gereinigte thermostabile DNS-Polymerase aus Pyrococcus-Arten. |
JPH067160A (ja) * | 1991-12-18 | 1994-01-18 | New England Biolabs Inc | 古細菌からの組換え熱安定性dnaポリメラーゼ |
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1994
- 1994-11-17 CN CN94113990.5A patent/CN1123328A/zh active Pending
-
1995
- 1995-10-18 US US08/544,643 patent/US5747298A/en not_active Expired - Lifetime
- 1995-11-14 EP EP95308132A patent/EP0712927A3/en not_active Withdrawn
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EP0712927A2 (en) | 1996-05-22 |
EP0712927A3 (en) | 1999-04-28 |
US5747298A (en) | 1998-05-05 |
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