CN1313893A - Fy7聚合酶 - Google Patents
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Abstract
本发明公开了一种纯化的重组热稳定性DNA聚合酶,它在50mM和50mM以上的盐浓度下表现出至少约80%的活性、在25mM和25mM以上的盐浓度下表现出至少约70%的活性、且每次结合具有约30个核苷酸的加工能力。本发明还公开了一种编码所述热稳定性DNA聚合酶的分离的核酸以及一种包括所述核酸的重组DNA载体和用该载体转化的重组宿主细胞。本发明还公开了一种使用所述DNA聚合酶测序DNA的方法以及一种用于测序DNA的试剂盒。
Description
相关申请的交叉参考
本申请要求1998年6月17日提交的美国临时申请顺序号60/089,556的优先权,将该文献的全部公开内容引入本文。
发明背景发明领域
本说明书涉及表现出改善的稳定性和功效的热稳定性DNA聚合酶。
背景
DNA聚合酶是用于许多重组DNA技术的酶类,所述的重组DNA技术诸如通过聚合酶链反应(“PCR”)的核酸扩增、自动维持序列扩增(“3SR”)和高温DNA测序。热稳定性聚合酶类是特别有用的。因为加热不会破坏聚合酶的活性,所以不需要在每次变性步骤后添加另外的聚合酶。
然而,已经发现许多热稳定性聚合酶显示出5’-3’外切核酸酶或结构依赖性单链内切核酸酶(“SDSSE”)活性,这种活性可以限制生成的产物的量或产生通常产物呈指数累积中的平台现象。这类5’-3’核酸酶活性可以使有效生成大于或等于10kb的长PCR产物的能力减弱,特别是就富含G+C的靶物而言。在DNA测序的应用和循环测序的应用中,5’-3’核酸酶活性的存在可使所需的带强度降低和/或生成假或背景带。
另外,目前可得到的许多酶类对高盐环境敏感,通常的条件是:
目前可得到的酶类勉强具有加工能力(更具有分布性-通常减退得更为明显-更具体地解释)
添加dITP以解决迫切的问题-通常杀灭酶的活性。
因此,需要持续存在一种对高盐条件、dITP的有效应用、高生产能力和对富含GC模板的改善特性具有增加的耐受性的改进的DNA聚合酶。
本发明的简要概括
本说明书教导了一种纯化的重组热稳定性DNA聚合酶,它包括附图1中所示的氨基酸序列;本说明书还教导了一种纯化的重组热稳定性DNA聚合酶,它在50mM和50mM以上的盐浓度下表现出至少约80%的活性。本说明书进一步教导了一种纯化的重组热稳定性DNA聚合酶,它在25mM和25mM以上的盐浓度下表现出至少约70%的活性;且本说明书还教导了一种纯化的重组热稳定性DNA聚合酶,它在每次结合时具有约30个核苷酸的加工(processivity)能力。
本说明书还教导了一种编码热稳定性DNA聚合酶的分离的核酸,其中所述的核酸由附图1中所示的核苷酸序列组成;本说明书还教导了一种包括所述核酸的重组DNA载体和用该载体转化的重组宿主细胞。
本说明书还教导了一种测序DNA的方法,该方法包括下列步骤:在有至少一种链终止剂和一种或多种核苷酸三磷酸存在的情况下从用所述的DNA聚合酶测序的DNA模板生成链终止的片段并根据所述片段的大小来确定所述DNA的序列。本说明书还教导了一种用于测序DNA的试剂盒,它包括所述的DNA聚合酶。
几个附图的简要说明
附图1描绘了FT7聚合酶的氨基酸序列(和编码它们的DNA序列)。
附图2描绘了使用在Mn条件下配制的FY7聚合酶测序的M13mp18DNA的DNA序列,正如从ABI 377型自动荧光DNA测序仪中打印出的结果所示。
附图3描绘了使用在Mg条件下配制的FY7聚合酶测序的M13mp18DNA的DNA序列,正如从ABI 377型自动荧光DNA测序仪中打印出的结果所示。
附图4描绘了在不同KCl浓度下Thermo SequenaseTM酶DNA聚合酶/FY7 DNA聚合酶的最大聚合酶活性的百分比。
附图5描绘了高盐浓度对在使用Thermo SequenaseTM酶DNA聚合酶/FY7 DNA聚合酶的放射性标记的DNA测序反应中的DNA测序能力的影响。
附图6-10描绘了增加的盐浓度对Thermo Sequenase特性的影响。在低至25mM的浓度下,数据的质量受到影响读取长度从至少600个碱基减少至约450个碱基。在50mM盐浓度下,读取的长度进一步减少至约350个碱基;在75mM盐浓度下,读取的长度进一步减少至约250个碱基,而浓度在100mM时读取长度可以忽略不计。
附图11-15描绘了增加的盐浓度对FY7 DNA聚合酶特性的影响。至少75mM KCl时,不存在对特性的不利影响,且仅在100mM KCl下数据的质量轻微降低。
附图16描绘了对Thermo Sequenase DNA聚合酶、Ampli Taq FS DNA聚合酶测定的加工能力/对FY7 DNA聚合酶测定的加工能力相比较的结果。
附图17描绘了在72℃下引入dGTP(鸟苷三磷酸)类似物dITP(肌苷三磷酸)的放射性标记测序反应中使用FY7聚合酶/热测序酶(ThermoSequenase)DNA聚合酶时获得的改进的读取长度。
附图18-22表示增加的延伸步骤时间对由荧光标记的终止子DNA测序反应中Thermo Sequenase DNA聚合酶产生的读取长度和数据质量的影响。
附图23-27表示增加的延伸步骤时间对由荧光标记的终止子DNA测序反应中FY7 DNA聚合酶产生的读取长度和数据质量的影响。
发明详述
为了获得一种用于DNA测序的改进的聚合酶、通过降低在全长嗜热栖热菌和水生栖热菌DNA聚合酶Ⅰ型酶类中发现的外切核酸酶活性来构建一系列聚合酶突变体。在polⅠ型聚合酶的氨基末端结构域中鉴定了6种保守基元,已证实在其中保留了5′至3′的外切核酸酯活性(Gutman和Minton(1993)《核酸研究》(Nucleic Acids Research)21,4406-4407)。此外,晶体结构表明在这些保守基元中的6个羧酸残基位于水生栖热菌DNA polⅠ的外切核酸酶结构域的活性位点上(Kim等(1995)《自然》(Nature)376,612-616)。通过对Tth和Taq聚合酶内6个保守基元中的3个中的羧酸和其它残基进行定点诱变来进行点突变,如下:
Taq D18A、Taq T140V、Taq D142N/D144N。所有这些均在外切核酸酶结构域的外部具有突变F667Y;
Tth D18A、Tth T141V、Tth D143N/D145N。所有这些均在外切核酸酶结构域的外部具有突变F669Y。
对所有的聚合酶评估外切核酸酶活性、加工能力、链置换、盐耐受性、热稳定性和测序质量。下面进一步具体地描述一种FY7聚合酶(Tth D18A、F669Y)。
实施例方法体外诱变
使用PCR在克隆的全长F669Y Tth的密码子18(D18A)上引入天冬氨酸-丙氨酸的改变(质粒pMR10)。诱变引物1(CTGTTCGAACCCAAAGGCCGTGTCCTCCTGGTGGCCGGCCACCAC)包括pMR10的19-60位核苷酸包括密码子18和BstBⅠ限制位点。寡核苷酸引物2(GAGGCTGCCGAATTCCAGCCTCTC)包括pMR10的EcoRⅠ位点,将pMR10用作模板DNA。将PCR产物用BstBⅠ和EcoRⅠ消化并与pMR10的两个片段即5000bp KpnⅠ/BstBⅠ和2057bp EcoRⅠ/KpnⅠ连接,从而生成质粒pMR12。将大肠杆菌菌株DH1λ+的细胞用于初级转化并将菌株M5248(λcI857)用于蛋白质表达,不过,可以使用携带cI+和cI857等位基因的类似一对大肠杆菌菌株。另一方面,用化学剂诸如萘啶酮酸可以将任意的rec+cI+菌株诱变以产生聚合酶。聚合酶的纯化
在30℃下,使含有质粒pMR12的M5248在1升的LB培养基(1%胰化蛋白胨、0.5%酵母提取物、1%NaCl)、优选在含有100mg/ml氨苄西林的2X LB培养基中生长。当OD600达到1.0时,在42℃下将培养物诱导1.5小时。然后使该培养物冷却至<20℃并在4℃下的SorvallRC-3B离心机中通过以500rpm离心15-30分钟来收集细胞。将所收集的细胞保存在-80℃下。
将细胞沉淀重新悬浮于25ml预热的裂解缓冲液(50mM Tris-HCl pH8.0;10mM MgCl2;16mM(NH4)2SO4;1mM EDTA;0.1%、优选0.2%Tween20;0.1%、优选0.2%NP40)中。优选所述的裂解缓冲液含有300mMNaCl。将重新悬浮的细胞在75-85℃下培养10-20分钟、超声处理1分钟并通过离心澄清。使澄清的裂解液通过用含有100mM、优选含有300mM NaCl的缓冲液A(50mM Tris-HCl pH 8.0;1mM EDTA;0.2%Tween20;0.1%NP40)平衡的300ml的二乙氨基乙基纤维素(Whatman DE 52)柱。分析级分的聚合酶活性并收集那些被证明具有聚合酶活性的峰、用50mM含有缓冲液A的NaCl稀释并上用50mM NaCl的缓冲液A的溶液平衡的肝素琼脂糖柱(20ml)。使用50mM-700mM NaCl的缓冲液A的溶液的线性盐梯度从该柱上洗脱聚合酶。分析级分的聚合酶活性并收集那些可证明具有活性峰的级分且对最终缓冲液(20mM Tris-HCl pH8.5、50%(v/v)甘油、0.1mM EDTA、0.5%Tween 20、0.5%NP40、1mM DTT、100mM KCl)透析。将纯化的蛋白质命名为FY7。氨基酸序列(和编码氨基酸序列的DNA序列)如附图1中所示。细菌菌株
大肠杆菌菌株:DHIλ+[gyrA96、recAl、relA1、endA1、thi-1、hsdR17、supE44、λ+];M5248[λ(bio275、cI857、cⅢ+、N+、λ(H1))]。PGR
通过SDS碱性裂解方法(Sambrook等《分子克隆》(MolecularCloning)第2版,Cold Spring Harbor Press,1989)来制备来自大肠杆菌DHIλ+的质粒DNA(pMR10)。反应条件如下:每100μl反应物中用10mM Tris-HCl pH 8.3、50mM KCl、1.5mM MgCl2、0.001%明胶、1uM各种引物、2.5 U Taq聚合酶。循环条件为94℃下2分钟;然后是94℃30秒、55℃30秒、72℃3分钟、随后是72℃7分钟组成的35个循环。实施例1在Mn条件下的酶制剂
在下列“预混合”方案中,在两种溶液中均包括所有试剂;试剂混合物A和试剂混合物B。试剂混合物A
合并下列试剂来制成10ml试剂混合物A:
2.5ml 1M HEPPS N-(2-羟乙基)哌嗪-N’-(3-丙磺酸),pH8.0;
500μl 1M酒石酸,pH8.0;
50,000单位的FY7 DNA聚合酶;
1单位的嗜酸热原体无机焦磷酸酶;
100μl 100mM dATP;
100μl 100mM dTTP;
100μl 100mM dCTP;
500μl 100mM dITP;
9.375μl 100μM C-7-炔丙基氨基-4-罗丹明-6-G-ddATP;
90μl 100μM C-5-炔丙基氨基-4-罗丹明-X-ddCTP;
6.75μl 100μM C-7-炔丙基氨基-4-罗丹明-I10-ddGTP;
165μl 100μM C-5-炔丙基氨基-4-四甲基罗丹明-ddUTP;
10μl 50mM EDTA;
1ml甘油;
用去离子H2O将体积加至10,000μl。试剂混合物B
合并下列试剂以制成10ml的试剂混合物B:
10μl 1M MES 2-(N-吗啉代)乙磺酸,pH6.0;
200μl 1M MgCl2
75μl 1M MnSO4;
用去离子H2O将体积加至10,000μl。实施例2:来自实施例1的制剂的应用
将两(2)μl试剂混合物A、2μl试剂混合物B、200ng M13mp18 DNA、5pmole引物(M13-40正向5’-GTTTTCCCAGTCACGTTGTA)和去离子水补充20μl总体积彼此混合并在热循环仪中进行25个循环(95℃30秒、60℃1分钟)。循环后,加入含有1.5M乙酸钠、250mM EDTA的4μl溶液。将该溶液混合并加入4个体积(100μl)的乙醇。通过在冰上培养15-20分钟、随后离心来沉淀DNA。除去上清液并用70%乙醇洗涤沉淀、将其干燥并重新悬浮于4μl的含有加样染料的甲酰胺中。然后使重新悬浮的DNA在自动荧光DNA测序仪(ABI 377型仪器)上运动。从该机器中打印出的DNA序列如附图2中所示。实施例3在Mg条件下的酶制剂
在下列“预混合物”方案中,在一种溶液中包括所有试剂。测序预混合物
合并下列试剂以制成800μl的测序预混合物:
200μl的500mM Tris-HCl pH 9.5、20mM MgCl2;
100μl 40单位/μl FY7 DNA聚合酶、0.0008单位/μl的嗜酸热原体无机焦磷酸酶;
100μl 10mM dITP、2mM dATP、2mM dTTP、2mM dCTP;
100μl 0.125μM C-7-炔丙基氨基-4-罗丹明-6-G-ddATP;
100μl 1.2μM C-5-炔丙基氨基-4-罗丹明-X-ddCTP;
100μl 0.09μM C-7-炔丙基氨基-4-罗丹明-110-ddGTP;
100μl 2.2μM C-5-炔丙基氨基-4-四甲基罗丹明-ddUTP。实施例4来自实施例3的制剂的应用
将四(4)μl测序预混合物、200ng M13mp18 DNA、5pmole引物(M13-40正向5’-GTTTTCCCAGTCACGTTGTA)和去离子水,至20μl总体积的彼此混合并在热循环仪中进行25个循环(95℃30秒、60℃2分钟)。循环后,加入7μl的7.5M乙酸铵。将该溶液混合并加入4个体积(100μl)的乙醇。通过在冰上培养15-20分钟、随后离心来沉淀DNA。除去上清液并用70%乙醇洗涤沉淀、将其干燥并重新悬浮于4μl的含有加样染料的甲酰胺中。然后将重新悬浮的DNA在自动荧光DNA测序仪(ABI 377型仪器)测序。从该机器中打印出的DNA序列如附图3中所示。实施例5 Thermo SequenaseTM酶与FY7酶的聚合酶活性/盐浓度(KCl)
在不同KCl浓度下测定Thermo SequenaseTM酶DNA聚合酶与FY7DNA聚合酶的最大聚合酶活性的百分比。结果如附图4中所示。数据表明FY7在反应混合物中具有远高于Thermo SequenaseTM的最适盐浓度和广范围的盐的耐受性。对于FY7来说,产生50%活性的盐浓度比Thermo Sequenase高5倍。
还实验了高盐浓度对放射性标记的DNA测序反应中DNA测序能力的影响。结果如附图5中所示。在50mM或50mM以上的KCl浓度下,ThermoSequenaseTM聚合酶特性降解至不能够获得有用数据的水平。然而,FY7DNA聚合酶能够在100mM的KCl浓度下得到极好的测序数据。实施例6荧光测序的盐耐受性
这些实验检验了上述证明的聚合酶活性在高盐浓度下对荧光标记的终止子DNA测序反应中DNA测序能力的影响。结果如附图6-15中所示。
附图6-10表示增加的盐浓度对Thermo Sequenase特性的影响。在低至25mM的浓度下,数据的质量受到从至少600个碱基减少至约450个碱基的读取长度的影响。在50mM盐浓度下,读取的长度进一步减少至约350个碱基;在75mM盐浓度下,读取的长度进一步减少至约250个碱基,而浓度在100mM时读取长度可以忽略不计。
附图11-15表示增加的盐浓度对FY7 DNA聚合酶特性的影响。至少75mM KCl时,对其特性无不利影响,且仅在100mM KCl下数据的质量轻度降低。
当认识到某些类型的DNA制剂可以受到盐污染时(对DNA测序数据的质量不利),FY7 DNA聚合酶的应用可使得在广范围模板条件中的测序反应更为稳定。实施例7聚合酶的加工能力
已经测定了不同DNA测序聚合酶的加工能力(每次DNA聚合酶结合时参入的核苷酸数量)。结果如附图16中所示。Thermo Sequenase DNA聚合酶每次结合时仅具有~4个核苷酸的加工能力。Ampli Taq FS DNA聚合酶在每次结合时具有~15个核苷酸的加工能力。FY7 DNA聚合酶具有比Thermo Sequenase DNA聚合酶高7倍的加工能力且在每次结合~30个核苷酸时具有比Ampli Taq FS DNA聚合酶高~2倍的加工能力。实施例8在72℃下使用dITP的聚合酶延伸反应
本系列实验检验了当在72℃下引入dGTP(鸟苷三磷酸)类似物dITP(肌苷三磷酸)的放射性标记测序反应中使用FY7聚合酶ThermoSequenase DNA聚合酶时获得的改进的读取长度。结果如附图17中所示。FY7能够在标准33P[α-aATP]测序条件下结合比Thermo Sequenase>50-100个以上的核苷酸。实施例9延伸步骤时间对读取长度的影响
这些系列实验检验了在放射性标记的终止子DNA测序反应中增加的延伸步骤时间对Thermo Sequenase和FY7 DNA聚合酶获得的读取长度和数据质量的影响。结果如附图18-27中所示。
附图18-22表示增加的延伸步骤时间对由Thermo SequenaseDNA聚合酶产生的读取长度和数据质量的影响。该数据表明热测序酶(Thermo Sequenase)要求最短2分钟的延伸步骤以便获得至少优质读取600个碱基。信号强度一般在4分钟的延伸时(该时间在应用这种酶和方法的商品中是特定的)增加到最大值。
附图23-27表示增加的延伸步骤时间对由FY7 DNA聚合酶产生的读取长度和数据质量的影响。该数据表明FY7要求最短30秒的延伸步骤以便获得至少优质读取600个碱基。信号强度在约1分钟的延伸时间时产生平台。FY7 DNA聚合酶可以在四分之一或二分之一的延伸反应时间中产生与Thermo Sequenase相同质量的数据。
尽管上述实施例具体地描述了本发明的各种实施方案,但是对于本领域技术人员来说显然可以对它们进行改变。因此,上述实施例用于解释目的且不应以任何方式用于限制所附权利要求的范围。
Claims (13)
1.一种纯化的重组热稳定性DNA聚合酶,它包括附图1中所示的氨基酸序列。
2.一种纯化的重组热稳定性DNA聚合酶,它在50mM和50mM以上的盐浓度下表现出至少约为80%的活性。
3.一种纯化的重组热稳定性DNA聚合酶,它在25mM和25mM以上的盐浓度下表现出至少约为70%的活性。
4.一种纯化的重组热稳定性DNA聚合酶,它在每次结合时具有约30个核苷酸的加工能力。
5.一种编码热稳定性DNA聚合酶的分离的核酸,其中所述的核酸由附图1所示的核苷酸序列组成。
6.一种重组DNA载体,它包括权利要求3所述的核酸。
7.一种权利要求4的重组DNA序列,它包括质粒pMR10。
8.一种用权利要求5的载体转化的重组宿主细胞。
9.权利要求6的重组宿主细胞,它是大肠杆菌。
10.权利要求7的重组宿主细胞,它是携带cI+和cI857等位基因的大肠杆菌。
11.权利要求7的重组宿主细胞,它选自DHIλ+[gyrA96、recA1、relA1、endA1、thi-1、hsdR17、supE44、λ+]和M5248[λ(bio275、cI857、cⅢ+、N+、λ(H1))]组成的组。
12.测序DNA的方法,该方法包括下列步骤:在有至少一种链终止剂和一种或多种核苷酸三磷酸存在的情况下,用权利要求1的DNA聚合酶从待测序的DNA模板生成链终止的片段,并根据所述片段的大小来确定所述DNA的序列。
13.一种用于测序DNA的试剂盒,它包括权利要求1所述的DNA聚合酶。
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US7875440B2 (en) | 1998-05-01 | 2011-01-25 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and DNA molecules |
US6780591B2 (en) | 1998-05-01 | 2004-08-24 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and DNA molecules |
WO2003004632A2 (en) * | 2001-07-06 | 2003-01-16 | Amersham Biosciences Corp | Novel dna polymerases having amino acid substitutions and homologs thereof |
AU2002347981A1 (en) * | 2001-11-07 | 2003-05-19 | Applera Corporation | Universal nucleotides for nucleic acid analysis |
US7169560B2 (en) | 2003-11-12 | 2007-01-30 | Helicos Biosciences Corporation | Short cycle methods for sequencing polynucleotides |
DE602005020421D1 (de) | 2004-02-19 | 2010-05-20 | Helicos Biosciences Corp | Verfahren zur analyse von polynukleotidsequenzen |
WO2005123957A2 (en) * | 2004-06-10 | 2005-12-29 | Ge Healthcare Bio-Sciences Corp. | Method for nucleic acid analysis |
KR20070093992A (ko) * | 2004-12-11 | 2007-09-19 | 사이토제닉, 인크. | 고품질 핵산의 무세포 생합성 및 그것의 사용 |
US7666593B2 (en) | 2005-08-26 | 2010-02-23 | Helicos Biosciences Corporation | Single molecule sequencing of captured nucleic acids |
JP6381628B2 (ja) | 2013-03-18 | 2018-08-29 | キアゲン ゲーエムベーハー | 生物試料の安定化 |
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US5618711A (en) | 1986-08-22 | 1997-04-08 | Hoffmann-La Roche Inc. | Recombinant expression vectors and purification methods for Thermus thermophilus DNA polymerase |
US5795762A (en) | 1986-08-22 | 1998-08-18 | Roche Molecular Systems, Inc. | 5' to 3' exonuclease mutations of thermostable DNA polymerases |
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CA2071196C (en) | 1989-12-22 | 2002-04-23 | David H. Gelfand | Recombinant expression vectors and purification methods for thermus thermophilus dna polymerase |
US5210015A (en) | 1990-08-06 | 1993-05-11 | Hoffman-La Roche Inc. | Homogeneous assay system using the nuclease activity of a nucleic acid polymerase |
US5593840A (en) | 1993-01-27 | 1997-01-14 | Oncor, Inc. | Amplification of nucleic acid sequences |
US5610066A (en) | 1993-12-10 | 1997-03-11 | Amersham Life Science, Inc. | Nucleic acid modifying proteins from Pyrococcus furiosus |
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US5885813A (en) | 1995-05-31 | 1999-03-23 | Amersham Life Science, Inc. | Thermostable DNA polymerases |
US5948614A (en) * | 1995-09-08 | 1999-09-07 | Life Technologies, Inc. | Cloned DNA polymerases from thermotoga maritima and mutants thereof |
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