CN114438053A - 一种dna聚合酶突变体及其应用 - Google Patents
一种dna聚合酶突变体及其应用 Download PDFInfo
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- CN114438053A CN114438053A CN202011203123.7A CN202011203123A CN114438053A CN 114438053 A CN114438053 A CN 114438053A CN 202011203123 A CN202011203123 A CN 202011203123A CN 114438053 A CN114438053 A CN 114438053A
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- leu
- dna polymerase
- ala
- glu
- taq dna
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Abstract
本申请公开了一种无5’至3’的核酸外切酶活性的Taq‑DNA聚合酶突变体及其应用。
Description
技术领域
本申请涉及生物技术领域，并具体涉及一种Taq DNA聚合酶的突变体及其应用。
背景技术
聚合酶链式反应(PCR)是一种应用十分广泛的体外扩增DNA技术，其自1985年问世以来，逐渐扩展到各个领域，并且除了在实验室分子生物学中运用，在临床疾病诊断和法医鉴定上也有着十分重要的作用。比如，PCR可以用于检测基因突变和微生物或病毒感染因子等，还可以用于检测抗生素耐药基因和生物威胁剂。PCR的最早的诊断用途之一是用于镰状细胞贫血的产前诊断试验，使用PCR检测镰状细胞突变比以前的方法更快速和敏感。随后开发了更多基于PCR的诊断方法，包括：通过检测低拷贝数的病毒靶点诊断病毒(如HIV)的感染；通过检测结核分枝杆菌诊断肺结核；以及通过检测胃肠道幽门螺杆菌的不同分离物诊断幽门螺杆菌的感染。1992年，Higuchi等人开发了实时PCR，这种增强的PCR方法通过荧光染料(如SYBR Green I)或荧光共振能量转移(FRET)探针实时检测反应过程中形成的产物量。与传统的培养方法相比，基于PCR的检测方法具有速度快、特异性强、灵敏度高等优点。
而PCR反应主要依赖于DNA聚合酶。最初PCR技术中扩增DNA的酶来源于大肠杆菌聚合酶的Klenow片段，它可以在短短几小时内就产生数十亿个拷贝的分子。随后在一些嗜热细菌中分离出了几种非常耐热的DNA聚合酶，包括Thermus aquaticus(Taq),Thermusthermophilus(Tth)和Pyrococcus furiosus(Pfu)，它们在95℃的高温下依旧不会失去活性。其中Taq DNA聚合酶是第一种应用于PCR的酶，它既有聚合酶活性又有外切酶活性。由于Taq DNA聚合酶具有高稳定性和高效性以及简单和经济的生产过程，这种聚合酶是大多数PCR应用中最受欢迎和最广泛使用的酶。
Taq DNA聚合酶具有5’至3’的聚合酶活性及5’至3’核酸外切酶活性(在本文中也简称为“外切酶活性”)。此外，还发现其具有焦磷酸酶活性，可以催化ddNMP和PPi反应生成ddNTP。Taq DNA聚合酶还具有非模板依赖活性，可以在DNA分子的3’端引入非模板互补的核苷酸A，从而产生3’端突出单个核苷酸A的PCR产物。Taq DNA聚合酶被证实具有逆转录活性，可以与Pfu DNA聚合酶组合用于获得真核基因的cDNA。并且，与常用的禽源AMV反转录酶和鼠源Mulv反转录酶相比，用Taq DNA聚合酶在高温下进行反转录可以避免mRNA二级结构所带来的不利影响。
Taq-DNA聚合酶所具有的5’至3’核酸外切酶活性使得能够从5’至3’方向水解DNA生长链前方的DNA链，主要产生5’-脱氧核苷酸。这种酶活性只对DNA上配对部分的磷酸二酯键有切割活力作用。这会导致对PCR产物的片段进行酶切，影响PCR扩增。在基因工程中，常需要进行DNA测序。将Taq DNA聚合酶用于DNA测序，与普通的测序酶相比，除了具有良好的链延伸性能外，Taq DNA聚合酶还有可在高温中进行反应的特点，这使得能够克服富含GC序列的模板形成自身二级结构对测序的影响。若将PCR反应与末端终止法测序结合进行，则测序反应只需少量模板，并且对双链模板的测序不需要碱变性。Taq DNA聚合酶的耐热性，使得进行PCR反应时可在高温下进行退火，增强引物与模板结合的特异性。
荧光标记的核苷酸大大简化并提高了分子生物学中许多程序的效用。在合成过程中，使用荧光标记的核苷酸来标记多核苷酸，在很大程度上已经取代了放射性标记的使用。然而，使用荧光标记的核苷酸的一个主要问题是DNA聚合酶对荧光标记核苷酸的掺入进行区分的能力。已经有人发现，在TET(6-羧基-47.2,7'-TET rachlorofluresein)标记的2’,3’-二脱氧核苷酸和相应的未标记的二脱氧核苷酸之间的竞争分析中，Taq DNA聚合酶将未标记的二脱氧核苷酸并入DNA的频率至少是对应标记核苷酸的85倍。标记和未标记核苷酸之间的这种区别对使用DNA聚合酶标记DNA的程序有着深远的影响。例如，在测序反应中必须使用大量荧光标记的核苷酸。这种大量荧光标记的核苷酸价格昂贵，并且会产生过量的背景荧光，从而降低序列信息的产量。DNA聚合酶对荧光标记核苷酸的这种辨别能力对许多需要使用酶添加荧光标记核苷酸的分子生物学程序有不良影响，例如标记双脱氧终止子测序。另外，Taq DNA聚合酶用于测序时对各种ddNTP具有不同的偏好性，这使得四种ddNTP掺入的速度明显不同，其中，ddGTP掺入的速率比ddATP、ddCTP和ddTTP快10倍，这就造成测序时四种带的强度和峰的高低不均衡，不利于结果判读，影响测序结果的准确性。
除此之外，随着研究的不断深入，开发了许多检测稀有突变的方法，如单链构象多态性(SSCP)、异源双链技术(HA)、变性的高效液相色谱法(DHPLC)、变性梯度凝胶电泳(DGGE)、化学错配裂解法(CMC)、质谱法、DNA芯片技术和焦磷酸水解激活的聚合酶反应(PAP)技术等。焦磷酸水解激活的聚合酶反应技术是目前对稀有突变检测最有效的技术之一。DNA聚合酶不仅有聚合活性，还有焦磷酸酸活性。聚合酶活性使得在PCR反应中有dNTP→dNIP+PPi反应，焦磷酸酶活性则使得有dNMP+PPi→dNTP反应。设计一段末端用ddNMP封闭的引物，只有当引物的3’端完全和模板DNA序列配对的时候，才可以发生焦磷酸酶反应即dNMP+PPi→ddNTP，从而使得末端的ddNMP脱落下来，并进而使得封闭被解除，引物得以延伸。而如果引物与模板不匹配，则无法发生焦磷酸酶反应，进而引物由于3’端被封闭而无法延伸，这样就可以将一个核昔酸的区别识别开来。但是，Taq DNA聚合酶对各种ddNTP具有不同的偏好性，这影响了PAP反应结果的准确性。
在测序和PAP中，由于DNA聚合酶对ddNTP和荧光标记核苷酸辨别能力的不一致，导致了一系列问题的产生。而这种辨别能力的不一致主要是由Taq DNA聚合酶的5’至3’外切酶活性过高引起的。因此，希望开发新的无5’至3’核酸外切酶活性、同时具有优异的聚合酶活性的Taq DNA聚合酶来应用于各种场景，例如PAP和测序技术。
发明内容
本申请的发明人经过深入的研究，开发获得了一种Taq DNA聚合酶的突变体，其不仅具有优异的聚合酶活性(即，能够在短时间内即可获得大量的DNA产物)，并且具有显著降低的5’至3’核酸外切酶活性。本申请的突变体是特别有利的，可以应用于临床诊断和试剂盒的开发，并且可特别有利地用于测序反应和PAP反应。
因此，在一个方面，本申请提供了一种突变的Taq DNA聚合酶或其变体，其中，所述突变的Taq DNA聚合酶与野生型Taq DNA聚合酶相比，具有下述突变：
(1)在与SEQ ID NO:1的第345位对应的位置处的氨基酸残基被缬氨酸残基替换；
(2)在与SEQ ID NO:1的第520位对应的位置处的氨基酸残基被甘氨酸残基替换；和
(3)在与SEQ ID NO:1的第578位对应的位置处的氨基酸残基被天冬酰胺残基替换；
其中，所述变体与所述突变的Taq DNA聚合酶相比，具有至少90％，例如至少95％，至少96％，至少97％，至少98％，至少99％的序列同一性；或者，具有一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸的置换(优选保守置换)、添加或缺失；且，
所述变体中与SEQ ID NO:1的第345位、第520位和第578位对应的位置处的氨基酸残基分别与所述突变的Taq DNA聚合酶相同；且
所述变体保留了所述突变的Taq DNA聚合酶的功能(例如，具有高于野生型TaqDNA聚合酶的DNA聚合酶活性，且具有低于野生型Taq DNA聚合酶的5’至3’核酸外切酶活性)。
本申请的突变的Taq DNA聚合酶或其变体具有优良的DNA聚合酶活性。例如，本申请的突变的Taq DNA聚合酶或其变体的聚合酶活性是野生型Taq DNA聚合酶的大约至少2倍，至少3倍，至少4倍，至少5倍，或6倍。此外，本申请的突变的Taq DNA聚合酶或其变体还具有显著降低的5’至3’核酸外切酶活性。例如，本申请的突变的Taq DNA聚合酶或其变体的核酸外切酶活性不超过野生型Taq DNA聚合酶的大约10％，5％，或1％。在某些优选的实施方案中，本申请的突变的Taq DNA聚合酶或其变体实质上不具有5’至3’核酸外切酶活性。
在某些优选的实施方案中，所述野生型Taq DNA聚合酶具有如SEQ ID NO:1所示的氨基酸序列。
在某些优选的实施方案中，所述突变的Taq DNA聚合酶具有如SEQ ID NO:2所示的氨基酸序列。
在另一个方面，本申请提供了一种分离的核酸，其包含编码如上所述的突变的TaqDNA聚合酶或其变体的核苷酸序列。在某些优选的实施方案中，本申请的分离的核酸具有如SEQ ID NO:3所示的核苷酸序列。
在另一个方面，本申请提供了一种载体，其包含所述分离的核酸。可用于插入目的多核苷酸的载体是本领域公知的，包括但不限于克隆载体和表达载体。在一个实施方案中，所述载体是例如质粒，粘粒，噬菌体等。
在另一个方面，本申请还涉及包含上述分离的核酸或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的宿主细胞还可以是细胞系，例如293T细胞。在某些优选的实施方案中，所述宿主细胞是大肠杆菌。
在另一个方面，本申请还涉及包含上述突变的Taq DNA聚合酶或其变体，或上述分离的核酸或载体或宿主细胞的组合物。在某些优选的实施方案中，所述组合物包含本发明的Taq DNA聚合酶或其变体。
在另一个方面，本申请涉及一种制备如上所述的突变的Taq DNA聚合酶或其变体的方法，其包括，在宿主细胞中表达所述突变的Taq DNA聚合酶或其变体，然后从所述宿主细胞的培养物中回收所述突变的Taq DNA聚合酶或其变体。
在某些优选的实施方案中，所述宿主细胞为大肠杆菌。
在某些优选的实施方案中，所述方法包括步骤：在大肠杆菌中表达所述突变的TaqDNA聚合酶或其变体，然后从所述大肠杆菌的裂解上清中纯化得到所述突变的Taq DNA聚合酶或其变体。在某些优选的实施方案中，通过色谱法(例如，阳离子交换色谱，羟基磷灰石色谱和/或疏水相互作用色谱)，从所述大肠杆菌的裂解上清中回收所述突变的Taq DNA聚合酶或其变体。
在另一个方面，本申请还涉及所述突变的Taq DNA聚合酶或其变体用于进行核酸合成或扩增(例如PCR)的用途。
在另一个方面，本申请还涉及所述突变的Taq DNA聚合酶或其变体用于进行焦磷酸水解激活的聚合酶反应(PAP)的用途。在一些优选的实施方案中，所述焦磷酸水解激活的聚合酶反应(PAP)用于检测稀有突变。
在另一个方面，本申请还涉及所述突变的Taq DNA聚合酶或其变体用于分析或测定核酸(例如DNA)分子的核苷酸序列的用途。在一个优选的实施方案中，所述核苷酸序列分析或测定过程包括以下步骤：将能与所述核酸分子杂交的引物分子，与所述核酸分子以及所述突变的Taq DNA聚合酶或其变体孵育；以及确定所述核酸分子至少一部分的核苷酸序列。
在另一个方面，本申请还涉及一种试剂盒，其包含所述突变的Taq DNA聚合酶或其变体。本申请的试剂盒可用于各种用途，例如进行核酸合成或扩增(例如PCR)，PAP或测序反应。在一些优选的实施方案中，所述试剂盒还包括选自下列的试剂：用于进行PCR的试剂(例如，缓冲液，dNTP，引物)；用于进行PAP的试剂(例如，缓冲液，dNTP，引物，焦磷酸或其类似物)；用于进行测序反应的试剂(例如，缓冲液，dNTP，引物，合成终止剂)；或其任何组合。
本申请中相关术语的说明及解释
在本申请中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。
根据本发明，术语“变体”是指这样的蛋白，其氨基酸序列与本发明的突变的TaqDNA聚合酶(如SEQ ID NO：2所示的蛋白)的氨基酸序列相比，具有一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸的置换(优选保守置换)、添加或缺失，或者具有至少90％，95％，96％，97％，98％，或99％的同一性，并且其保留了所述突变的Taq DNA聚合酶的功能。本申请的突变的Taq DNA聚合酶具有高于野生型Taq DNA聚合酶的DNA聚合酶活性。例如，本申请的突变的Taq DNA聚合酶的聚合酶活性是野生型Taq DNA聚合酶的大约至少2倍，至少3倍，至少4倍，至少5倍，或6倍。此外，本申请的突变的Taq DNA聚合酶还具有低于野生型Taq DNA聚合酶的的5’至3’核酸外切酶活性。例如，本申请的突变的Taq DNA聚合酶的核酸外切酶活性不超过野生型Taq DNA聚合酶的大约10％，5％，或1％。在某些优选的实施方案中，本申请的突变的Taq DNA聚合酶实质上不具有5’至3’核酸外切酶活性。
术语“同一性”是对核苷酸序列或氨基酸序列的相似性的量度。通常将序列排列起来，以获得最大限度的匹配。“同一性”本身具有本领域公知的意义并且可用公开的算法(例如BLAST)来计算。
根据本发明，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.ApplBiosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoIBiol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，表述“与SEQ ID NO:1的第X位对应的位置”是指，当将目标序列与SEQ ID NO:1进行比对以产生最大同一性时，目标序列中与SEQ ID NO:1的第X位处于等同位置的位置。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，保守置换通常是指，用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA94:412-417(1997)，其通过引用并入本文)。
本文将“显著降低的5'至3'核酸外切酶活性”定义为:(1)具有约或低于10％的，或优选约或低于5％或1％，相应的未突变的野生型酶(例如野生型Taq DNA聚合酶)的5'至3'核酸外切酶活性。
根据本发明，术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸所编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌体；柯斯质粒等等。
根据本发明，术语“裂解上清”是指通过下述步骤所产生的溶液：将宿主细胞(例如大肠杆菌)在裂解液中破碎，然后将含有经破碎的宿主细胞的裂解液中的不溶物去除。各种裂解液是本领域技术人员公知的，包括但不限于Tris缓冲液，磷酸盐缓冲液，HEPES缓冲液，MOPS缓冲液等等。此外，可通过本领域技术人员熟知的各种方法来实现宿主细胞的破碎，包括但不限于匀浆器破碎、均质机破碎、超声波处理、研磨、高压挤压、溶菌酶处理等等。去除裂解液中的不溶物的方法也是本领域技术人员公知的，包括但不限于过滤和离心。
本文中“表达”是指由结构基因产生多肽的方法。它包括将该基因转录成信使RNA(mRNA)和将这种mRNA翻译成多肽。
本文中“焦磷酸水解激活的聚合酶反应”是指聚合酶利用其焦磷酸酶活性将封闭在引物末端的ddNMP焦磷酸化产生ddNTP，使得末端的ddNMP脱落下来，引物封闭被解除，从而使得引物得以延伸，DNA聚合反应得以继续的过程。
发明的有益效果
(1)本申请的突变的Taq DNA聚合酶(例如Taq 07突变体)或其变体具有优异的聚合酶活性，其是野生型Taq DNA聚合酶的大约至少2倍，至少3倍，至少4倍，至少5倍，或6倍；
(2)本申请的突变的Taq DNA聚合酶(例如Taq 07突变体)或其变体具有显著降低的5'至3'核酸外切酶活性(例如，低于野生型Taq DNA聚合酶的5'至3'核酸外切酶活性的大约10％，5％，或1％)。例如，本申请的Taq 07突变体实质上不具有5’至3’的核酸外切酶活性。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1为利用荧光探针检测Taq DNA聚合酶5’至3’核酸外切酶活性的原理图。所使用的荧光探针是一种寡核苷酸探针，其5’末端可以携带荧光基团(例如FAM)，5’端下游可以携带淬灭基团(例如BHQ)。该探针在55℃条件下仍然会保持稳定的发夹结构，当反应刚开始时，报告基团发射的荧光信号被淬灭基团吸收；随着反应的进行，Taq DNA聚合酶会跟随3’端延伸而移动，当移动到探针5’端荧光基团处时，Taq DNA聚合酶的5’至3’外切酶活性将探针酶切，使报告荧光基团和淬灭荧光基团分离，从而荧光监测系统可接收到荧光信号。因此，在反应过程中，每扩增一条DNA链，就有一个荧光分子形成，实现了荧光信号的累积与荧光产物的形成完全同步。
图2为利用Picogreen荧光染料检测Taq DNA聚合酶的聚合酶活性的原理图。荧光染料Picogreen是一种高灵敏的插入到DNA分子的荧光染料。在72℃条件下，Taq DNA聚合酶对引物的延伸伴随着荧光染料的插入，从而实时报告DNA产物的生成。
图3为利用图1和图2所示的检测系统绘制的野生型Taq DNA聚合酶在不同浓度条件下的聚合酶活性及5’至3’外切酶活性的标准曲线。利用该曲线可以定量计算其他突变体的聚合酶活性及5’至3’外切酶活性。
图4为以野生型Taq DNA聚合酶为标准所定量出的WT(野生型)、突变体E507K和Taq07的相对聚合酶活性(图4A)及相对5’至3’外切酶活性(图4B)。
序列信息
本发明涉及的序列的信息提供于下面的表1中。
表1
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本申请中所使用的分子生物学实验方法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wi ley&Sons,Inc.，1995中所述的方法进行；酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本申请，且不意欲限制本申请所要求保护的范围。
本实施例所用野生型Taq-DNA聚合酶，聚合酶突变体E507K和Taq07的氨基酸序列分别如SEQ ID NO：1，SEQ ID NO：4和SEQ ID NO：2，所述三种聚合酶制备方法如下：
1.挑取含编码聚合酶的DNA的菌种于5mL LB培养基中,37℃过夜；
2.种子液按1:100转接至500mL新鲜培养基中,待OD600到达0.4-0.6之间时加入终浓度为0.2mM的IPTG，37℃诱导过夜；
3.将菌液倒入500mL离心瓶中，2500g 4℃离心20min，弃去上清,于-80℃冰箱中放置30min；
4.用30mL预冷的Banding buffer(β-ME 1:1000，PMSF 1:100)重悬菌体(若为不含溶菌酶的菌种可以自行加入终浓度为0.1mg/mL的溶菌酶)；
5.将菌悬液在冰水混合物中超声破碎，6Ω功率50％，10-30min(程序设置为每超声2s暂停3s)；
6.将超声后的悬液在75℃水浴锅热处理30min；
7.将加热后的悬液12,000rpm 4℃离心20min除去细胞碎片，收集上清液S，取出100uL制样；
8.取1mL-2mL Ni-NTA Agarose(购于QIAGEN公司，货号：30210)，先用15mL双蒸水冲去乙醇，后分别用5mL Elution buffer、5mL Wash buffer、15mL Banding buffer过柱洗脱(每ml填料可以结合蛋白大约20-30mg)；
9.将步骤7中离心后的上清与beads充分混合，4℃孵育1h以上，随后让液体自然流出，收集穿透液FT，取出100uL制样；
10.用15个Ni-NTA Agarose体积的Banding buffer洗Ni-NTA Agarose，随后让液体自然流出，收集洗脱液B，并取出100uL制样；
11.用1个Ni-NTA Agarose体积的Wash buffer洗Ni-NTA Agarose,收集洗脱液W1,取出100uL制样；重复洗涤一次，收集洗脱液W2，取出100uL制样；
12.用0.5个Ni-NTA Agarose体积的Elution buffer洗Ni-NTA Agarose,收集洗脱液E1，取出100uL制样；重复洗脱四次，分别收集洗脱液E2、E3、E4，各取出100uL制样；
13.将收集好的S、FT、B、W1、W2、E1、E2、E3、E4置于4℃保存；将各自取出的100uL进行制样，通过SDS-PAGE分析确定含有聚合酶的级分；而后对相应的级分进行过夜透析，获得经纯化的聚合酶。
实施例1：检测野生型Taq-DNA聚合酶(WT)的5’至3’外切酶活性及聚合酶活性
1.野生型Taq-DNA聚合酶的5’至3’外切酶活性的检测
设计一段可形成双端发夹结构的单链DNA，该单链DNA在延伸过程中可以形成稳定的发夹结构。在该发夹结构中设计可以与该单链DNA结合的荧光探针，该探针5’端核苷酸携带了FAM荧光基团，5’端下游核苷酸携带了BHQ猝灭基团(如图1所示)，该探针在55℃条件下仍然会保持稳定的发夹结构。在Taq DNA聚合酶开始延伸之前，报告基团发射的荧光信号被淬灭基团吸收；随着延伸反应的进行，Taq DNA聚合酶会跟随3’端延伸而移动，当移动到探针5’端荧光基团处时，Taq DNA聚合酶的5’至3’外切酶活性将探针酶切，使报告荧光基团和淬灭荧光基团分离，从而荧光监测系统可接收到荧光信号。因此，在反应过程中，每扩增一条DNA链，就有一个荧光分子形成，实现了荧光信号的累积与荧光产物的形成完全同步。由此，通过检测荧光信号的强弱，可以确定DNA聚合酶的5’至3’外切酶活性的高低。本实验所使用的形成双端发夹结构的单链DNA的序列如SEQ ID NO:5。
本实验所使用的反应体系及反应程序如表2-3所示：
表2：外切酶活性检测体系
| 组分 | 20μL反应混合物 |
| 双蒸水 | 添加至20μL |
| 10×Taq缓冲液 | 2μL |
| 2.5mM dNTP | 2μL |
| 100μM发夹结构DNA(SEQ ID NO:5) | 1μL |
| Taq-DNA聚合酶(1U/uL) | 1/2/3/4/5μL |
表3：外切酶活性检测程序:
| 反应过程 | 反应条件 | 循环数 |
| 扩增程序 | 55℃15s | 99 |
测量Taq-DNA聚合酶不同浓度下反应的荧光强度，可以绘制野生型Taq DNA聚合酶的5’至3’外切酶活性的标准曲线。
2.野生型Taq-DNA聚合酶的聚合酶活性的检测
设计一段可形成单端发夹结构的单链DNA，该单链DNA在72℃延伸过程中可以形成稳定的发夹结构(如图2所示)。形成单端发夹结构的单链DNA的序列如SEQ ID NO:6。
Picogreen(购于赛默飞公司，货号：P7589)是一种极为灵敏的荧光核酸染料，其仅在与DNA双链结合后才发出荧光，且所产生的荧光与双链DNA浓度呈正比。往反应体系中加入Picogreen，当引物延伸时随着DNA分子的增加，荧光信号也会逐渐增强，从而实时报告DNA分子的增加。由此，通过检测荧光信号的强弱，可以确定DNA分子的浓度/数量，进而可确定聚合酶活性的高低(如图2所示)。
本实验所使用的聚合酶活性测定体系及反应程序如表4-5所示：
表4：聚合酶活性体系
| 组分 | 20μL反应混合物 |
| 双蒸水 | 添加至20μL |
| 10×Taq缓冲液 | 2μL |
| 2.5mM dNTP | 2μL |
| 100μM发夹结构DNA(SEQ ID NO:6) | 1μL |
| Taq-DNA聚合酶(1U/uL) | 1/2/3/4/5μL |
| 40X Picogreen | 0.5uL |
表5：聚合酶活性测定程序:
| 反应过程 | 反应条件 | 循环数 |
| 反应程序 | 72℃15s | 99 |
测量Taq-DNA聚合酶不同浓度下反应的荧光强度，可以绘制Taq DNA聚合酶的聚合酶活性的标准曲线。
标准曲线的绘制方法如下。简言之，如上所述使用指定用量的Taq DNA聚合酶进行酶活性测定反应，测定并记录各个聚合酶用量条件下的1分钟反应时所改变的荧光信号FU值如ΔFU1，ΔFU2，ΔFU3，ΔFU4，ΔFU5。随后，以Taq DNA聚合酶(1U/uL)用量为1uL时测定的ΔFU1值为基底，利用公式RFU＝ΔFUX/ΔFU1，计算各个聚合酶用量条件下的RFU。随后，以聚合酶用量为横坐标，以RFU为纵坐标，绘制酶活的标准曲线。
实验结果如图3所示。图3显示了利用上述两个检测系统绘制的野生型Taq DNA聚合酶的聚合酶活性及5’至3’外切酶活性的标准曲线，其中，横坐标为野生型Taq DNA聚合酶的浓度，纵坐标为RFU值。利用该曲线可以定量计算Taq DNA聚合酶突变体的相对聚合酶活性及相对5’至3’外切酶活性。
实施例2：检测Taq-DNA聚合酶突变体(E507K和Taq07)的5’至3’外切酶活性及聚合酶活性
Taq-DNA聚合酶突变体E507K和Taq07的5’至3’外切酶活性及聚合酶活性的检测方法同实施例1。如下计算突变体E507K和Taq07相对于野生型Taq-DNA聚合酶的相对酶活。简言之，取1U/uL的E507K和Taq 07聚合酶各1uL，如实施例1所述进行反应，测定并记录ΔFU值。随后，如实施例1所述计算E507K和Taq 07聚合酶在该用量下的RFU值，并基于标准曲线方程，确定E507K和Taq 07聚合酶相对于野生型Taq-DNA聚合酶的相对酶活。
实验结果如图4所示。图4A显示了野生型Taq DNA聚合酶(WT)、E507K、Taq 07的相对聚合酶活性(以WT的聚合酶活性为参照)；图4B显示了WT、E507K、Taq 07的相对5’至3’外切酶活性(以WT的5’至3’外切酶活性为参照)。结果表明Taq 07的聚合酶活性是WT的6倍左右，是E507K的1.5倍左右；且外切酶活性检测结果表明，Taq 07无5’至3’外切酶活性。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
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Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu
195 200 205
Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu
210 215 220
Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys
225 230 235 240
Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val
245 250 255
Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe
260 265 270
Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu
275 280 285
Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly
290 295 300
Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp
305 310 315 320
Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro
325 330 335
Glu Pro Tyr Lys Ala Leu Arg Asp Val Lys Glu Ala Arg Gly Leu Leu
340 345 350
Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro
355 360 365
Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn
370 375 380
Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu
385 390 395 400
Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu
405 410 415
Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu
420 425 430
Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly
435 440 445
Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala
450 455 460
Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His
465 470 475 480
Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp
485 490 495
Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg
500 505 510
Ser Thr Ser Ala Ala Val Leu Gly Ala Leu Arg Glu Ala His Pro Ile
515 520 525
Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr
530 535 540
Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu
545 550 555 560
His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser
565 570 575
Ser Asn Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln
580 585 590
Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala
595 600 605
Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly
610 615 620
Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr
625 630 635 640
Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro
645 650 655
Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly
660 665 670
Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
675 680 685
Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg
690 695 700
Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val
705 710 715 720
Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg
725 730 735
Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro
740 745 750
Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu
755 760 765
Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His
770 775 780
Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala
785 790 795 800
Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro
805 810 815
Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu
820 825 830
<210> 3
<211> 2499
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码突变的Taq 07聚合酶的核苷酸序列
<400> 3
atggcgggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60
cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120
gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180
gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240
tacaaggcgg gccgggcccc cacgccagag gactttcccc ggcaactcgc cctcatcaag 300
gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360
gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420
gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480
tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540
gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600
gagaagacgg cgaggaagct cctggaggag tgggggagcc tggaagccct cctcaagaac 660
ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720
ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780
aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840
ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900
ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960
cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020
gccctcaggg acgtgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080
ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140
gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200
gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260
gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320
ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380
ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440
cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500
cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctgggg 1560
gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag 1620
ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680
cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc caatcccaac 1740
ctccagaaca tccccgtccg caccccgctt gggcagagga tcaggcgggc cttcatcgcc 1800
gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860
cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920
gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg 1980
gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040
gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100
cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160
gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220
cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280
atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340
cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400
cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460
gtggggatag gggaggactg gctctccgcc aaggagtaa 2499
<210> 4
<211> 832
<212> PRT
<213> Artificial Sequence
<220>
<223> 突变的Taq E507K聚合酶的氨基酸序列
<400> 4
Met Ala Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu
1 5 10 15
Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly
20 25 30
Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala
35 40 45
Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val
50 55 60
Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly
65 70 75 80
Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu
85 90 95
Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu
100 105 110
Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys
115 120 125
Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp
130 135 140
Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly
145 150 155 160
Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175
Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn
180 185 190
Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu
195 200 205
Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu
210 215 220
Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys
225 230 235 240
Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val
245 250 255
Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe
260 265 270
Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu
275 280 285
Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly
290 295 300
Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp
305 310 315 320
Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro
325 330 335
Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu
340 345 350
Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro
355 360 365
Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn
370 375 380
Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu
385 390 395 400
Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu
405 410 415
Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu
420 425 430
Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly
435 440 445
Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala
450 455 460
Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His
465 470 475 480
Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp
485 490 495
Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Lys Lys Thr Gly Lys Arg
500 505 510
Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile
515 520 525
Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr
530 535 540
Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu
545 550 555 560
His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser
565 570 575
Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln
580 585 590
Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala
595 600 605
Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly
610 615 620
Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr
625 630 635 640
Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro
645 650 655
Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly
660 665 670
Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
675 680 685
Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg
690 695 700
Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val
705 710 715 720
Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg
725 730 735
Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro
740 745 750
Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu
755 760 765
Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His
770 775 780
Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala
785 790 795 800
Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro
805 810 815
Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu
820 825 830
<210> 5
<211> 92
<212> DNA
<213> Artificial Sequence
<220>
<223> 形成双端发夹结构的寡核苷酸探针的序列
<400> 5
caccgctggg cgcgatctgc cgcgcccagc ggtgacgtat aggtcctagc tacatgaacc 60
ccggcgccgt agatctgcct acggcgccgg gg 92
<210> 6
<211> 72
<212> DNA
<213> Artificial Sequence
<220>
<223> 形成单端发夹结构的寡核苷酸的序列
<400> 6
cgcgcccagc ggtgacgtat aggtcctagc tacatgaacc ccggcgccgt agatctgcct 60
acggcgccgg gg 72
Claims (10)
1.一种突变的Taq DNA聚合酶或其变体，其中，所述突变的Taq DNA聚合酶与野生型TaqDNA聚合酶相比，具有下述突变：
(1)在与SEQ ID NO:1的第345位对应的位置处的氨基酸残基被缬氨酸残基替换；
(2)在与SEQ ID NO:1的第520位对应的位置处的氨基酸残基被甘氨酸残基替换；和
(3)在与SEQ ID NO:1的第578位对应的位置处的氨基酸残基被天冬酰胺残基替换；
其中，所述变体与所述突变的Taq DNA聚合酶相比，具有至少90％，例如至少95％，至少96％，至少97％，至少98％，至少99％的序列同一性；或者，具有一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸的置换(优选保守置换)、添加或缺失；且，
所述变体中与SEQ ID NO:1的第345位、第520位和第578位对应的位置处的氨基酸残基分别与所述突变的Taq DNA聚合酶相同；且
所述变体保留了所述突变的Taq DNA聚合酶的功能(例如，具有DNA聚合酶活性，且具有低于野生型Taq DNA聚合酶的5’至3’核酸外切酶活性)。
2.权利要求1所述的Taq DNA聚合酶或其变体，其聚合酶活性是野生型Taq DNA聚合酶的大约至少2倍，至少3倍，至少4倍，至少5倍，或6倍；和/或，所述Taq DNA聚合酶或其变体的核酸外切酶活性不超过野生型Taq DNA聚合酶的大约10％，5％，或1％；
优选地，所述Taq DNA聚合酶或其变体实质上不具有5’至3’核酸外切酶活性。
3.权利要求1或2所述的Taq DNA聚合酶或其变体，其中，所述野生型Taq DNA聚合酶具有如SEQ ID NO:1所示的氨基酸序列；或者，所述突变的Taq DNA聚合酶具有如SEQ ID NO:2所示的氨基酸序列。
4.一种核酸，其包含编码如权利要求1-3任一项所述的突变的Taq DNA聚合酶或其变体的核苷酸序列；
优选地，所述核酸具有如SEQ ID NO:3所示的核苷酸序列。
5.载体，其包含权利要求4所述的核酸；
优选地，所述载体是例如质粒，粘粒，噬菌体。
6.宿主细胞，其包含权利要求4所述的核酸或权利要求5所述的载体；
优选地，所述宿主细胞选自：原核细胞例如大肠杆菌细胞，真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞)；
优选地，所述宿主细胞是大肠杆菌。
7.制备权利要求1-3任一项所述的突变的Taq DNA聚合酶或其变体的方法，其包括：在宿主细胞中表达所述突变的Taq DNA聚合酶或其变体，然后从所述宿主细胞的培养物中回收所述突变的Taq DNA聚合酶或其变体；
优选地，所述宿主细胞是大肠杆菌。
8.权利要求1-3任一项所述突变的Taq DNA聚合酶或其变体的用途，其用于：
1)进行核酸合成或扩增(例如PCR)；或
2)进行焦磷酸水解激活的聚合酶反应(PAP)；或
3)分析或测定核酸(例如DNA)分子的核苷酸序列；
优选地，所述焦磷酸水解激活的聚合酶反应(PAP)用于检测稀有突变；
优选地，所述核苷酸序列分析或测定过程包括以下步骤：将能与所述核酸分子杂交的引物分子，与所述核酸分子以及所述突变的Taq DNA聚合酶或其变体孵育；以及确定所述核酸分子至少一部分的核苷酸序列。
9.一种试剂盒，其包含权利要求1-3任一项所述的突变的Taq DNA聚合酶或其变体；
优选地，所述试剂盒可用于进行核酸合成或扩增(例如PCR)，PAP或测序反应；
优选地，所述试剂盒还包括选自下列的试剂：用于进行PCR的试剂(例如，缓冲液，dNTP，引物)；用于进行PAP的试剂(例如，缓冲液，焦磷酸或其类似物)；用于进行测序反应的试剂(例如，缓冲液，dNTP，引物，合成终止剂)；或其任何组合。
10.一种组合物，其包含权利要求1-3任一项所述的突变的Taq DNA聚合酶或其变体，或权利要求4所述的核酸，或权利要求5所述的载体，或权利要求6所述的宿主细胞。
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