CN117693581A - 聚合酶突变体以及与3'-oh未封闭的可逆终止子一起使用 - Google Patents
聚合酶突变体以及与3'-oh未封闭的可逆终止子一起使用 Download PDFInfo
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- CN117693581A CN117693581A CN202180099775.7A CN202180099775A CN117693581A CN 117693581 A CN117693581 A CN 117693581A CN 202180099775 A CN202180099775 A CN 202180099775A CN 117693581 A CN117693581 A CN 117693581A
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Abstract
提供了突变聚合酶,其具有改进的掺入包括3'‑OH未封闭的可逆终止子在内的修饰的核苷酸的能力。所述突变聚合酶可用于多种应用,例如用于多核苷酸测序、引物延伸反应和非模板依赖性酶促寡核苷酸合成。
Description
相关申请的交叉引用
无。
技术领域
本发明涉及突变聚合酶和使用此类突变聚合酶用于多核苷酸测序、引物延伸反应和其他应用的方法。
背景技术
聚合酶天然存在于生物体中以复制和维持其基因组。聚合酶已经在生物技术领域中广泛用于多种应用,包括PCR和测序。聚合酶通过检测核苷酸碱基之间的互补性和/或识别寡核苷酸链的结构特征,并充当用于核苷酸和链的3′端之间的反应的酶,使得能够复制DNA或RNA。仍然需要用于各种生物技术应用的聚合酶,其具有改进的核苷酸掺入,特别是被修饰以在核酸聚合过程中充当可逆终止子的核苷酸的掺入。
Therminator DNA聚合酶是B家族嗜热球菌属物种(Thermococcus sp.)9°N-7DNA聚合酶的衍生物。Therminator可从New England Biolabs,Inc.(伊普斯威奇,马塞诸塞州)商购获得,并且其特性和应用最近综述于Gardner等人“Therminator DNA Polymerase:Modified Nucleotides and Unnatural Substrates”,Front.Mol.Biosci.,2019年4月24日(doi.org/10.3389/fmolb.2019.00028)中。
激烈火球菌(Pyrococcus furiosus,Pfu)是一种极端嗜热的古细菌物种,最初是从温度在90℃与100℃之间的地热加热的海洋沉积物中分离出来的。激烈火球菌拥有B家族DNA聚合酶,所述聚合酶已用于聚合酶链式反应(PCR)和其他生物技术应用。参见PfuTurboDNA聚合酶指导手册,修订版G0,Agilent Technologies,Inc.2015,2020;Elshawadfy等人,“DNA polymerase hybrids derived from the family-B enzymes of Pyrococcusfuriosus and Thermococcus kodakarensis:improving performance in thepolymerase chain reaction.”Front Microbiol.2014年5月27日;5:224.doi:10.3389/fmicb.2014.00224。
许多野生型和突变DNA聚合酶已经用于或有潜力用于多种生物技术应用中,特别是在它们具有掺入修饰的核苷酸的能力时。此类DNA聚合酶可用于多核苷酸测序、克隆、PCR或其他扩增、单核苷酸多态性(SNP)检测、全基因组扩增(WGA)、合成生物学、分子诊断和其他应用。
DNA聚合酶的一个潜在应用是酶介导的寡核苷酸合成(TiEOS,非模板依赖性酶促寡聚物合成)。TiEOS是一种从天然和修饰的核苷酸产生长核酸聚合物的方法。参见Jensen等人,“Template-Independent Enzymatic Oligonucleotide Synthesis(TiEOS):ItsHistory,Prospects,and Challenges”,(2018)Biochemistry 57:1821-32。目前的方法将非模板依赖性DNA聚合酶与可逆修饰的终止子结合以将寡核苷酸延长控制为每个循环添加单个碱基。TiEOS的优选酶是末端脱氧核苷酸转移酶(TdT)。TdT被归类为X家族DNA聚合酶,在体内以非模板依赖性方式添加核苷酸以增加哺乳动物的抗原受体多样性。TdT以低于100%的效率掺入可逆终止子,这限制了所得合成寡核苷酸的长度和保真度。迄今为止,最长的酶促合成的寡核苷酸由DNA Script使用工程化的X家族DNA聚合酶报道(280mer,99.4%逐步产率)。参见Eisenstein(2020)Nature Biotechnology 38:1113-1115;和美国专利号10,752,887。此外,许多开发用于边合成边测序(sequencing-by-synthesis)的可逆终止子在从核苷酸的糖(3′-OH封闭)或碱基(3′-OH未封闭)去除终止基团后留下疤痕或修饰。
需要用于多核苷酸测序和其他生物技术应用的聚合酶,其具有改进的掺入修饰的核苷酸的能力,包括修饰为用作可逆终止子的核苷酸。还需要以高掺入和终止效率以非模板依赖性方式掺入无疤可逆终止子的酶。
发明内容
作为本发明的一个方面,提供了突变聚合酶。所述突变聚合酶包含与SEQ ID NO:1至少80%相同的氨基酸序列,并且还包含在与本文鉴定的Pfu聚合酶中的氨基酸位置功能等同的一个或多个位置处的至少一个氨基酸突变。在一些实施方案中,所述聚合酶包含在与Pfu聚合酶中的位置486功能等同的位置处的突变,和/或包含在与Pfu聚合酶中的位置546功能等同的位置处的突变,和/或包含在与Pfu聚合酶中的位置477功能等同的位置处的突变。示例性突变聚合酶包括那些包含SEQ ID NO:2、SEQ ID NO:9、SEQ ID NO:10、SEQ IDNO:4或SEQ ID NO:5的氨基酸序列的聚合酶。
作为本发明的另一个方面,提供了包含如本文所述的可逆终止子和突变聚合酶的组合物。在一些实施方案中,所述可逆终止子是3′-OH未修饰的可逆终止子(例如闪电终止子(Lightning Terminator))。参见例如,例如,Weidong Wu等人,“Termination of DNAsynthesis by N6-alkylated,not 3′-O-alkylated,photocleavable 2′-deoxyadenosinetriphosphates,”Nucleic Acids Research,第35卷,第19期,2007年10月1日,6339-6349页;Vladislav A.Litosh等人,“Improved nucleotide selectivity and termination of3′-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzylalkylated HOMedU triphosphates,”Nucleic Acids Research,第39卷,第6期,2011年3月1日,第e39页;和Brian P.Stupi等人,“Stereochemistry of Benzylic CarbonSubstitution Coupled with Ring Modification of 2-Nitrobenzyl Groups as KeyDeterminants for Fast-Cleaving Reversible Terminators”,Angew.Chem.Int.Ed.2012,51,1724 -1727;Andrew F.Gardner等人,“Rapidincorporation kinetics and improved fidelity of a novel class of 3′-OHunblocked reversible terminators”,Nucleic Acids Research,第40卷,第15期,2012年8月1日,7404-7415页。
作为另一个方面,提供了包含3′-OH未封闭的可逆终止子和突变聚合酶的组合物。所述突变聚合酶包含与SEQ ID NO:2至少96%相同的氨基酸序列,并且包含在与Pfu聚合酶中的K477、A486和Y546处的氨基酸位置功能等同的位置处的氨基酸突变。
作为另一个方面,提供了一种将核苷酸掺入包含核酸的引发链的方法。所述方法包括在足以进行掺入反应的条件下,使所述引发链与核苷酸和突变聚合酶接触。所述突变聚合酶包含与SEQ ID NO:2至少96%相同的氨基酸序列,并且包含在与Pfu聚合酶中的K477、A486和Y546处的氨基酸位置功能等同的位置处的氨基酸突变。
本发明的另一方面涉及一种多核苷酸测序的方法。所述方法包括(a)形成包含模板和引发链的双链体,其中所述模板包含待测序的靶核酸和与所述引发链的至少一部分互补的引物结合位点;(b)将所述引发链与可逆终止子核苷酸和突变聚合酶组合,其中所述突变聚合酶包含与SEQ ID NO:2至少96%相同的氨基酸序列,并且包含在与Pfu聚合酶中的K477、A486和Y546处的氨基酸位置功能等同的位置处的氨基酸突变;(c)在模板依赖性反应中在引发链的3′端掺入所述可逆终止子;和(d)鉴定掺入的可逆终止子核苷酸,从而确定所述模板的序列。
作为本发明的另一个方面,提供了一种包含引发链、3′-OH未封闭的可逆终止子和突变聚合酶的组合物。所述突变聚合酶包含与SEQ ID NO:1至少80%(可替代地,至少85%、90%或95%)相同的氨基酸序列。所述突变聚合酶还包含在与Pfu聚合酶中的位置L270、E330、Q332、L333、L409、P451、L453、L457、E476、L489、L490、N492、F494、Y497和E581功能等同的位置处的一个或多个突变。所述突变聚合酶的掺入活性比SEQ ID NO:11的DNA聚合酶的掺入活性高至少4倍。
作为又一方面,提供了一种将3′-OH未修饰的可逆终止子掺入引发链中的方法。所述方法包括在足以进行掺入反应的条件下,使引发链与3′-OH未修饰的可逆终止子和突变聚合酶接触。所述突变聚合酶包含与SEQ ID NO:1至少80%相同的氨基酸序列和在与Pfu聚合酶中的位置L270、E330、Q332、L333、L409、P451、L453、L457、E476、L489、L490、N492、F494、Y497和E581功能等同的位置处的一个或多个突变。所述方法还包括在引发链的3′端掺入3′-OH未修饰的可逆终止子。
作为另一个方面,提供了一种包含引发链、3′-OH未修饰的可逆终止子和突变聚合酶的组合物。所述突变聚合酶与SEQ ID NO:2至少96%相同,并且包含:在与Pfu聚合酶中的位置546功能等同的位置处的Y546H突变;在与Pfu聚合酶中的位置409功能等同的位置处的L409Y、L409H或L409F突变;以及在与Pfu聚合酶中的位置486功能等同的位置处的A486X突变,其中X是除丙氨酸之外的任何氨基酸。
作为本发明的另一个方面,提供了一种在非模板依赖性反应中将单个核苷酸掺入引发链的方法。所述方法包括将引发链与3′-OH未修饰的可逆终止子和突变聚合酶组合。所述突变聚合酶与SEQ ID NO:2至少96%相同,并且包含:在与Pfu聚合酶中的位置546功能等同的位置处的Y546H突变;在与Pfu聚合酶中的位置409功能等同的位置处的L409Y、L409H或L409F突变;以及在与Pfu聚合酶中的位置486功能等同的位置处的A486X突变,其中X是除丙氨酸之外的任何氨基酸。终止子的掺入比SEQ ID NO:11的突变DNA聚合酶高至少2倍(可替代地,4倍或10倍)。
作为另一个方面,提供了一种用于3′-OH非模板依赖性寡核苷酸合成的方法。所述方法包括将引发链与3′-OH未修饰的可逆终止子和突变DNA聚合酶组合。所述突变DNA聚合酶包含:与SEQ ID NO:2至少96%相同的氨基酸序列;在与Pfu聚合酶中的位置546功能等同的位置处的变为组氨酸的Y546H突变;在与Pfu聚合酶中的位置409功能等同的位置处的L409Y、L409H或L409F突变;以及在与Pfu聚合酶中的位置486功能等同的位置处的A486X突变,其中X是除丙氨酸之外的任何氨基酸。所述方法还包括将3′-OH未封闭的未修饰的可逆终止子掺入引发链。
结合所附权利要求,从下面的详细描述中,本发明的方法和组合物的这些和其他特征和优点将是清楚的。
附图说明
图1A显示了用于DNA测序的具有5-羟甲基尿嘧啶碱基和附接的染料的可逆(3′-OH未封闭)闪电终止子。图1B和图1C显示了3-OH封闭的终止子与未修饰的3-OH终止子(在本公开文本中使用)之间的概念差异。图1D显示了天然dATP。图1E和图1F显示了用于使用3′-OH未封闭的可逆终止子的TiEOS的可逆dATP终止子。图1E(“LTA-1”)在腺苷的N6位置用硝基苄基部分修饰。图1F(“LTA-2”)在7-羟甲基-7-脱氮-脱氧腺苷上被α-叔丁基硝基苄基部分修饰,并且任选地在三磷酸酯部分上具有α-硫代修饰。
图2显示了来自实施例4中用突变嵌合聚合酶测序的测序度量。Pfu A486L/Y546HDNA聚合酶(“对照”;Pfu 2)的以下结构域被9°N DNA聚合酶的相应区段取代:A-1-99;B-100-199;C-400-449;和D-(20-40个氨基酸的4个区段,跨越500-599)。对于结构域D,ARL和L+L表示为由4种子嵌合聚合酶展现出的平均值。
图3显示了具有在Pfu聚合酶的位置486处的突变的突变聚合酶用于掺入可逆终止子(LTA)的活性。
图4A和图4B显示了在实施例5中测试的各种F494X突变聚合酶用于掺入可逆终止子(LTA)的活性。
图5显示了在实施例6中用天然核苷酸测试的各种突变聚合酶的活性。
图6显示了在实施例6中测试的各种突变聚合酶对可逆终止子的相对掺入情况。
图7A-图7B显示了在实施例6中测试的Y546X突变体(不含A486L)缺乏可逆终止子的掺入。
图8显示了突变聚合酶与修饰的核苷酸之间的相互作用的模型。
图9A至图9C显示了实施例7中通过TdT的市售制剂(Promega)和各种突变聚合酶进行的天然dATP的非模板依赖性掺入的测量结果。
图10A至图10E显示了实施例8中通过TdT的市售制剂(Promega)和各种突变聚合酶进行的可逆终止子在引发链中的非模板依赖性掺入的测量结果。
图11A至图11F显示了实施例9中通过TdT的市售制剂(Promega)进行的可逆终止子的非模板依赖性掺入的测量结果。
图12A和图12B显示了实施例9中通过突变聚合酶(Pfu26)进行的可逆终止子在引发链中的非模板依赖性掺入的测量结果。
图13A至图13C显示了实施例10中通过突变聚合酶(Pfu26)进行的3′-OH未封闭的可逆终止子(LTA-2)的多循环添加的测量结果。
当结合附图阅读时,从以下详细描述中可以最好地理解本教导。特征不一定按比例绘制。
具体实施方式
本公开文本提供了用于改进的3′-OH未封闭的可逆终止子核苷酸(例如闪电终止子)的掺入的突变聚合酶。诸位发明人已经出人意料地鉴定了某些突变聚合酶,其展现出改进的可逆终止子的掺入并且具有许多其他相关优点,例如改进的测序性能和较低的DNA结合亲和力。
本公开文本还提供了编码本文所述的突变聚合酶的核酸分子。根据已知密码子和氨基酸之间的对应关系,可以很容易地根据本文所披露的氨基酸序列设想出此类核酸分子。本公开文本还提供了包含此类核酸分子的表达载体。本公开文本还提供了包含此类表达载体的宿主细胞。
本公开文本还提供了用于将一个或多个可逆终止子核苷酸掺入引发链中的方法,所述引发链能够充当核苷酸的掺入点并从其3′端延伸。所述方法包括允许以下组分相互作用:(i)如本文所述的突变聚合酶,(ii)引发链;和(iii)包含可逆终止子如闪电终止子的核苷酸溶液。
本公开文本还提供了一种用于进行核苷酸掺入反应的试剂盒,其包含如本文所述的突变聚合酶和核苷酸溶液。在一些实施方案中,所述核苷酸溶液包含3′-OH未封闭的可逆终止子。
本文所述的突变聚合酶针对修饰的核苷酸,尤其是3′-OH未封闭的可逆终止子的掺入有所改进。诸位发明人已经鉴定了某些突变聚合酶,其展现出改进的可逆终止子的掺入并且具有许多其他相关优点,包括较低的DNA结合亲和力和在边合成边测序反应中改进的测序度量。较低的结合亲和力将允许突变聚合酶在延伸的和未延伸的主链之间快速循环,与具有较高亲和力的聚合酶相比,预计这将产生更高的掺入效率。
如下文更详细描述的,已经发现聚合酶中一个或多个残基的一个或多个突变导致更替速率的显著增加和焦磷酸解作用的降低。这些突变聚合酶在DNA边合成边测序(SBS)中具有改进的性能,并导致定相和/或预定相减少,以及在边合成边测序反应中总体改进的质量度量。“定相”是指由于在测序循环期间,未能在簇内的一些链中掺入核苷酸,而在SBS期间在该簇内失去同步性。“预定相”是指SBS簇中的一种情况,其中没有有效3′终止子的核苷酸被掺入一些链中,导致它们比所述簇的结果提前1个循环。
在一些实施方案中,突变聚合酶包含与SEQ ID NO:1至少60%、70%、80%、85%、90%、95%、96%、97%、98%或99%相同的氨基酸序列,所述重组DNA聚合酶包含在与PfuDNA聚合酶氨基酸序列中的某些位置功能等同的一个或多个位置处的至少一个氨基酸突变。野生型Pfu DNA聚合酶氨基酸序列示于SEQ ID NO:1中。
在一些实施方案中,本发明的突变聚合酶包含与SEQ ID NO:1至少80%、85%、90%、95%、96%、97%、98%或99%相同的氨基酸序列,并且还包含在与Pfu聚合酶中的409、477、486或546处的氨基酸位置残基功能等同的一个或多个位置处的至少一个氨基酸突变。在一些实施方案中,聚合酶包含在与Pfu聚合酶中的位置486功能等同的位置处的突变,并且还可以包含在与Pfu聚合酶中的位置546功能等同的位置处的突变,并且可以仍进一步包含在与Pfu聚合酶中的位置477功能等同的位置处的突变。在一些实施方案中,突变聚合酶与SEQ ID NO:2至少96%相同并且包含突变A486X,其中X可以是除丙氨酸之外的任何氨基酸。例如,突变聚合酶可以包含具有突变Y546H、K477W和A486X的SEQ ID NO:1的氨基酸序列,其中X可以是除丙氨酸之外的任何氨基酸。在一些实施方案中,突变聚合酶还包含突变D141A和E143A。
在一些实施方案中,突变聚合酶还包含在与Pfu聚合酶中的位置270、330、332、333、409、451、453、457、476、489、490、492、494、497和581功能等同的位置处的一个或多个突变。在一些实施方案中,所述突变聚合酶不包含在与Pfu聚合酶中的位置266、267、268、269、329、336、399、400、403、404、407、408、410、411、450、452、455、456、458、459、460、461、462、463、464、465、466、475、477、478、479、480、481、482、483、485、487、488、491、493、495、496、498、499、500、515、522、545、546、577、579、580、582、584、591、595、603、606、607、608、612、613、614、664、665、666、668、669、674、675和676功能等同的任一位置处的突变。
上文所述的突变聚合酶可包含已知在3′封闭核苷酸的存在下和/或在DNA测序应用中增强聚合酶活性的一个或多个方面的另外的突变。
在一些实施方案中,与野生型聚合酶相比,突变聚合酶包含降低的外切核酸酶活性。例如,突变聚合酶可以包含在与9°N DNA聚合酶的氨基酸序列中的Asp141和/或Glu143功能等同的位置处的突变。
在一些实施方案中,突变聚合酶可以包含另外的突变以去除内部甲硫氨酸。例如,在一些实施方案中,突变聚合酶包含在与Pfu和9°N DNA聚合酶氨基酸序列中的Met129功能等同的位置处的变为不同氨基酸的突变。在一些实施方案中,突变聚合酶包含与Pfu和9°NDNA聚合酶氨基酸序列中的Met129Ala功能等同的突变。
在一些实施方案中,突变包括突变为具有非极性侧链的残基。具有非极性侧链的氨基酸是本领域熟知的,并且包括例如:丙氨酸、半胱氨酸、甘氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、色氨酸、酪氨酸和缬氨酸。
在一些实施方案中,突变包括突变为具有极性侧链的残基。具有极性侧链的氨基酸是本领域熟知的,并且包括例如:精氨酸、天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、组氨酸、赖氨酸、丝氨酸和苏氨酸。
在一些实施方案中,突变包括突变为具有疏水性侧链的残基。具有疏水侧链的氨基酸是本领域熟知的,并且包括例如:甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸和色氨酸。
在一些实施方案中,突变包括突变为具有不带电侧链的残基。具有不带电侧链的氨基酸是本领域熟知的,并且包括例如:甘氨酸、丝氨酸、半胱氨酸、天冬酰胺、谷氨酰胺、酪氨酸和苏氨酸。
在一些实施方案中,突变聚合酶是DNA聚合酶的衍生物。在一些实施方案中,DNA聚合酶是古细菌DNA聚合酶。在一些实施方案中,DNA聚合酶是B家族DNA聚合酶。B家族古细菌DNA聚合酶是本领域已知的,如通过Arezi等人的美国专利申请公开号20030228616的公开内容所例证。在一些实施方案中,古细菌DNA聚合酶来自极端嗜热古细菌,这意味着所述聚合酶通常是热稳定的。因此,在进一步优选的实施方案中,突变聚合酶源自选自Vent、DeepVent、9°N和Pfu聚合酶的DNA聚合酶。Vent和Deep Vent分别是从极端嗜热古细菌嗜热高温球菌(Thermococcus litoralis)和火球菌属物种GB-D中分离的B家族DNA聚合酶使用的商业名称。9°N聚合酶也是从独特的嗜热球菌属物种(T.sp.9°N)分离的。如上所述,Pfu聚合酶是从激烈火球菌分离的。在一些实施方案中,本发明的突变聚合酶是火球菌属聚合酶或嗜热球菌属聚合酶的衍生物。
在一些实施方案中,B家族古细菌DNA聚合酶来自一个属,如例如嗜热球菌属、火球菌属或甲烷球菌属(Methanococcus)。嗜热球菌属的成员包括但不限于嗜热球菌4557(Thermococcus 4557)、Thermococcus barophilus、耐伽玛射线热球菌(Thermococcusgammatolerans)、Thermococcus onnurineus、西伯利亚嗜热球菌(Thermococcussibiricus)、Thermococcus kodakarensis、高加索嗜热球菌(Thermococcusgorgonarius)。火球菌属的成员包括但不限于火球菌NA2(Pyrococcus NA2)、深海火球菌(Pyrococcus abyssi)、激烈火球菌、堀越氏火球菌(Pyrococcus horikoshii)、亚氏火球菌(Pyrococcus yayanosii)、Pyrococcus endeavori、甘氏火球菌(Pyrococcusglycovorans)、沃氏火球菌(Pyrococcus woesei)。甲烷球菌属的成员包括但不限于杂色甲烷球菌(Methanococcus aeolicus)、海沼甲烷球菌(Methanococcus maripaludis)、万氏甲烷球菌(Methanococcus vannielii)、沃氏甲烷球菌(Methanococcus voltae)、热自养甲烷球菌(Methanococcus thermolithotrophicus)和詹氏甲烷球菌(Methanococcusjannaschii)。
例如,聚合酶可选自Vent、Deep Vent、9°N和Pfu聚合酶。在一些实施方案中,B家族古细菌DNA聚合酶是Pfu聚合酶。关于Pfu聚合酶的其他信息可以在美国专利号5,789,166;5,932,419;5,948,663;6,183,997;6,391,548;6,444,428;6,734,293;7,132,265;和7,176,004中找到。也可以使用来自火球菌属菌株的其他聚合酶,例如来自菌株GB-D的“DeepVent”(Q51334)和Pwo DNA聚合酶。
术语
应当理解,本文使用的术语仅仅是出于描述具体实施方案的目的,而不意图限制。除了定义的术语的技术和科学含义之外,所定义的术语是本传授的技术领域中通常理解和接受的术语。
术语“核酸”和“多核苷酸”在本文中可互换使用以描述由核苷酸(例如,脱氧核糖核苷酸或核糖核苷酸)构成的任何长度(例如,多于约10个碱基、多于约100个碱基、多于约500个碱基、多于1000个碱基、多达约10,000个或更多个碱基)的聚合物,或者可以与天然存在的核酸以序列特异性方式杂交的合成产生的化合物,所述序列特异性杂交方式类似于两个天然存在的核酸的杂交方式,例如,可以参与沃森-克里克碱基配对相互作用。天然存在的核苷酸包括鸟嘌呤、胞嘧啶、腺嘌呤、胸腺嘧啶和尿嘧啶(分别为G、C、A、T和U)。
如本文定义的术语“核苷”是一种包括在1′位置或等同位置连接到糖或糖替代物(如碳环或无环接头)的嘌呤、脱氮嘌呤或嘧啶碱基的化合物,并且包括2′-脱氧和2′-羟基、2′,3′-二脱氧形式,以及其他取代。
如本文所用的术语“多磷酸核苷”是指核苷与两个或更多个磷酸基团的磷酸酯。脱氧腺苷三磷酸是多磷酸核苷的一个例子。多磷酸核苷可以含有附接至末端磷酸酯或内部磷酸酯的化学基团。例如,多磷酸核苷可以包括具有附接至多磷酸酯链中的末端磷酸酯或内部磷酸酯的电化学标记、质量标签、电荷阻断标记、或显色标记、化学发光标记、荧光染料或荧光淬灭标记的分子。可用作标记的化学基团的其他例子包括生色团、酶、抗原、重金属、磁性探针、磷光基团、放射性材料、散射或荧光纳米颗粒、拉曼信号产生部分和电化学检测部分。另外,如本文所用的术语“多磷酸核苷”是指核苷的磷酸酯,其可包含硫原子、亚氨基或对磷酸酯链的其他修饰。
如本文所用,术语“核苷酸”是指核苷的磷酸酯,其中酯化位点通常对应于附接到戊糖或糖取代基的C-5位置的羟基。在一些情况下,核苷酸包括多磷酸核苷。然而,术语“添加的核苷酸(added nucleotide)”、“掺入的核苷酸”、“添加的核苷酸(nucleotide added)”和“掺入后的核苷酸”都是指作为寡核苷酸或多核苷酸链的一部分的核苷酸残基。
术语“核苷”、“核苷酸”、“脱氧核苷”和“脱氧核苷酸”旨在包括那些不仅含有已知嘌呤和嘧啶碱基,还含有已经被修饰的其他杂环碱基的部分。此类修饰包括甲基化嘌呤或嘧啶、酰化嘌呤或嘧啶、烷基化核糖或其他杂环。此外,“核苷”、“核苷酸”、“脱氧核苷”和“脱氧核苷酸”包括那些不仅含有常规核糖和脱氧核糖,还含有其他糖的那些部分。经修饰的核苷、核苷酸、脱氧核苷或脱氧核苷酸还包括糖部分上的修饰,例如,其中一个或多个羟基被卤素原子或脂族基团取代,或被官能化为醚、胺等。
天然存在的核苷酸或核苷在本文中被定义为腺苷(A)、胸苷(T)、鸟苷(G)、胞苷(C)和尿苷(U)。人们认识到这些核苷酸或核苷的某些修饰存在于自然界中。然而,在自然界中发生的影响氢键碱基配对的A、T、G和C的修饰被认为不是天然存在的。例如,2-氨基腺苷发现于自然界中,但不是“天然存在的”核苷酸或核苷,如该术语在本文中使用的那样。在自然界中存在的不影响碱基配对并且被认为是天然存在的修饰的核苷酸或核苷的其他非限制性例子是5-甲基胞嘧啶、3-甲基腺嘌呤、O(6)-甲基鸟嘌呤和8-氧代鸟嘌呤等。核苷酸包括任何核苷酸或核苷酸类似物,无论是天然存在的还是合成的。
术语“互补”、“互补体”或“互补核酸序列”意指通过Watson-Crick碱基配对规则与另一条核酸链中的碱基序列相关的核酸链。通常,当一个序列可以与另一个序列以反向平行的意义杂交时,这两个序列是互补的,其中每个序列的3′端与另一个序列的5′端杂交,然后一个序列中的每个A、T、G和C分别与另一个序列的T、A、C和G对齐。
术语“双链体”意指至少两个完全或部分互补的序列在其全部或大部分核苷酸之间进行Watson-Crick型碱基配对,从而形成稳定的复合物。术语“退火”和“杂交”可互换使用,意指稳定双链体的形成。
在核苷酸序列的背景下,术语“杂交(hybridization)”和“杂交(hybridizing)”在本文中可互换使用。两个核苷酸序列相互杂交的能力基于两个核苷酸序列的互补程度,而互补程度又基于匹配的互补核苷酸对的分数。给定序列中与另一序列互补的核苷酸越多,杂交条件可能越严格,并且两个序列的杂交特异性将越强。可以通过升高温度、增加共溶剂的比率、降低盐浓度等来实现严格性的增加。
术语“引发链”意指酶法制备的或合成的核酸,其能够作为核苷酸掺入点并从其3′端延伸。在一些实施方案中,引发链是与模板形成双链体的引物,并且其通过掺入与模板互补的核苷酸而从其3′端沿着模板延伸;在延伸过程中添加的核苷酸序列由模板多核苷酸的序列决定。在一些实施方案中,引发链是能够充当用于单碱基延伸反应或测定或用于非模板依赖性寡核苷酸合成的掺入点的核酸。引发链用作由DNA聚合酶、RNA聚合酶或逆转录酶催化的核苷酸掺入的起始点。引发链的长度可以是2-1000个碱基或更长,例如10-500个碱基。
术语“模板”表示可由核酸聚合酶用于根据Watson-Crick碱基配对规则指导与模板互补的核酸分子的合成的核酸分子。例如,DNA聚合酶利用DNA合成具有与模板DNA链互补的序列的另一DNA分子。RNA聚合酶利用DNA作为模板来指导具有与DNA模板链互补的序列的RNA的合成。DNA逆转录酶利用RNA来指导具有与RNA模板链互补的序列的DNA的合成。
短语“引物延伸条件”表示允许通过使用模板链作为模板向引物分子的末端添加核苷酸来进行聚合酶介导的引物延伸的条件。
短语“单碱基延伸”是指其中将单个可逆终止子核苷酸掺入引发链中的程序。本发明的方法和组合物可以用于单碱基延伸测定,所述测定可以用于确定沿着核酸的特定位置处的核苷酸碱基的身份。例如,单碱基延伸测定可用于鉴定单核苷酸多态性(SNP)或测量DNA甲基化水平。
如果引物“对应于”或“针对”某一核酸模板,则所述引物与该核酸模板碱基配对,即特异性杂交。如下面将更详细讨论的,针对特定核酸模板的引物和所述特定核酸模板或其互补体通常含有至少一个序列相同的连续核苷酸区域。
术语“终止子”和“终止子核苷酸”可互换使用,并且是指不能充当通过聚合酶进行核苷酸添加的底物,或以其他方式抵抗延伸的核苷酸。双脱氧核苷酸、3′叠氮基核苷酸和3′氨基核苷酸是终止子核苷酸的例子,但许多其他终止子核苷酸是已知的。其他非限制性例子包括3′-磷酸标记的核苷酸或虚拟终止子核苷酸。
术语“可逆终止子”是指如下终止子核苷酸,其不能用作核苷酸添加的底物或对延伸的抗性被配置为可逆转的。例如,可逆终止子可以具有封闭部分,所述封闭部分可以被去除,使得所述核苷酸变得可用作聚合酶的底物。在一些情况下,封闭部分在核苷酸的糖的3′-OH位置上,并且所述核苷酸被称为“3′-OH封闭的核苷酸”,并且去除所述封闭部分产生3′-OH。
可替代地,一些可逆终止子在糖的3′-OH位置上不具有封闭部分,并且此类终止子在本文中称为3′-OH未封闭的可逆终止子。闪电终止子是3′-OH未封闭的可逆终止子的例子,其中所述核苷酸的特征在于核糖部分上的游离3′-OH和附接至嘌呤(C7)或嘧啶(C5)碱基的光可切割的封闭基团。图1A、图1E和图1F提供了闪电终止子的例子的图示。在一些实施方案中,3′-OH未封闭的可逆终止子包含光可切割的封闭基团。这些可逆终止子可以被掺入链中,但在一段时间内被阻止延伸。在它们被解除封闭后,它们变得能够在引物延伸反应中延伸。包含光可切割的封闭基团的3′-OH未封闭的可逆终止子对于PCR扩增基本上是无活性的,直到它们通过暴露于紫外光或其他光切割技术而被解除封闭并被激活。在后期引物中可以包括很多种光可切割的封闭基团,例如那些在美国专利号8,969,535;9,200,319;和10,041,115中所描述的。在一些实施方案中,光可切割的封闭基团具有从约90%至约100%的封闭效率。
光可切割的封闭基团被设计为可逆地封闭和终止DNA合成,然后通过暴露于紫外光而被有效地切割,从而启动引物。在一些实施方案中,光可切割的封闭基团呈含有如下碱基的核苷酸化合物的形式:腺嘌呤、胞嘧啶、鸟嘌呤、胸腺嘧啶、尿嘧啶,或其修饰的嘧啶和嘌呤衍生物,如7-羟基-7-脱氮-腺嘌呤/鸟嘌呤。在其他实施方案中,可切割基团可以被衍生化以包括报告物,例如染料。在一些实施方案中,碱基腺嘌呤、胞嘧啶、鸟嘌呤、胸腺嘧啶、尿嘧啶或其修饰的嘧啶和嘌呤衍生物可共价附接至光可切割的保护基团,例如2-硝基苄基。在一些实施方案中,2-硝基苄基被衍生化以增强其对DNA合成的终止。光可切割的保护基团,例如2-硝基苄基,也可以被衍生化,在一些实施方案中,用荧光染料通过与所述光可切割的保护基团共价连接来衍生化。
在一些实施方案中,光可切割的封闭基团包含与2-硝基苄基共价附接的核苷的碱基,并且所述2-硝基苄基的α碳位置任选地被一个烷基或芳基取代。在其他实施方案中,2-硝基苄基被官能化以增强终止和封闭特性以及光催化的脱保护速率。在其他实施方案中,即使当核糖上的3′-OH基团被解除封闭时,附接至碱基的2-硝基苄基和α碳取代的2-硝基苄基的终止和封闭特性也会发生。在一些实施方案中,α碳取代的2-硝基苄基也可以被衍生化以包括选择的荧光染料或其他报告物。
术语“报告物”是指能够直接或间接产生可检测信号的化学部分。报告物的例子包括荧光染料基团、放射性标记或通过化学发光或生物发光手段产生信号的基团。荧光染料基团的例子包括呫吨、荧光素、罗丹明、BODIPY、花青、香豆素、芘、酞菁、藻胆蛋白、ALEXAFLUOR 350、ALEXA FLUOR 405、ALEXA FLUOR 430、ALEXA FLUOR 488、ALEXA FLUOR 514、ALEXA FLUOR 532、ALEXA FLUOR 546、ALEXA FLUOR 555、ALEXA FLUOR 568、ALEXA FLUOR568、ALEXA FLUOR 594、ALEXA FLUOR 610、ALEXA FLUOR 633、ALEXA FLUOR 647、ALEXAFLUOR 660、ALEXA FLUOR680、ALEXA FLUOR 700、ALEXA FLUOR 750和方酸菁染料。在本发明的一些实施方案中可以用作报告物的放射性标记的例子是本领域熟知的,例如35S、3H、32P或33P。
术语“引物延伸试剂”是指对多核苷酸分子(例如多核苷酸靶标)进行引物延伸反应(例如聚合酶链式反应(PCR))需要的或适合的任何试剂。引物延伸试剂通常包括引物、热稳定聚合酶或逆转录酶和核苷酸它们处于与适当缓冲液(如Tris-HCl或其他缓冲液)的混合物中。在一些实施方案中,引物延伸试剂还可以包括盐或离子、洗涤剂、有机溶剂、聚合物和/或其他添加剂。例如,引物延伸试剂可以包括离子(例如,Mg2+、Mn2+或K+)或其盐,洗涤剂如Triton X-100、Tween 20或NP40,血清或血清蛋白组分如牛血清白蛋白(BSA),多元醇如甘油、甘露糖醇或山梨糖醇,和/或还原剂(例如,二硫苏糖醇(DTT)或三(2-羧乙基)膦(TCEP))。cDNA合成由反向引物引发,所述反向引物与RNA转录物的3′聚A尾退火(寡(dT)),或者与RNA内的多个序列特异性位点退火(随机物、靶特异性引物)。
在比较两种或更多种聚合酶的背景下,术语“功能等同”意指一种聚合酶含有被认为出现在另一种聚合酶的氨基酸位置上的氨基酸,所述氨基酸在所述聚合酶中具有相同的功能作用。例如,在Vent DNA聚合酶中位置412处从酪氨酸到缬氨酸的突变(Y412V)将与9°N聚合酶中位置409处从酪氨酸到缬氨酸的取代(Y409V)是功能等同的。通常,两种或更多种不同聚合酶中的功能等同突变发生在所述聚合酶的氨基酸序列中的同源氨基酸位置处。因此,本文使用的术语“功能等同”还包括与给定突变“位置等同”或“同源”的突变,而不管突变氨基酸的特定功能是否是已知的。可以基于序列比对和/或分子建模鉴定两种或更多种不同聚合酶的氨基酸序列中的位置等同或同源的氨基酸残基。
在两个或更多个核酸或多肽序列的背景下,术语“相同”或“同一性百分比”是指这样的两个或更多个序列或子序列,当为了最大对应性进行对比和比对时,它们是相同的或者具有指定百分比的相同的氨基酸残基或核苷酸,如使用合适的序列对比算法或通过目视检查所测量的。
在两个核酸或多肽(例如,编码聚合酶的DNA或聚合酶的氨基酸序列)的背景下,短语“基本上相同的”是指这样的两个或更多个序列或子序列,当为了最大对应性进行对比和比对时,它们具有至少约60%、或至少约70%、或至少约80%、或至少约90%、或至少约95%、或至少约98%、或至少约99%或更高的核苷酸或氨基酸残基同一性,如使用序列对比算法或通过目视检查所测量的。此类“基本上相同”的序列通常被认为是“同源的”,不论实际祖先如何。优选地,“实质同一性”存在于长度为至少约50个残基的序列区域上,更优选地存在于至少约100个残基的区域上,且最优选地,序列在至少约150个残基上基本上相同,或在要比较的两个序列的全长上基本上相同。
当多肽(如聚合酶)和/或氨基酸序列天然地或人工地源自共同的祖先蛋白质或蛋白质序列时,所述多肽和/或氨基酸序列是“同源的”。类似地,当多核苷酸和/或核酸序列天然地或人工地源自共同的祖先核酸或核酸序列时,所述多核苷酸和/或核酸序列是同源的。同源性通常从两个或更多个核酸或蛋白质(或其序列)之间的序列相似性推断。可用于确立同源性的序列之间的相似性的精确百分比随所讨论的核酸和蛋白质而变化,但是常规使用在50、100、150或更多个残基上的低至25%的序列相似性来确立同源性。也可以使用更高水平的序列相似性(例如30%、40%、50%、60%、70%、80%、90%、95%或99%或更多)来确立同源性。用于确定序列相似性百分比的方法是容易获得的。适合于确定序列同一性和序列相似性百分比的算法的例子是BLAST算法,并且用于进行BLAST分析的基于软件的界面可通过国家生物技术信息中心公开获得。
如说明书和所附权利要求中所使用的,并且除了它们的普通含义之外,术语“基本”或“基本上”意指在本领域普通技术人员可接受的限度或程度之内。例如,“基本上取消”意味着本领域技术人员认为取消是可接受的。
如在说明书和所附权利要求中所使用的,并且除了其普通含义之外,术语“大致”和“约”意指在本领域普通技术人员可接受的限度或量之内。术语“约”通常是指所示数字的正负15%。例如,“约10”可以指示8.5到11.5的范围。例如,“大致相同”意味着本领域普通技术人员认为所比较的项目是相同的。
在本公开文本中,数值范围包括限定所述范围的数字。应当认识到,为了说明的目的,化学结构和化学式可以被延长或放大。
除非另外定义,本文所使用的全部技术术语和科学术语具有与本披露所涉及领域中的技术人员通常所理解的相同含义。
应理解的是,本文使用的术语仅用于描述特定实施方案的目的,并且不旨在是限制性的,因为本教导的范围将仅由所附权利要求限定。
如本文所公开的,提供了值的多个范围。应当理解的是,除非上下文明确另有指示,还明确公开该范围的上限与下限之间的每个中间值,精确到下限单位的小数点后一位。
除非另外定义,否则本文所用的全部技术和科学术语具有与本公开文本所属领域的普通技术人员通常所理解的相同的含义。尽管类似于或等同于在此所描述的方法和材料的任何方法和材料也可以用于对本传授内容的实践或测试,但是现在将对一些示例方法和材料进行描述。
在此引用的所有专利和出版物均明确地通过引用并入。
如说明书和所附权利要求中所使用的,术语“一个/一种(a)”、“一个/一种(an)”和“所述”同时包括单数和复数指代物,除非上下文另有明确规定。因此,例如,“一个部分”包括一个部分和多个部分。
使用方法和应用
本文呈现的突变聚合酶可用于多核苷酸测序,例如边合成边测序(SBS)技术。简言之,可以通过使用靶核酸作为合成互补链的模板来进行SBS。引物与模板结合,并通过DNA聚合酶的活性用一个或多个标记的核苷酸延伸。使用靶核酸作为模板延伸主链(即,与模板杂交的引物),并掺入可检测的标记的核苷酸。任选地,标记的核苷酸可以是可逆终止子,其在核苷酸已添加至引物后,终止进一步的引物延伸。例如,可将可逆终止子添加至引物中,使得随后的延伸不能发生,直到递送解封闭剂以逆转终止。因此,对于使用可逆终止子的实施方案,可以将解封闭试剂递送至测序仪器(在标记的检测发生之前或之后)。
一些可逆终止子(例如,闪电终止子)具有光可切割的封闭基团,所述封闭基团被设计为终止DNA合成以及快速切割。闪电终止子与主链组合并在主链的3′端掺入,例如通过与模板退火的链的单碱基延伸,或者可替代地通过主链以非模板依赖性方式用末端脱氧核苷酸转移酶(TdT)进行的单碱基延伸来实现。
在一些实施方案中,本发明的突变聚合酶用于非模板依赖性酶促寡核苷酸合成(TiEOS)。在一些实施方案中,本发明的方法包括使引发链与突变聚合酶(例如Pfu26或Pfu48)和一个或多个3′-OH未封闭的可逆终止子接触。本发明的方法可以包括通过每个循环中添加单个碱基来延长引发链的3′端。在一些实施方案中,本发明的突变聚合酶以高掺入和终止效率以非模板依赖性方式将可逆终止子掺入引发链。
编码突变聚合酶的核酸
本文进一步呈现了编码本文呈现的突变聚合酶的核酸。对于本公开文本所公开或教导的任何给定的突变聚合酶,可以根据分子生物学的已知原理获得编码该突变聚合酶的核苷酸序列。因此,本公开文本提供了编码突变聚合酶的核酸以及突变聚合酶本身的描述。
编码本文公开的重组聚合酶的核酸也是本文呈现的实施方案的特征。特定氨基酸可以由多个密码子编码,并且某些翻译系统(例如,原核或真核细胞)通常展现出密码子偏倚,例如,不同的生物体通常偏好编码相同氨基酸的几个同义密码子中的一个。因此,本文呈现的核酸任选地被“密码子优化”,这意味着所述核酸被合成为包括用于表达聚合酶的特定翻译系统偏好的密码子。例如,当期望在细菌细胞(或甚至细菌的特定菌株)中表达聚合酶时,可以合成核酸以包括在该细菌细胞的基因组中最常发现的密码子,用于有效表达聚合酶。当期望在真核细胞中表达聚合酶时,可以采用类似的策略,例如,核酸可以包括该真核细胞偏好的密码子。
多种蛋白质分离和检测方法是已知的,并且可用于分离聚合酶,例如从表达本文呈现的重组聚合酶的细胞的重组培养物中分离聚合酶。
鉴于编码9°N聚合酶的野生型核苷酸序列是已知的,可以推导出使用标准遗传密码编码具有一个或多个氨基酸取代的9°N的任何给定突变体形式的核苷酸序列。类似地,可以容易地导出其他聚合酶(例如Vent、Pfu、T.sp.JDF-3、Taq等)的突变体形式的核苷酸序列。然后可以使用本领域已知的标准分子生物学技术构建具有所需核苷酸序列的核酸分子。
根据本文呈现的实施方案,所定义的核酸不仅包括相同的核酸,而且包括任何微小的碱基变异,特别地包括由于保守氨基酸取代中的简并密码子而导致同义密码子(指定相同氨基酸残基的不同密码子)的情况下的取代。术语“核酸序列”还包括关于碱基变异给出的任何单链序列的互补序列。
本文所述的核酸分子还可以有利地包含在合适的表达载体中以在合适的宿主中表达由其编码的聚合酶蛋白。将克隆的DNA掺入合适的表达载体中用于随后转化所述细胞和随后选择转化细胞是本领域技术人员熟知的,如Sambrook等人(1989),Molecularcloning:A Laboratory Manual,Cold Spring Harbor Laboratory中所提供,所述文献通过引用以其整体并入。
这种表达载体包括具有根据本文呈现的实施方案的核酸的载体,所述核酸可操作地连接至能够实现所述DNA片段的表达的调节序列,例如启动子区。术语“可操作地连接”是指并置,其中所述组分处于允许它们以其预期方式起作用的关系中。可以将此类载体转化到合适的宿主细胞中以提供根据本文呈现的实施方案的蛋白质的表达。
核酸分子可以编码成熟蛋白或具有前序列的蛋白,包括编码前蛋白上的前导序列的核酸分子,然后所述前导序列被宿主细胞切割以形成成熟蛋白。载体可以是例如质粒、病毒或噬菌体载体,其具有复制起点以及任选地用于表达所述核苷酸的启动子和任选地所述启动子的调节子。载体可以含有一种或多种选择标记,如例如抗生素抗性基因。
表达所需的调节元件包括结合RNA聚合酶和指导适当水平的转录起始的启动子序列以及用于核糖体结合的翻译起始序列。例如,细菌表达载体可以包括启动子例如lac启动子和用于翻译起始的Shine-Dalgarno序列和起始密码子AUG。类似地,真核表达载体可包括RNA聚合酶II的异源或同源启动子、下游多腺苷酸化信号、起始密码子AUG和用于核糖体脱离的终止密码子。这样的载体可商购获得或通过本领域熟知的方法从所述序列组装。
可以通过在载体中包括增强子序列来优化高等真核生物对编码聚合酶的DNA的转录。增强子是作用于启动子以提高转录水平的DNA的顺式作用元件。除了选择标记之外,载体通常还将包括复制起点。
鉴于本公开文本,可以按照本教导来实现各种方法。此外,各种组分、材料、结构和参数仅通过说明和举例的方式被包括在内,并且不具有任何限制意义。鉴于本公开文本,本教导可以在其他应用中实现,并且可以确定实现这些应用的组分、材料、结构和设备,同时保持在所附权利要求书的范围内。
实施例
实施例的通用方法和条件
实施例中使用的实验程序和条件大体上描述如下。
A.突变聚合酶的产生和生产
此部分大体上描述了如何通过产生突变基因序列来产生本发明的突变聚合酶,并且表达突变基因以获得以下实施例中使用的各种突变聚合酶。使用来自Agilent的QuikChange Lightning多位点定点诱变试剂盒的试剂和方案制备突变DNA聚合酶。对突变质粒进行序列确认,然后将其转化到表达宿主BL21-Gold(DE3)(Agilent)中。使细胞生长至指数期(OD600约0.4)并用1mM IPTG诱导。热处理的细菌裂解物和纯化的Pfu突变体如Hansen等人(2011)NAR 39:1801-1810中所述来制备。
B.使用突变聚合酶的测序和测序度量
此部分大体上描述了如何将本发明的突变聚合酶用于边合成边测序,以及如何测量它们在测序中的性能。在实验电路板仪器上进行测序,基本上如Hertzog D等人,“Ahigh-performance,low-cost approach to next-generation sequencing”,BioOpticsWorld.2011期2011年11月/12月中所述。
实施例1
在此实施例中,将突变引入SEQ ID NO:12的DNA聚合酶(Therminator)中,以试图改善其在用3′-OH未封闭的终止子测序中的能力。对嗜热球菌属物种9°N DNA聚合酶的此类突变披露于以下文献中:美国专利号9,273,352;美国专利号9,677,059;美国专利号9,765,309;美国专利申请公开号20160032377。下表总结了使用闪电终止子的测序度量。
#在相同的单位浓度下比较纯化的突变体(循环1中为0.08U/ul且循环2-n中为0.008U/ul)。产生“无”对照并与Therminator突变体一起筛选。
如在此实施例和以下那些实施例中所使用的,“超前+滞后”是指在边合成边测序技术中由于相对于当前碱基信号读取下一个碱基信号(超前)或读取前一个碱基信号(滞后)而引起的移相误差。此类误差可能是由于聚合酶对可逆终止子的不完善掺入而引起的。发现聚合酶的这些突变在改善Therminator聚合酶的测序性能方面是无效的,因为对于掺入闪电终止子,相对于Therminator,平均读长(ARL)没有增加。这些结果表明了鉴定碱基修饰的可逆终止子的替代聚合酶和/或突变的困难和不可预测性。
实施例2
在此实施例中,通过引入存在于Therminator中的某些突变(D141A/E143A/A485L)产生突变聚合酶。在JDF3(嗜热球菌属物种JDF3)和Pfu(激烈火球菌)DNA聚合酶的等同位置处引入所述三个突变。然后评价新的突变聚合酶在用闪电终止子测序中的性能,如上所述。表2总结了显示自然变异对测序度量的影响的结果。所得ARL与Therminator相比低超过20bp,表明不同聚合酶之间的变异可对使用3′-OH未封闭的可逆终止子的测序具有显著影响。
此外,构建了一系列嵌合聚合酶和单点突变体,以鉴定9°N、JDF3和Pfu DNA聚合酶之间影响使用3′-OH未封闭的可逆终止子的测序的氨基酸差异。下表指示对那些聚合酶进行的突变和来自它们在用闪电终止子测序中的使用的测序度量。在测序中评价突变聚合酶的结果也示于图2中。
#在相同的蛋白质浓度下比较替代聚合酶(循环1中为0.02ug/uL且循环2-n中为0.002ug/uL)
发现那三种聚合酶在494和546(Pfu编号)处的自然变异对测序度量具有显著影响。当在JDF3中在与Pfu中的494等同的位置处引入苯丙氨酸(F;天然存在于9°N和Pfu中)时,ARL改善了17bp(JDF3 A485L/Y493F;实验#2-3)。当在Pfu中的密码子546处引入组氨酸(H;天然存在于9°N和JDF3中)时,注意到甚至更大的改善(实验#2-5)。事实上,使用PfuA486L/Y546H(实验#2-5)的测序度量与Therminator几乎相同,但是与Therminator相比,Pfu A486L/Y546H展现出更低的超前值和更高的滞后值(示于下表中)。
3′-OH未封闭实施例3
在此实施例中,突变DNA聚合酶通过产生针对古细菌聚合酶鉴定的各种突变而源自Pfu聚合酶。突变选自研究和专利文献(示于下文中)中描述的那些突变。将突变引入Pfu聚合酶中,所述Pfu聚合酶还具有突变A486L和Y546H。突变连同针对各种聚合酶讨论此类突变的文献一起示于下表中。
突变聚合酶用于如上所述的测序。在添加到Pfu聚合酶的这些突变中,只有K477W对序列度量有显著影响,最显著的是在更高百分比的全长读段中(实验#2-6)。该突变体(Pfu D141A/E143A/K477W/A486L/Y546H)被赋予任意名称Pfu10。Pfu10的氨基酸序列示于SEQ ID NO:2中。此实施例显示,Pfu10是一种改进的测序酶,在使用3′-OH未封闭的可逆终止子的边合成边测序中使用。
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如在此实施例和其他实施例中使用的,“FracQ30”是指Q得分为30(Q30)的测序碱基读段在总测序读段中的百分比。K477W改善Pfu的发现是出乎意料的,因为Therminator中的等同突变未能改善使用闪电终止子的测序(参见实验#1-8;K476W),即使Therminator分别在与pfu10中的位置486、494和546等同的位置处含有L、F和H。这些结果表明,使用3′-OH未封闭的可逆终止子,可以通过与Pfu比与Therminator更密切相关的测序聚合酶实现K477W的益处。BLASTP比对表明,Pfu10与9°N DNA聚合酶(Therminator的亲本)之间的氨基酸序列同一性百分比为79.9%。
实施例4
在此实施例中,通过多位点诱变将多个突变引入Pfu10中,或通过结构域取代将多个突变引入Pfu A486L/Y546H中,以探索降低的同一性的可接受程度,并鉴定含有L486、F494、H546和W477的其他突变Pfu聚合酶。SEQ ID NO:3显示Pfu10变体(称为“PFU10-N12”)的氨基酸序列,所述Pfu10变体具有在古细菌DNA聚合酶中出现的15个保守突变,加上消除潜在翻译起始位点的M129A取代(与Pfu10的同一性为97.9%)。如上所述,评价Pfu10和Pfu10-N12在用闪电终止子测序中的性能。
下表总结了显示Pfu10变体的测序性能的结果。该变体(Pfu10-N12)在用闪电终止子测序方面的表现与Pfu10相当,不然略好于Pfu10。
此外,可以使用任意结构域取代来增加多样性,同时保持所需的活性水平。结构域取代对测序度量的影响如图2所示。例如,用Therminator中的相应多肽序列替代PfuL486L/Y546H中的氨基酸区段1-99、100-199、400-449和500-599对测序度量的影响极小,证明Pfu10可以容纳至少16-29个另外的突变(96.3-97.9%变异),而不会损害其掺入3′-OH未封闭的可逆终止子的能力。
实施例5
此实施例在Pfu D141A/E143A中的位置486和494处使用饱和诱变以产生另外的突变聚合酶。在采用闪电终止子-A(LTA)的基于板的单碱基延伸测定中筛选细菌提取物。对产生最高荧光信号的阳性克隆进行测序,以鉴定氨基酸替代。如图3和图4中所示,A486和F494是高度可突变的,并且多个取代改善闪电终止子的掺入,包括A486F、A486Y、A486N、A486R、A486H、F494C、F494I、F494N和F494T。基于筛选结果,提交选择的裂解物以供测序,并且基于选择进行氨基酸鉴定。
图3示出了Pfu聚合酶中位置486的饱和。使用QuikChange试剂盒与简并NDT密码子引物产生Pfu A486突变体的文库。NDT密码子包括在第一位置处的A、C、G或T,在第二位置处的A、G或T,以及在第三位置处的T,从而将变异性引入引物中。12种可能的NDT密码子代表12种氨基酸(Phe、Leu、Ile、Val、Tyr、His、Asn、Asp、Cys、Arg、Ser和Gly)。从32个随机选择的菌落制备热澄清提取物。在采用固定化dsDNA底物的微量滴定板测定中针对LTA的掺入筛选提取物。“A”突变体的荧光信号对应于野生型Pfu(密码子486处的野生型丙氨酸)的背景。
图4A和图4B示出了Pfu聚合酶中位置494的饱和。使用QuikChange试剂盒与2个简并密码子引物库(A、B)产生Pfu F494突变体的文库。从32个随机选择的菌落制备热澄清提取物。在采用固定化DNA底物的微量滴定板测定中针对LTA的掺入筛选提取物。“F”突变体的荧光信号对应于野生型Pfu(位置494处的野生型苯丙氨酸)的背景。
实施例6
在此实施例中,另外的突变聚合酶源自Pfu聚合酶。为了产生突变体,使用QuikChange Lightning诱变试剂盒和含有“NDT”密码子的寡核苷酸使Pfu pol B基因中的87个密码子饱和,以产生具有相等频率的R、N、D、C、G、H、I、L、F、S、Y和V取代。在一些情况下,用编码其余突变(A、T、K、Q、E、P、K、W)的诱变寡核苷酸的等摩尔混合物在单独的QuikChange反应中制备NDT文库中缺失的突变体。从每个QuikChange文库中随机选择32个克隆(T267、Y403、Y410、I475、G499和K675文库例外,它们产生更少的转化体)。制备细菌裂解物并使用具有固定化引物模板的基于板的测定针对LTA和LTC的掺入进行筛选。还针对LTG和LTU的利用筛选有限数量的突变体(类似的趋势;数据未显示)。在洗涤和UV切割(以最小化染料淬灭效应)后,读取板并将荧光信号与Pfu对照进行比较。下表鉴定了用作对照的四种突变Pfu聚合酶,它们被赋予任意名称Pfu5、Pfu1和Pfu2以及Pfu10(其在上文讨论)。
D141A/E143A | A486L | Y546H | K477W | |
Pfu5 | + | - | - | - |
Pfu1 | + | + | - | - |
Pfu2 | + | + | + | - |
Pfu10 | + | + | + | + |
基于筛选结果,提交选择的裂解物以供测序,并且基于选择进行氨基酸鉴定。对照提取物显示与天然核苷酸相似的活性(U/ul)(图5),但只有含有A486L的突变聚合酶(Pfu1、Pfu2、Pfu10)可以利用闪电终止子(图6)。Y546H本身不允许闪电终止子掺入(图7A-图7B)。然而,发现A486L和Y546H的组合(在Pfu2和Pfu10中)使LTC的掺入最大化(图6),这表明LTC和Y546之间的不利相互作用,这可以通过用酪氨酸取代组氨酸来克服。如实施例3所示,K477W因增加全长读段的分数而有益于测序。然而,所述机制显现与掺入效率无关,因为K477W(在Pfu10中)对LTA和LTU掺入具有负面影响,或者可替代地,由于附接的荧光团的色氨酸(W)淬灭而对最终荧光具有负面影响。
使以下密码子经历饱和:266、267、268、269、270、329、330、332、333、336、399、400、403、404、407、408、409、410、411、450、451、452、453、455、456、457、458、459、460、461、462、463、464、465、466、475、476、477、478、479、480、481、482、483、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、515、522、545、546、577、579、580、581、582、584、591、595、603、606、607、608、612、613、614、664、665、666、668、669、674、675、676。筛选的87个位置中的16个可以突变以实现掺入LTA和/或LTC的显著(>4X野生型Pfu)改善。下表显示了赋予改善的LTA和LTC掺入的突变。四个位置显现是高度可取代的(L409、A486、F494、Y497),并且某些氨基酸替代(L409H、L409F、A486Y、A486R、A486H、A486N、F494C、F494N、F494I、F494T)与Pfu A486L对照(Pfu10)相比产生更好的掺入信号。
*未测序
观察到显示N492处的突变(N492I/V/P)仅有益于LTC掺入。此外,L409(L409D/C/V/H/F)、A486(A486Y/R/F/I/H/N)和Y497(Y497H/I)处的某些取代显现对LTC摄取的影响大于对LTA摄取的影响。这些结果表明,在Y546和N492处(其与核苷酸的三磷酸部分形成H键)的取代改变了LTC在结合袋中的取向,以更好地适应其独特的碱基、接头和/或染料部分。图8显示了使用基于网络的iCn3D结构观察器可视化的9°NDNA聚合酶(5OMV)结构中有益突变的位置。仅有益于LTC的突变位于N492和Y546处。相对于LTA,提供相对更高的LTC掺入的突变位于L409、A486和Y497处。
实施例7
本实施例评价了本发明的突变Pfu聚合酶的实施方案在非模板依赖性酶促寡核苷酸合成中的性能。在此实施例中,在非模板依赖性DNA合成测定中比较聚合酶TdT和Pfu26(Pfu10+L409Y)(SEQ ID NO:4)和Pfu48(Pfu26,但具有A486R代替A486L)(SEQ ID NO:5)。用dATP(图1D)、LTA-1(图1E)或LTA-2(图1F)进行测定。LTA-1和LTA-2不包括与所述分子连接的任何染料或其他报告物。
非模板依赖性DNA合成反应使用7.5U TdT(Promega)或180nM(引物的3X)Pfu26(D141A/E143A/A486L/Y546H/K477W/L409Y)或Pfu48(D141A/E143A/A486R/Y546H/K477W/L409Y)。在含有1X TdT缓冲液与CoCl2(Promega)或1X ThermoPol缓冲液(NEB)的一式两份反应中,将不同浓度的dATP加入到2μM(TdT)或60nM(Pfu)T7 FOR引物(6′FamTAATACGACTCACTATAGGG)(SEQ ID NO:6)中。反应在37℃(TdT)或60℃(Pfu)孵育30min,然后用热(TdT;70℃下10min)或EDTA(1ul 500uM EDTA)灭活。使用Stratalinker(15瓦灯泡,365nM,10min)将反应产物暴露于UV。将反应物在水中稀释(TdT,1:333;Pfu,1:10),并用HiDI/LIZ 120尺寸标准品使1μl(TdT)或0.5μl(Pfu)等分试样达到10μl。将产物在95℃加热5min,在冰上冷却2min,然后在ABI 3500毛细管电泳系统(50cM毛细管,过滤器组#5,测定=GE5 LIZ120)上进行分析。
通过非模板依赖性dATP添加的数量来测量末端转移酶活性。TdT的结果示于图9A中,Pfu26的结果示于图9B中,且Pfu48的结果示于图9C中。发现末端转移酶活性分别随着dATP或引物浓度的变化增加或减少。这些结果表明,L409Y突变赋予末端转移酶活性,因为缺乏L409Y的Pfu突变体(例如,Pfu10)不能进行非模板化的dA添加(参见下图10C)。
实施例8
在此实施例中,使用实施例7中所述的条件,用作为α-硫代三磷酸酯的Rp+Sp外消旋混合物的LTA-2(图1F)进行引物延伸。图10A显示了在四种不同浓度的LTA-2(5μM、10μM、50μM和100μM)下的结果。比较图9A至图10A显示,即使在最高100μM浓度下,TdT也没有像dATP那样有效地掺入LTA-2终止子。
在进一步的实验中,用两种单链寡核苷酸的等摩尔混合物(每种3uM)进行单碱基延伸反应(10ul):
CE2:6Fam-TAATACGACTCACTATAGGGCAGGAAACAGCTATGACCAGGGGATC AGC(SEQ IDNO:7)和
T7:6Fam-TAATACGACTCACTATAGGG(SEQ ID NO:8)
两者都用Fam标记,但可使用毛细管电泳通过其长度加以区分。以18nM聚合酶浓度使用以下三种Pfu突变体进行引物延伸反应:
Pfu10:具有D141A/E143A/A486L/Y546H/K477W突变的Pfu聚合酶(SEQ ID NO:2)
Pfu26:具有D141A/E143A/A486L/Y546H/K477W/L409Y突变的Pfu聚合酶(SEQ IDNO:4)
Pfu48:具有D141A/E143A/A486R/Y546H/K477W/L409Y突变的Pfu聚合酶(SEQ IDNO:5)
延伸反应还以5uM的终浓度采用Rp+Sp外消旋混合物中的单独αS修饰的LT(即LTA-2、LTU-2、LTG-2和LTC-2)。LTU-2、LTG-2和LTC-2都是C7或C5-羟甲基-α-叔丁基-2-硝基苄基修饰的可逆终止子,像LTA-2一样,但是用尿嘧啶、鸟嘌呤和胞嘧啶代替腺嘌呤。在这些实验中,可逆终止子不包括染料。如实施例7中所述,将反应孵育、终止、UV处理并加以分析。
图10B显示了不包括任何聚合酶的对照延伸反应设置的结果。图10C显示了使用Pfu10聚合酶时的延伸反应的结果,并且图10D和图10E分别显示了使用Pfu26聚合酶和Pfu48聚合酶的结果。比较图10A至图10C至图10E显示,在掺入3′-OH未封闭的可逆终止子LTA-2、LTU-2、LTG-2和LTC-2方面,含有L409Y的Pfu突变体(Pfu26、Pfu48)优于小牛胸腺末端转移酶(TdT)。此外,初步数据显示引物延伸效率受引物序列(CE2>P7)和碱基(LTA、LTU>LTG>LTC)的影响。
Pfu26也可用于直接末端标记某些寡核苷酸(例如,用于原位杂交应用,如美国专利申请公开号20200216841中所披露),这避免了使用(以及随后去除)具有5′突出端的互补模板。
实施例9
本实施例测试了向单链引发链的3′端添加3′-OH未封闭的可逆终止子(LTA-1,示于图1E中,或LTA-2,示于图1F中)。用2uM或80nM的引发链和不同浓度(100/50/10/5/1μM)的可逆终止子进行延伸反应。将Pfu26聚合酶的性能与TdT聚合酶进行比较。
在含有7.5U小牛胸腺TdT、1X Promega TdT缓冲液(含CoCl2)、不同浓度的LTA-1(100uM、50uM、10uM、5uM或1uM)和2uM(图11A至图11C)或80nM(图11D至图11F)的引发链(Fam标记的T7 F引物(6Fam TAATACGACTCACTATAGGG),SEQ ID NO:8)的一式两份25ul反应中进行引物延伸。在不添加聚合酶的情况下进行对照。还在含有Pfu26突变聚合酶、60nM的引发链和不同浓度(10μM或5μM或1μM)的LTA-1或LTA-2的反应中进行引物延伸。
反应物在37℃下孵育30min,然后在70℃下热灭活10min。将反应产物分为两半,并且如实施例7中所述用UV处理一半。然后将反应物在水中1:333(2uM引物)或1:13(80nM引物)稀释,并使用实施例7中提供的条件通过毛细管电泳分析0.5ul部分。
使用TdT聚合酶的反应的结果示于图11A至图11F中。使用Pfu26聚合酶的反应的结果示于图12A和图12B中。
使用LTA-1证明将“无疤的”LT用于TiEOS的潜力,LTA-1是一种用于NGS中的原型可逆终止子,其缺乏2-硝基苄基部分上的α-硫代三磷酸酯和α-叔丁基修饰,以分别消除“chewback活性”并改善切割/终止效率。如图11A至图AC所示,TdT比更高度取代的LTA-2(图10A)显著更有效地掺入LTA-1。然而,终止效率较差,并且在单个合成循环中掺入3个碱基(在2uM引物下)或更多个碱基(在80nM引物下)。
与TdT相比,基于在1、5和10uM浓度下添加的碱基数量,Pfu26显现不太有效地掺入LTA-1(图11D至图11F2相比于图12A)。然而,与未能掺入LTA-2的TdT不同,Pfu26在单个孵育步骤中提供了有效的掺入和终止(参见图12B)。该结果证明了使用专门的Pfu突变体与“无疤的”和修饰的可逆终止子组合以分别实现天然和非同源核酸聚合物的酶介导的合成的可行性。
实施例10
此实施例测试了使用Pfu26突变聚合酶向单链引发链的3′端多循环添加3′-OH未阻断的可逆终止子(LTA-2,示于图1F中)。如实施例7中所述,使用Pfu26和修饰的LTA-2进行单碱基延伸测定,所述修饰的LTA-2具有附接至7-脱氮-C7羟甲基的α-叔丁基取代的2-硝基苄基终止基团。修饰的LTA-2任选地含有αS三磷酸酯修饰,并经HPLC纯化以分离Sp异构体(图13A和图13C)。在每个掺入循环(3个循环-图13A和图13B;6个循环-图13C),取出1ul并保存在冰上用于ABI 3500分析。在每个循环,使用MyOne T1生物素化的珠如下纯化剩余的反应体积(对于3个循环的实验)。将127.5ul珠在1ml洗涤溶液中洗涤2X,并且用洗涤溶液重悬在初始体积中。以180nM的终浓度添加具有间隔子的生物素化的反义T7(下划线)引物(5’-CCC TAT AGT GAG TCG TAT ACG GAG CAT A-生物素)。将珠和捕获寡核苷酸在室温下以300rpm混合30min,在500ul洗涤溶液中洗涤2X,并重悬于150ul的相同溶液中。将10ul珠/捕获寡核苷酸混合物添加到9ul的每个掺入反应(循环2-6)中,并在室温下以300rpm孵育30min。然后将混合物在室温下用500ul洗涤溶液洗涤2x,并重悬于10ul Thermopol缓冲液中。为了释放Fam-T7寡核苷酸,将反应在95℃孵育5分钟,并快速施加到磁体上,然后取出8ul的每种洗脱液用于下一轮掺入。纯化(循环2-6)后,加入2ul新鲜的酶/LTA混合物,如上所述进行引物延伸和分析。
结果(示于图13A至图13C中)表明,在2-3轮TiEOS后,与7-脱氮-7-羟甲基-α-叔丁基-2-硝基苄基修饰的LTA组合的Pfu26没有以100%效率产生单独产物。去除αS修饰(图13B)或掺入7-脱氮-7-羟甲基-α-叔丁基-2-硝基苄基修饰的LTA的手性纯αS(Sp异构体)(图13A、图13C)未能克服次级产物的问题。然而,结果证明了设计“无疤的”、非异构可逆终止子的概念验证,所述可逆终止子可以与本发明的突变聚合酶的实施方案组合用于实现长的天然和修饰的核酸聚合物的酶促合成。
示例性实施方案
尽管描述了各种实施方案,应当理解,本公开文本的教导不限于所描述的特定实施方案,并且因此当然可以变化。
实施方案1.一种包含3′-OH未封闭的可逆终止子和突变聚合酶的组合物,其中所述突变聚合酶包含与SEQ ID NO:2至少96%相同的氨基酸序列,并且包含在与Pfu聚合酶中的K477、A486和Y546的氨基酸位置功能等同的位置处的氨基酸突变。
实施方案2.根据实施方案1所述的组合物,其中所述突变聚合酶包含在与Pfu聚合酶中的位置486功能等同的位置处的A486X突变,其中X是除丙氨酸之外的任何氨基酸。
实施方案3.根据实施方案2所述的组合物,其中所述A486X突变是A486F、A486Y、A486N、A486R或A486H。
实施方案4.根据实施方案1至3中任一项所述的组合物,其中所述突变聚合酶还包含在与Pfu聚合酶中的位置546功能等同的位置处的Y546H突变。
实施方案5.根据实施方案1至4中任一项所述的组合物,其中所述突变聚合酶还包含在与Pfu聚合酶中的位置477功能等同的位置处的K477W突变。
实施方案6.根据实施方案1至5中任一项所述的组合物,其中所述突变聚合酶还包含在与Pfu聚合酶中的位置F494功能等同的位置处的突变。
实施方案7.根据实施方案6所述的组合物,其中所述F494突变是F494C、F494I、F494N或F494T。
实施方案8.根据前述实施方案中任一项所述的组合物,其中所述突变聚合酶是火球菌属聚合酶的衍生物。
实施方案9.根据实施方案8所述的组合物,其中所述突变聚合酶包含SEQ ID NO:2的氨基酸序列。
实施方案10.根据实施方案1至7中任一项所述的组合物,其中所述突变聚合酶是嗜热球菌属聚合酶的衍生物。
实施方案11.一种将核苷酸掺入包含核酸的引发链的方法,所述方法包括:
在足以进行掺入反应的条件下,使所述引发链与核苷酸和突变聚合酶接触,
其中所述突变聚合酶包含与SEQ ID NO:2至少96%相同的氨基酸序列,并且包含在与Pfu聚合酶中的K477、A486和Y546的氨基酸位置功能等同的位置处的氨基酸突变。
实施方案12.根据实施方案11所述的方法,其中所述核苷酸是3′-OH未封闭的可逆终止子。
实施方案13.一种多核苷酸测序的方法,所述方法包括:
(a)形成包含模板和引发链的双链体,其中所述模板包含待测序的靶核酸和与所述引发链的至少一部分互补的引物结合位点;
(b)将所述引发链与可逆终止子核苷酸和突变聚合酶组合,其中所述突变聚合酶包含与SEQ ID NO:2至少96%相同的氨基酸序列,并且包含在与Pfu聚合酶中的K477、A486和Y546的氨基酸位置功能等同的位置处的氨基酸突变;
(c)在模板依赖性反应中,在所述引发链的3′端掺入所述可逆终止子;和
(d)鉴定掺入的可逆终止子核苷酸,从而确定所述模板的序列。
实施方案14.根据实施方案13所述的方法,其中所述3′-OH方法还包括重复步骤(c)和(d)至少80次。
实施方案15.一种包含引发链、3′-OH未封闭的可逆终止子3′-OH和突变聚合酶的组合物,其中:
所述突变聚合酶包含与SEQ ID NO:1至少80%相同的氨基酸序列,
所述突变聚合酶还包含在与Pfu聚合酶中的位置L270、E330、Q332、L333、L409、P451、L453、L457、E476、L489、L490、N492、F494、Y497和E581功能等同的位置处的一个或多个突变;并且
所述突变聚合酶对所述3′-OH未封闭的可逆终止子的掺入活性比SEQ ID NO:11的DNA聚合酶的掺入活性高至少4倍。
实施方案16.根据实施方案15所述的组合物,其中所述氨基酸序列与SEQ ID NO:1至少85%相同。
实施方案17.根据实施方案15所述的组合物,其中所述氨基酸序列与SEQ ID NO:1至少90%相同。
实施方案18.根据实施方案15所述的组合物,其中所述氨基酸序列与SEQ ID NO:1至少95%相同。
实施方案19.根据实施方案15至18中任一项所述的组合物,其中所述突变聚合酶不包含在与Pfu聚合酶中的位置266、267、268、269、329、336、399、400、403、404、407、408、410、411、450、452、455、456、458、459、460、461、462、463、464、465、466、475、477、478、479、480、481、482、483、485、487、488、491、493、495、496、498、499、500、515、522、545、546、577、579、580、582、584、591、595、603、606、607、608、612、613、614、664、665、666、668、669、674、675和676功能等同的任一位置处的突变。
实施方案20.根据实施方案15至19中任一项所述的组合物,其中所述突变聚合酶是火球菌属聚合酶的衍生物。
实施方案21.根据实施方案15至19中任一项所述的组合物,其中所述突变聚合酶是嗜热球菌属聚合酶的衍生物。
实施方案22.根据实施方案15至21中任一项所述的组合物,其中所述组合物还包含模板,所述模板包含与所述引发链的至少一部分互补的引物结合位点。
实施方案23.根据实施方案15至22中任一项所述的组合物,其中所述组合物还包含546H和486X突变。
实施方案24.根据实施方案15至23中任一项所述的组合物,其中所述组合物不含与所述引发链互补的模板。
实施方案25.一种将3′-OH未修饰的可逆终止子掺入引发链中的方法,所述方法包括:
在足以进行掺入反应的条件下,使引发链与3′-OH未修饰的可逆终止子和突变聚合酶接触,
其中所述突变聚合酶包含与SEQ ID NO:1至少80%相同的氨基酸序列和在与Pfu聚合酶中的位置L270、E330、Q332、L333、L409、P451、L453、L457、E476、L489、L490、N492、F494、Y497和E581功能等同的位置处的一个或多个突变;以及
在所述引发链的3′端掺入所述3′-OH未修饰的可逆终止子。
实施方案26.根据实施方案25所述的方法,其中所述3′-OH未修饰的可逆终止子是2-硝基苄基修饰的核苷酸。
实施方案27.根据实施方案25所述的方法,其中所述3′-OH未修饰的可逆终止子是C7-或C5-羟甲基-α-叔丁基-2-硝基苄基修饰的核苷酸及其α-硫代衍生物。
实施方案28.根据实施方案25至27中任一项所述的方法,其中所述突变聚合酶包含在与Pfu聚合酶中的位置492功能等同的位置处的至少一个氨基酸突变,并且所述方法包括选择性地掺入所述终止子。
实施方案29.根据实施方案28所述的方法,其中所述突变选自N492I、N492V或N492P。
实施方案30.根据实施方案28所述的方法,其中通过所述突变聚合酶选择性地掺入包含胞嘧啶碱基的3′-OH未封闭的可逆终止子。
实施方案31.一种包含引发链、3′-OH未修饰的可逆终止子和突变聚合酶的组合物,所述突变聚合酶与SEQ ID NO:2至少96%相同并且包含:
在与Pfu聚合酶中的位置546功能等同的位置处的Y546H突变;
在与Pfu聚合酶中的位置409功能等同的位置处的L409Y、L409H或L409F突变;和
在与Pfu聚合酶中的位置486功能等同的位置处的A486X突变,其中X是除丙氨酸之外的任何氨基酸。
实施方案32.根据实施方案31所述的组合物,其中所述组合物不含与所述引发链互补的模板。
实施方案33.根据实施方案31或32所述的组合物,其中所述突变聚合酶还包含在与Pfu聚合酶中的位置L270、E330、Q332、L333、P451、L453、L457、E476、L489、L490、N492、F494、Y497和E581功能等同的位置处的一个或多个突变。
实施方案34.根据实施方案31或32所述的组合物,其中所述突变聚合酶包含SEQID NO:4或SEQ ID NO:5的氨基酸序列。
实施方案35.根据实施方案31至34中任一项所述的组合物,其中所述突变聚合酶的掺入活性比SEQ ID NO:11的DNA聚合酶的掺入活性高至少2倍。
实施方案36.根据实施方案31至35中任一项所述的组合物,其中所述突变聚合酶是火球菌属聚合酶的衍生物。
实施方案37.根据实施方案31至36中任一项所述的组合物,其中所述突变聚合酶包含SEQ ID NO:2的氨基酸序列。
实施方案38.根据实施方案31至35中任一项所述的组合物,其中所述突变聚合酶是嗜热球菌属聚合酶的衍生物。
实施方案39.一种用于在非模板依赖性反应中将单个核苷酸掺入引发链中的方法,所述方法包括:
将引发链与3′-OH未修饰的可逆终止子和突变聚合酶组合,其中所述突变聚合酶与SEQ ID NO:2至少96%相同,并且包含:
在与Pfu聚合酶中的位置546功能等同的位置处的Y546H突变;
在与Pfu聚合酶中的位置409功能等同的位置处的L409Y、L409H或L409F突变;和
在与Pfu聚合酶中的位置486功能等同的位置处的A486X突变,其中X是除丙氨酸之外的任何氨基酸;
其中所述终止子的掺入比SEQ ID NO:11的突变DNA聚合酶高至少2倍。
实施方案40.一种非模板依赖性寡核苷酸合成的方法,所述方法包括:
将引发链、3′-OH未修饰的可逆终止子和突变DNA聚合酶组合,其中突变DNA聚合酶包含:
与SEQ ID NO:2至少96%相同的氨基酸序列;
在与Pfu聚合酶中的位置546功能等同的位置处的变为组氨酸的Y546H突变;
在与Pfu聚合酶中的位置409功能等同的位置处的L409Y、L409H或L409F突变;和
在与Pfu聚合酶中的位置486功能等同的位置处的A486X突变,其中X是除丙氨酸之外的任何氨基酸;
将所述3′-OH未修饰的可逆终止子掺入所述引发链。
实施方案41.根据实施方案39或40所述的方法,其中所述聚合酶还包含在与Pfu聚合酶中的位置L270、E330、Q332、L333、P451、L453、L457、E476、L489、L490、N492、F494、Y497和E581功能等同的位置处的一个或多个突变。
实施例42.根据实施方案39至41中任一项所述的方法,其中所述3′-OH未修饰的可逆终止子是2-硝基苄基修饰的核苷酸。
实施方案43.根据实施方案39至41中任一项所述的方法,其中所述3′-OH未修饰的可逆终止子是C7-或C5-羟甲基-α-叔丁基-2-硝基苄基修饰的核苷酸及其α-硫代衍生物。
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序列表
<110> 安捷伦科技有限公司
<120> 聚合酶突变体以及与3'-OH未封闭的可逆终止子一起使用
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Trp Leu Asn Ile Lys Lys Ser
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Ile Val Asp Val Glu Lys Val Glu Lys Lys Phe Leu Gly Lys Pro Val
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Thr Val Trp Arg Leu Tyr Leu Glu His Pro Gln Asp Val Pro Thr Ile
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Arg Glu Lys Val Lys Glu His Pro Ala Val Ile Asp Ile Phe Glu Tyr
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Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile
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Ser Tyr Ala Asp Glu Asn Glu Ala Lys Val Ile Thr Trp Lys Asn Ile
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Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys
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Arg Phe Val Lys Ile Ile Lys Glu Lys Asp Pro Asp Ile Ile Val Thr
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Lys Leu Gly Val Lys Leu Thr Ile Gly Arg Asp Gly Ser Glu Pro Lys
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Met Gln Arg Ile Gly Asp Met Thr Ala Val Glu Val Lys Gly Arg Ile
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His Phe Asp Leu Tyr His Val Ile Thr Arg Thr Ile Asn Leu Pro Thr
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Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu
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Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu Ser Gly Glu Asn
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Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr
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Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ile Gln Leu Ser Arg Leu
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Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Val Ala
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Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gln Arg Arg Leu Arg Glu Ser
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Ile Val Tyr Leu Asp Phe Arg Ala Leu Tyr Pro Ser Ile Ile Ile Thr
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530 535 540
Leu His Ala Thr Ile Pro Gly Gly Glu Ser Glu Glu Ile Lys Lys Lys
545 550 555 560
Ala Leu Glu Phe Val Lys Tyr Ile Asn Ser Lys Leu Pro Gly Leu Leu
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Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
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Val Arg Ile Val Lys Glu Val Ile Gln Lys Leu Ala Asn Tyr Glu Ile
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690 695 700
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Tyr Asp Pro Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
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740 745 750
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Met Ile Leu Asp Val Asp Tyr Ile Thr Glu Glu Gly Lys Pro Val Ile
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Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys
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His Phe Asp Leu Tyr His Val Ile Thr Arg Thr Ile Asn Leu Pro Thr
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Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu Ser Gly Glu Asn
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325 330 335
Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Val Ala
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Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gln Arg Arg Leu Arg Glu Ser
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Ile Val Tyr Leu Asp Phe Arg Ala Tyr Tyr Pro Ser Ile Ile Ile Thr
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435 440 445
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485 490 495
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500 505 510
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515 520 525
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530 535 540
Leu His Ala Thr Ile Pro Gly Gly Glu Ser Glu Glu Ile Lys Lys Lys
545 550 555 560
Ala Leu Glu Phe Val Lys Tyr Ile Asn Ser Lys Leu Pro Gly Leu Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Arg Tyr Ala Val Ile Asp Glu Glu Gly Lys Val Ile Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
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625 630 635 640
Val Arg Ile Val Lys Glu Val Ile Gln Lys Leu Ala Asn Tyr Glu Ile
645 650 655
Pro Pro Glu Lys Leu Ala Ile Tyr Glu Gln Ile Thr Arg Pro Leu His
660 665 670
Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Lys Leu Ala
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690 695 700
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705 710 715 720
Tyr Asp Pro Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
725 730 735
Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Gly Phe Gly Tyr Arg
740 745 750
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755 760 765
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770 775
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Arg Leu Phe Lys Lys Glu Asn Gly Lys Phe Lys Ile Glu His Asp Arg
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35 40 45
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Ile Val Asp Val Glu Lys Val Glu Lys Lys Phe Leu Gly Lys Pro Ile
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Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
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Met Glu Gly Glu Glu Glu Leu Lys Ile Leu Ala Phe Ala Ile Ala Thr
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Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile
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Ser Tyr Ala Asp Glu Asn Glu Ala Lys Val Ile Thr Trp Lys Asn Ile
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Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys
180 185 190
Arg Phe Leu Arg Ile Ile Arg Glu Lys Asp Pro Asp Ile Ile Val Thr
195 200 205
Tyr Asn Gly Asp Ser Phe Asp Phe Pro Tyr Leu Ala Lys Arg Ala Glu
210 215 220
Lys Leu Gly Ile Lys Leu Thr Ile Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Met Gln Arg Ile Gly Asp Met Thr Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr His Val Ile Thr Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu
275 280 285
Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu Ser Gly Glu Asn
290 295 300
Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr
305 310 315 320
Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ile Gln Leu Ser Arg Leu
325 330 335
Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Val Ala
355 360 365
Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gln Arg Arg Leu Arg Glu Ser
370 375 380
Tyr Thr Gly Gly Phe Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn
385 390 395 400
Ile Val Tyr Leu Asp Phe Arg Ala Tyr Tyr Pro Ser Ile Ile Ile Thr
405 410 415
His Asn Val Ser Pro Asp Thr Leu Asn Leu Glu Gly Cys Lys Asn Tyr
420 425 430
Asp Ile Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Ile Pro Gly
435 440 445
Phe Ile Pro Ser Leu Leu Gly His Leu Leu Glu Glu Arg Gln Lys Ile
450 455 460
Lys Thr Lys Met Lys Glu Thr Gln Asp Pro Ile Glu Trp Ile Leu Leu
465 470 475 480
Asp Tyr Arg Gln Lys Arg Ile Lys Leu Leu Ala Asn Ser Phe Tyr Gly
485 490 495
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
500 505 510
Ser Val Thr Ala Trp Gly Arg Lys Tyr Ile Glu Leu Val Trp Lys Glu
515 520 525
Leu Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly
530 535 540
Leu His Ala Thr Ile Pro Gly Gly Glu Ser Glu Glu Ile Lys Lys Lys
545 550 555 560
Ala Leu Glu Phe Val Lys Tyr Ile Asn Ser Lys Leu Pro Gly Leu Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Arg Tyr Ala Val Ile Asp Glu Glu Gly Lys Val Ile Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
Ala Arg Val Leu Glu Thr Ile Leu Lys His Gly Asp Val Glu Glu Ala
625 630 635 640
Val Arg Ile Val Lys Glu Val Ile Gln Lys Leu Ala Asn Tyr Glu Ile
645 650 655
Pro Pro Glu Lys Leu Ala Ile Tyr Glu Gln Ile Thr Arg Pro Leu His
660 665 670
Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Lys Leu Ala
675 680 685
Ala Lys Gly Val Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Ile Val
690 695 700
Leu Arg Gly Asp Gly Pro Ile Ser Asn Arg Ala Ile Leu Ala Glu Glu
705 710 715 720
Tyr Asp Pro Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
725 730 735
Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Gly Phe Gly Tyr Arg
740 745 750
Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Thr Ser
755 760 765
Trp Leu Asn Ile Lys Lys Ser
770 775
<210> 6
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 用于单碱基延伸反应的引物
<400> 6
taatacgact cactataggg 20
<210> 7
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 用于单碱基延伸反应的引物
<400> 7
taatacgact cactataggg caggaaacag ctatgaccag gggatcagc 49
<210> 8
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 用于单碱基延伸反应的引物
<400> 8
taatacgact cactataggg 20
<210> 9
<211> 775
<212> PRT
<213> 人工序列
<220>
<223> 激烈火球菌聚合酶的突变体
<400> 9
Met Ile Leu Asp Val Asp Tyr Ile Thr Glu Glu Gly Lys Pro Val Ile
1 5 10 15
Arg Leu Phe Lys Lys Glu Asn Gly Lys Phe Lys Ile Glu His Asp Arg
20 25 30
Thr Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Lys Ile
35 40 45
Glu Glu Val Lys Lys Ile Thr Gly Glu Arg His Gly Lys Ile Val Arg
50 55 60
Ile Val Asp Val Glu Lys Val Glu Lys Lys Phe Leu Gly Lys Pro Ile
65 70 75 80
Thr Val Trp Lys Leu Tyr Leu Glu His Pro Gln Asp Val Pro Thr Ile
85 90 95
Arg Glu Lys Val Arg Glu His Pro Ala Val Val Asp Ile Phe Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Glu Glu Glu Leu Lys Ile Leu Ala Phe Ala Ile Ala Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Asn Glu Ala Lys Val Ile Thr Trp Lys Asn Ile
165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys
180 185 190
Arg Phe Leu Arg Ile Ile Arg Glu Lys Asp Pro Asp Ile Ile Val Thr
195 200 205
Tyr Asn Gly Asp Ser Phe Asp Phe Pro Tyr Leu Ala Lys Arg Ala Glu
210 215 220
Lys Leu Gly Ile Lys Leu Thr Ile Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Met Gln Arg Ile Gly Asp Met Thr Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr His Val Ile Thr Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu
275 280 285
Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu Ser Gly Glu Asn
290 295 300
Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr
305 310 315 320
Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ile Gln Leu Ser Arg Leu
325 330 335
Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Val Ala
355 360 365
Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gln Arg Arg Leu Arg Glu Ser
370 375 380
Tyr Thr Gly Gly Phe Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn
385 390 395 400
Ile Val Tyr Leu Asp Phe Arg Ala Leu Tyr Pro Ser Ile Ile Ile Thr
405 410 415
His Asn Val Ser Pro Asp Thr Leu Asn Leu Glu Gly Cys Lys Asn Tyr
420 425 430
Asp Ile Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Ile Pro Gly
435 440 445
Phe Ile Pro Ser Leu Leu Gly His Leu Leu Glu Glu Arg Gln Lys Ile
450 455 460
Lys Thr Lys Met Lys Glu Thr Gln Asp Pro Ile Glu Lys Ile Leu Leu
465 470 475 480
Asp Tyr Arg Gln Lys Leu Ile Lys Leu Leu Ala Asn Ser Phe Tyr Gly
485 490 495
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
500 505 510
Ser Val Thr Ala Trp Gly Arg Lys Tyr Ile Glu Leu Val Trp Lys Glu
515 520 525
Leu Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly
530 535 540
Leu Tyr Ala Thr Ile Pro Gly Gly Glu Ser Glu Glu Ile Lys Lys Lys
545 550 555 560
Ala Leu Glu Phe Val Lys Tyr Ile Asn Ser Lys Leu Pro Gly Leu Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Arg Tyr Ala Val Ile Asp Glu Glu Gly Lys Val Ile Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
Ala Arg Val Leu Glu Thr Ile Leu Lys His Gly Asp Val Glu Glu Ala
625 630 635 640
Val Arg Ile Val Lys Glu Val Ile Gln Lys Leu Ala Asn Tyr Glu Ile
645 650 655
Pro Pro Glu Lys Leu Ala Ile Tyr Glu Gln Ile Thr Arg Pro Leu His
660 665 670
Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Lys Leu Ala
675 680 685
Ala Lys Gly Val Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Ile Val
690 695 700
Leu Arg Gly Asp Gly Pro Ile Ser Asn Arg Ala Ile Leu Ala Glu Glu
705 710 715 720
Tyr Asp Pro Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
725 730 735
Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Gly Phe Gly Tyr Arg
740 745 750
Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Thr Ser
755 760 765
Trp Leu Asn Ile Lys Lys Ser
770 775
<210> 10
<211> 775
<212> PRT
<213> 人工序列
<220>
<223> 人工序列
<400> 10
Met Ile Leu Asp Val Asp Tyr Ile Thr Glu Glu Gly Lys Pro Val Ile
1 5 10 15
Arg Leu Phe Lys Lys Glu Asn Gly Lys Phe Lys Ile Glu His Asp Arg
20 25 30
Thr Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Lys Ile
35 40 45
Glu Glu Val Lys Lys Ile Thr Gly Glu Arg His Gly Lys Ile Val Arg
50 55 60
Ile Val Asp Val Glu Lys Val Glu Lys Lys Phe Leu Gly Lys Pro Ile
65 70 75 80
Thr Val Trp Lys Leu Tyr Leu Glu His Pro Gln Asp Val Pro Thr Ile
85 90 95
Arg Glu Lys Val Arg Glu His Pro Ala Val Val Asp Ile Phe Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Glu Glu Glu Leu Lys Ile Leu Ala Phe Ala Ile Ala Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Asn Glu Ala Lys Val Ile Thr Trp Lys Asn Ile
165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys
180 185 190
Arg Phe Leu Arg Ile Ile Arg Glu Lys Asp Pro Asp Ile Ile Val Thr
195 200 205
Tyr Asn Gly Asp Ser Phe Asp Phe Pro Tyr Leu Ala Lys Arg Ala Glu
210 215 220
Lys Leu Gly Ile Lys Leu Thr Ile Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Met Gln Arg Ile Gly Asp Met Thr Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr His Val Ile Thr Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu
275 280 285
Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu Ser Gly Glu Asn
290 295 300
Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr
305 310 315 320
Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ile Gln Leu Ser Arg Leu
325 330 335
Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Val Ala
355 360 365
Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gln Arg Arg Leu Arg Glu Ser
370 375 380
Tyr Thr Gly Gly Phe Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn
385 390 395 400
Ile Val Tyr Leu Asp Phe Arg Ala Leu Tyr Pro Ser Ile Ile Ile Thr
405 410 415
His Asn Val Ser Pro Asp Thr Leu Asn Leu Glu Gly Cys Lys Asn Tyr
420 425 430
Asp Ile Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Ile Pro Gly
435 440 445
Phe Ile Pro Ser Leu Leu Gly His Leu Leu Glu Glu Arg Gln Lys Ile
450 455 460
Lys Thr Lys Met Lys Glu Thr Gln Asp Pro Ile Glu Lys Ile Leu Leu
465 470 475 480
Asp Tyr Arg Gln Lys Leu Ile Lys Leu Leu Ala Asn Ser Phe Tyr Gly
485 490 495
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
500 505 510
Ser Val Thr Ala Trp Gly Arg Lys Tyr Ile Glu Leu Val Trp Lys Glu
515 520 525
Leu Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly
530 535 540
Leu His Ala Thr Ile Pro Gly Gly Glu Ser Glu Glu Ile Lys Lys Lys
545 550 555 560
Ala Leu Glu Phe Val Lys Tyr Ile Asn Ser Lys Leu Pro Gly Leu Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Arg Tyr Ala Val Ile Asp Glu Glu Gly Lys Val Ile Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
Ala Arg Val Leu Glu Thr Ile Leu Lys His Gly Asp Val Glu Glu Ala
625 630 635 640
Val Arg Ile Val Lys Glu Val Ile Gln Lys Leu Ala Asn Tyr Glu Ile
645 650 655
Pro Pro Glu Lys Leu Ala Ile Tyr Glu Gln Ile Thr Arg Pro Leu His
660 665 670
Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Lys Leu Ala
675 680 685
Ala Lys Gly Val Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Ile Val
690 695 700
Leu Arg Gly Asp Gly Pro Ile Ser Asn Arg Ala Ile Leu Ala Glu Glu
705 710 715 720
Tyr Asp Pro Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
725 730 735
Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Gly Phe Gly Tyr Arg
740 745 750
Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Thr Ser
755 760 765
Trp Leu Asn Ile Lys Lys Ser
770 775
<210> 11
<211> 775
<212> PRT
<213> 人工序列
<220>
<223> 人工序列
<400> 11
Met Ile Leu Asp Val Asp Tyr Ile Thr Glu Glu Gly Lys Pro Val Ile
1 5 10 15
Arg Leu Phe Lys Lys Glu Asn Gly Lys Phe Lys Ile Glu His Asp Arg
20 25 30
Thr Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Lys Ile
35 40 45
Glu Glu Val Lys Lys Ile Thr Gly Glu Arg His Gly Lys Ile Val Arg
50 55 60
Ile Val Asp Val Glu Lys Val Glu Lys Lys Phe Leu Gly Lys Pro Ile
65 70 75 80
Thr Val Trp Lys Leu Tyr Leu Glu His Pro Gln Asp Val Pro Thr Ile
85 90 95
Arg Glu Lys Val Arg Glu His Pro Ala Val Val Asp Ile Phe Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Glu Glu Glu Leu Lys Ile Leu Ala Phe Ala Ile Ala Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Asn Glu Ala Lys Val Ile Thr Trp Lys Asn Ile
165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys
180 185 190
Arg Phe Leu Arg Ile Ile Arg Glu Lys Asp Pro Asp Ile Ile Val Thr
195 200 205
Tyr Asn Gly Asp Ser Phe Asp Phe Pro Tyr Leu Ala Lys Arg Ala Glu
210 215 220
Lys Leu Gly Ile Lys Leu Thr Ile Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Met Gln Arg Ile Gly Asp Met Thr Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr His Val Ile Thr Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu
275 280 285
Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu Ser Gly Glu Asn
290 295 300
Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr
305 310 315 320
Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ile Gln Leu Ser Arg Leu
325 330 335
Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Val Ala
355 360 365
Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gln Arg Arg Leu Arg Glu Ser
370 375 380
Tyr Thr Gly Gly Phe Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn
385 390 395 400
Ile Val Tyr Leu Asp Phe Arg Ala Leu Tyr Pro Ser Ile Ile Ile Thr
405 410 415
His Asn Val Ser Pro Asp Thr Leu Asn Leu Glu Gly Cys Lys Asn Tyr
420 425 430
Asp Ile Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Ile Pro Gly
435 440 445
Phe Ile Pro Ser Leu Leu Gly His Leu Leu Glu Glu Arg Gln Lys Ile
450 455 460
Lys Thr Lys Met Lys Glu Thr Gln Asp Pro Ile Glu Lys Ile Leu Leu
465 470 475 480
Asp Tyr Arg Gln Lys Ala Ile Lys Leu Leu Ala Asn Ser Phe Tyr Gly
485 490 495
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
500 505 510
Ser Val Thr Ala Trp Gly Arg Lys Tyr Ile Glu Leu Val Trp Lys Glu
515 520 525
Leu Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly
530 535 540
Leu Tyr Ala Thr Ile Pro Gly Gly Glu Ser Glu Glu Ile Lys Lys Lys
545 550 555 560
Ala Leu Glu Phe Val Lys Tyr Ile Asn Ser Lys Leu Pro Gly Leu Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Arg Tyr Ala Val Ile Asp Glu Glu Gly Lys Val Ile Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
Ala Arg Val Leu Glu Thr Ile Leu Lys His Gly Asp Val Glu Glu Ala
625 630 635 640
Val Arg Ile Val Lys Glu Val Ile Gln Lys Leu Ala Asn Tyr Glu Ile
645 650 655
Pro Pro Glu Lys Leu Ala Ile Tyr Glu Gln Ile Thr Arg Pro Leu His
660 665 670
Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Lys Leu Ala
675 680 685
Ala Lys Gly Val Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Ile Val
690 695 700
Leu Arg Gly Asp Gly Pro Ile Ser Asn Arg Ala Ile Leu Ala Glu Glu
705 710 715 720
Tyr Asp Pro Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
725 730 735
Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Gly Phe Gly Tyr Arg
740 745 750
Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Thr Ser
755 760 765
Trp Leu Asn Ile Lys Lys Ser
770 775
<210> 12
<211> 775
<212> PRT
<213> 人工序列
<220>
<223> 人工序列
<400> 12
Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile
1 5 10 15
Arg Val Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg
20 25 30
Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile
35 40 45
Glu Asp Val Lys Lys Val Thr Ala Lys Arg His Gly Thr Val Val Lys
50 55 60
Val Lys Arg Ala Glu Lys Val Gln Lys Lys Phe Leu Gly Arg Pro Ile
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Asn His Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Asp Arg Ile Arg Ala His Pro Ala Val Val Asp Ile Tyr Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asp Glu Glu Leu Thr Met Leu Ala Phe Ala Ile Ala Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile
145 150 155 160
Ser Tyr Ala Asp Gly Ser Glu Ala Arg Val Ile Thr Trp Lys Lys Ile
165 170 175
Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys
180 185 190
Arg Phe Leu Arg Val Val Arg Glu Lys Asp Pro Asp Val Leu Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu
210 215 220
Glu Leu Gly Ile Lys Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu
275 280 285
Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Ser Gly Glu Gly
290 295 300
Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320
Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu
325 330 335
Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Lys Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr
370 375 380
Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile
385 390 395 400
Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His
405 410 415
Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp
420 425 430
Val Ala Pro Glu Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe
435 440 445
Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys
450 455 460
Arg Lys Met Lys Ala Thr Val Asp Pro Leu Glu Lys Lys Leu Leu Asp
465 470 475 480
Tyr Arg Gln Arg Leu Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr
485 490 495
Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser
500 505 510
Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu Leu
515 520 525
Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu
530 535 540
His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala
545 550 555 560
Lys Glu Phe Leu Lys Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu
565 570 575
Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys
580 585 590
Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu
595 600 605
Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala
610 615 620
Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val
625 630 635 640
Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro
645 650 655
Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Arg Asp
660 665 670
Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala
675 680 685
Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu
690 695 700
Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Ala Asp Glu Phe
705 710 715 720
Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln
725 730 735
Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Arg Lys
740 745 750
Glu Asp Leu Arg Tyr Gln Lys Thr Lys Gln Val Gly Leu Gly Ala Trp
755 760 765
Leu Lys Val Lys Gly Lys Lys
770 775
Claims (43)
1.一种包含3'-OH未封闭的可逆终止子和突变聚合酶的组合物,其中所述突变聚合酶包含与SEQ ID NO:2至少96%相同的氨基酸序列,并且包含在与Pfu聚合酶中的K477、A486和Y546的氨基酸位置功能等同的位置处的氨基酸突变。
2.根据权利要求1所述的组合物,其中所述突变聚合酶包含在与Pfu聚合酶中的位置486功能等同的位置处的A486X突变,其中X是除丙氨酸之外的任何氨基酸。
3.根据权利要求2所述的组合物,其中所述A486X突变为A486F、A486Y、A486N、A486R或A486H。
4.根据权利要求1至3中任一项所述的组合物,其中所述突变聚合酶还包含在与Pfu聚合酶中的位置546功能等同的位置处的Y546H突变。
5.根据权利要求1至4中任一项所述的组合物,其中所述突变聚合酶还包含在与Pfu聚合酶中的位置477功能等同的位置处的K477W突变。
6.根据权利要求1至5中任一项所述的组合物,其中所述突变聚合酶还包含在与Pfu聚合酶中的位置F494功能等同的位置处的突变。
7.根据权利要求6所述的组合物,其中所述F494突变为F494C、F494I、F494N或F494T。
8.根据前述权利要求中任一项所述的组合物,其中所述突变聚合酶是火球菌属聚合酶的衍生物。
9.根据权利要求8所述的组合物,其中所述突变聚合酶包含SEQ ID NO:2的氨基酸序列。
10.根据权利要求1至7中任一项所述的组合物,其中所述突变聚合酶是嗜热球菌属聚合酶的衍生物。
11.一种将核苷酸掺入包含核酸的引发链的方法,所述方法包括:
在足以进行掺入反应的条件下,使所述引发链与核苷酸和突变聚合酶接触,其中所述突变聚合酶包含与SEQ ID NO:2至少96%相同的氨基酸序列,并且包含在与Pfu聚合酶中的K477、A486和Y546的氨基酸位置功能等同的位置处的氨基酸突变。
12.根据权利要求11所述的方法,其中所述核苷酸是3'-OH未封闭的可逆终止子。
13.一种多核苷酸测序的方法,所述方法包括:
(a)形成包含模板和引发链的双链体,其中所述模板包含待测序的靶核酸和与所述引发链的至少一部分互补的引物结合位点;
(b)将所述引发链与可逆终止子核苷酸和突变聚合酶组合,其中所述突变聚合酶包含与SEQ ID NO:2至少96%相同的氨基酸序列,并且包含在与Pfu聚合酶中的K477、A486和Y546的氨基酸位置功能等同的位置处的氨基酸突变;
(c)在模板依赖性反应中,在所述引发链的3'端掺入所述可逆终止子;和
(d)鉴定掺入的可逆终止子核苷酸,从而确定所述模板的序列。
14.根据权利要求13所述的方法,其中所述方法还包括重复步骤(c)和(d)至少80次。
15.一种包含引发链、3'-OH未封闭的可逆终止子3'-OH和突变聚合酶的组合物,其中:
所述突变聚合酶包含与SEQ ID NO:1至少80%相同的氨基酸序列,
所述突变聚合酶还包含在与Pfu聚合酶中的位置L270、E330、Q332、L333、L409、P451、L453、L457、E476、L489、L490、N492、F494、Y497和E581功能等同的位置处的一个或多个突变;并且
所述突变聚合酶对所述3'-OH未封闭的可逆终止子的掺入活性比SEQ ID NO:11的DNA聚合酶的掺入活性高至少4倍。
16.根据权利要求15所述的组合物,其中所述氨基酸序列与SEQ ID NO:1至少85%相同。
17.根据权利要求15所述的组合物,其中所述氨基酸序列与SEQ ID NO:1至少90%相同。
18.根据权利要求15所述的组合物,其中所述氨基酸序列与SEQ ID NO:1至少95%相同。
19.根据权利要求15至18中任一项所述的组合物,其中所述突变聚合酶不包含在与Pfu聚合酶中的位置266、267、268、269、329、336、399、400、403、404、407、408、410、411、450、452、455、456、458、459、460、461、462、463、464、465、466、475、477、478、479、480、481、482、483、485、487、488、491、493、495、496、498、499、500、515、522、545、546、577、579、580、582、584、591、595、603、606、607、608、612、613、614、664、665、666、668、669、674、675和676功能等同的任一位置处的突变。
20.根据权利要求15至19中任一项所述的组合物,其中所述突变聚合酶是火球菌属聚合酶的衍生物。
21.根据权利要求15至19中任一项所述的组合物,其中所述突变聚合酶是嗜热球菌属聚合酶的衍生物。
22.根据权利要求15至21中任一项所述的组合物,其中所述组合物还包含模板,所述模板包含与所述引发链的至少一部分互补的引物结合位点。
23.根据权利要求15至22中任一项所述的组合物,其中所述组合物还包含546H和486X突变。
24.根据权利要求15至23中任一项所述的组合物,其中所述组合物不含与所述引发链互补的模板。
25.一种将3'-OH未修饰的可逆终止子掺入引发链中的方法,所述方法包括:
在足以进行掺入反应的条件下,使引发链与3'-OH未修饰的可逆终止子和突变聚合酶接触,
其中所述突变聚合酶包含与SEQ ID NO:1至少80%相同的氨基酸序列和在与Pfu聚合酶中的位置L270、E330、Q332、L333、L409、P451、L453、L457、E476、L489、L490、N492、F494、Y497和E581功能等同的位置处的一个或多个突变;以及
在所述引发链的3'端掺入所述3'-OH未修饰的可逆终止子。
26.根据权利要求25所述的方法,其中所述3'-OH未修饰的可逆终止子是2-硝基苄基修饰的核苷酸。
27.根据权利要求25所述的方法,其中所述3'-OH未修饰的可逆终止子是C7-或C5-羟甲基-α-叔丁基-2-硝基苄基修饰的核苷酸及其α-硫代衍生物。
28.根据权利要求25至27中任一项所述的方法,其中所述突变聚合酶包含在与Pfu聚合酶中的位置492功能等同的位置处的至少一个氨基酸突变,并且所述方法包括选择性地掺入所述终止子。
29.根据权利要求28所述的方法,其中所述突变选自N492I、N492V或N492P。
30.根据权利要求28所述的方法,其中通过所述突变聚合酶选择性地掺入包含胞嘧啶碱基的3'-OH未封闭的可逆终止子。
31.一种包含引发链、3'-OH未修饰的可逆终止子和突变聚合酶的组合物,所述突变聚合酶与SEQ ID NO:2至少96%相同并且包含:
在与Pfu聚合酶中的位置546功能等同的位置处的Y546H突变;
在与Pfu聚合酶中的位置409功能等同的位置处的L409Y、L409H或L409F突变;和
在与Pfu聚合酶中的位置486功能等同的位置处的A486X突变,其中X是除丙氨酸之外的任何氨基酸。
32.根据权利要求31所述的组合物,其中所述组合物不含与所述引发链互补的模板。
33.根据权利要求31或32所述的组合物,其中所述突变聚合酶还包含在与Pfu聚合酶中的位置L270、E330、Q332、L333、P451、L453、L457、E476、L489、L490、N492、F494、Y497和E581功能等同的位置处的一个或多个突变。
34.根据权利要求31或32所述的组合物,其中所述突变聚合酶包含SEQ ID NO:4或SEQID NO:5的氨基酸序列。
35.根据权利要求31至34中任一项所述的组合物,其中所述突变聚合酶的掺入活性比SEQ ID NO:11的DNA聚合酶的掺入活性高至少2倍。
36.根据权利要求31至35中任一项所述的组合物,其中所述突变聚合酶是火球菌属聚合酶的衍生物。
37.根据权利要求31至36中任一项所述的组合物,其中所述突变聚合酶包含SEQ IDNO:2的氨基酸序列。
38.根据权利要求31至35中任一项所述的组合物,其中所述突变聚合酶是嗜热球菌属聚合酶的衍生物。
39.一种用于在非模板依赖性反应中将单个核苷酸掺入引发链中的方法,所述方法包括:
将引发链与3'-OH未修饰的可逆终止子和突变聚合酶组合,其中所述突变聚合酶与SEQID NO:2至少96%相同,并且包含:
在与Pfu聚合酶中的位置546功能等同的位置处的Y546H突变;
在与Pfu聚合酶中的位置409功能等同的位置处的L409Y、L409H或L409F突变;和
在与Pfu聚合酶中的位置486功能等同的位置处的A486X突变,其中X是除丙氨酸之外的任何氨基酸;
其中所述终止子的掺入比SEQ ID NO:11的突变DNA聚合酶高至少2倍。
40.一种非模板依赖性寡核苷酸合成的方法,所述方法包括:
将引发链、3'-OH未修饰的可逆终止子和突变DNA聚合酶组合,其中突变DNA聚合酶包含:
与SEQ ID NO:2至少96%相同的氨基酸序列;
在与Pfu聚合酶中的位置546功能等同的位置处的变为组氨酸的Y546H突变;
在与Pfu聚合酶中的位置409功能等同的位置处的L409Y、L409H或L409F突变;和
在与Pfu聚合酶中的位置486功能等同的位置处的A486X突变,其中X是除丙氨酸之外的任何氨基酸;
将所述3'-OH未修饰的可逆终止子掺入所述引发链。
41.根据权利要求39或40所述的方法,其中所述聚合酶还包含在与Pfu聚合酶中的位置L270、E330、Q332、L333、P451、L453、L457、E476、L489、L490、N492、F494、Y497和E581功能等同的位置处的一个或多个突变。
42.根据权利要求39至41中任一项所述的方法,其中所述3'-OH未修饰的可逆终止子是2-硝基苄基修饰的核苷酸。
43.根据权利要求39至41中任一项所述的方法,其中所述3'-OH未修饰的可逆终止子是C7-或C5-羟甲基-α-叔丁基-2-硝基苄基修饰的核苷酸及其α-硫代衍生物。
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