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CN1152140C - 核酸序列分析 - Google Patents

核酸序列分析 Download PDF

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CN1152140C
CN1152140C CNB988076233A CN98807623A CN1152140C CN 1152140 C CN1152140 C CN 1152140C CN B988076233 A CNB988076233 A CN B988076233A CN 98807623 A CN98807623 A CN 98807623A CN 1152140 C CN1152140 C CN 1152140C
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D·H·登斯哈姆
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Abstract

本发明涉及测定多聚核苷酸序列的方法，该方法包括步骤：(i)在聚合酶反应足以进行的条件下，使靶多聚核苷酸与固定在固体支持物上的聚合酶及不同的核苷酸进行反应；和(ii)通过测量辐射来检测与靶多聚核苷酸互补的特定核苷酸的掺入。

Description

核酸序列分析
发明范围
本发明涉及测定多聚核苷酸序列的方法。
发明背景
测定多聚核苷酸有重要的科学意义。例如，全球正在雄心勃勃地进行的人类基因组计划，便是为人类基因组中编码DNA的30亿个碱基进行绘图和测序。倘若完成，得到的序列数据库可作为生物医学研究空前有力的工具。成功完成该计划的主要障碍与测序过程中使用的技术有关。
一般用于大范围DNA测序的重要方法是链终止法。该方法由Sanger和Coulson提出(Sanger等人，美国国家科学院院报(Proc.Natl.Acad.Sci.USA)1977；74：5463-5467)，需使用在聚合酶反应中掺入到新生的多聚核苷酸链中的四种核苷三磷酸的双脱氧衍生物。掺入后双脱氧衍生物使聚合酶反应终止，然后通过凝胶电泳将产物分离，分析特定双脱氧衍生物在链中的掺入位置。
虽然该方法得到广泛使用且结果可靠，但是此方法速度慢、工作量大而且昂贵。
EP-A-0471732提出了另一种测序方法，即通过光谱方法对核苷酸掺入到与靶链互补的新生多聚核苷酸链中的情况进行检测。该方法需使用模板和引物的固定化复合物，该复合物与仅含有不同核苷酸的一种的液体接触。然后采用光谱技术对随聚合酶催化的模板拷贝的延长而增强的时间依赖性信号进行测量。上述光谱技术为可在倏逝波场内测量分析物中的变化的表面等离子体共振(SPR)光谱法，和荧光测量技术。然而该方法有局限性；SPR技术最严重的问题在于，当拷贝链的长度增加时，由于链的运动使信号的绝对值也超出了倏逝波场，不易检测到增加。荧光测量技术的缺点是掺入到延长的新生多聚核苷酸链中的荧光团会造成背景干扰。随着链的延伸，背景“噪音”增加，检测每个核苷酸掺入的所需时间也要增加。这就严重限制了该方法在大片段多聚核苷酸测序中的应用。
因此需要多聚核苷酸测序的改进方法，其可明显提高对多聚核苷酸的测序速率，并优选通过自动化过程进行，从而降低现有方法的复杂性和成本。
发明概述
本发明建立在以下认识的基础上，即通过测量电磁或其他辐射，可以检测核苷酸掺入到新生多聚核苷酸链中时聚合酶发生的构象和/或质量变化。
根据本发明，对多聚核苷酸测序的方法包括步骤：
(i)在聚合酶反应足以进行的条件下，使靶多聚核苷酸与固定
   在固体支持物上的聚合酶及不同的核苷酸进行反应；和
(ii)通过测量辐射来检测与靶多聚核苷酸互补的特定核苷酸
    的掺入。
可用多种技术测量样品的辐射，包括表面敏感的检测技术，其中用固体光学表面的光反应的变化表示在该表面的结合作用。在本发明的优选实施方案中，采用的技术为瞬间波光谱法，尤其是表面等离子体共振(SPR)光谱法。
在本发明的一实施方案中，本方法采用的核苷酸包括在3’端任选在5’端，有防止该核苷酸掺入到多聚核苷酸链中的封闭基团，但可将封闭基团选择性地切除以允许进一步进行掺入。通过使用封闭的核苷酸，本方法可在任何时间内用反应中存在的全部核苷酸进行反应。以确保每个已掺入的核苷酸可被检测的方式选择性切除封闭基团。因此本方法可以在“实时”的基础上进行，以实现快速序列分析。
附图描述
通过举例的方式来描述本发明，仅参考下列附图，其中：
图1是采用SPR光谱法进行多聚核苷酸序列分析的图解性说明；和
图2表示每种不同的核苷酸聚合时检测到的不同反应信号。
图3表示双封闭核苷酸的合成步骤。
发明描述
用于测定多聚核苷酸序列的本方法包括对聚合酶、靶多聚核苷酸和互补核苷酸之间的动力学相互作用的分析。通过监测反应进行时造成的电磁或其他辐射的变化或吸收，来测量这种动力学相互作用。
此处使用的术语“多聚核苷酸”广义上讲，包括DNA和RNA，包括修饰的DNA和RNA，也包括其他杂交的核酸样分子如肽核酸(PNA)。
一般地，通过采用电磁辐射、使用表面等离子体共振或核磁共振技术来实施本方法。也可考虑测量辐射变化的其他技术，例如通过全内反射荧光(TIRF)、衰减全反射(ATR)、破坏全反射(FTR)、Brewster角反射光度法、散射全内反射(STIR)或倏逝波椭圆偏振测量术进行光谱分析。
除了需要电磁辐射的技术外，也可考虑其他技术，尤其是光化学技术如化学发光法，和包括共振系统如表面声波(SAW)技术和石英晶体微量秤(QCM)技术在内的比重测量技术。
一种优选方法是表面等离子体共振(SPR)光谱法，其通过检测溶液的体相与倏逝波域之间折射指数的差异对溶液性质进行测量。入射的单色光以一定角度自对侧的固体光学(传感器芯片)表面反射到待测样品。该光进入样本中很短的一段距离，并受表面相互作用的影响。
合适的传感器芯片在本领域已知。一般地，其包括光学透明材料如玻璃，和反射薄片如银片或金片。SPR光谱法综述见欧洲专利公开文本0648328(在此全部引入供参考)。
另一优选方法是核磁共振(NMR)光谱法，测量化合物的磁性。通过施加磁场和射频辐射的结合使化合物的核能量定向。当施加给核的能量与自旋状态之间的能差(与施加的场的方向平行或反平行取向之间的差别)相同时，即达到所谓的共振状态。通过射频接收器检测与自旋状态的变化有关的能量的吸收和随后的发射。
本发明方法的一个重要方面是对固定在固体支持物上的聚合酶的使用。固定该酶为本方法获得成功提供了几点优势。首先，大大减少了测量可溶性分子中的能量吸收时出现的随机“噪音”问题。其次，由于聚合酶可维持在相对于测量区域的特定限制范围内，减少了由任何无直接关系的底物(如核苷酸)与聚合酶作用而产生的噪音问题。这在用来测量辐射变化的技术需要测量荧光时尤为重要，如在TIRF中，其中背景荧光会随新生链的延伸而增加。还有，使用SPR光谱法时，是在倏逝波场内维持聚合酶反应，因此不论多聚核苷酸的长度如何均可进行准确测量。最后，由于靶多聚核苷酸和寡聚核苷酸引物均非不可逆地与固体表面结合，更新该表面便变得相对简单，从而可用同样的固定化聚合酶进行更多的测序反应。
可用本领域已知的标准方法进行固定。具体地，可用标准的胺偶合方法进行固定，使连有配基的胺与，如，葡聚糖或N-羟基琥珀酰亚胺酯-活化的表面结合。在本发明的优选实施方案中，将聚合酶固定在维持该酶和传感器表面紧密相连的SPR传感器芯片表面上，该表面可测量折射指数的变化。将生物分子固定到光学传感器上的方法示例见EP-A-0589867，和Lofas等人，生物传感器与生物电子(Biosens.Bioelectron.)(1995) 10：813-822。
本发明使用的聚合酶可以是任何已知类型。例如，聚合酶可以是任何DNA依赖的DNA聚合酶。如果靶多聚核苷酸是RNA分子，则聚合酶可以是RNA依赖的DNA聚合酶，即逆转录酶，或RNA依赖的RNA聚合酶，即RNA复制酶。在本发明的一个优选实施方案中，聚合酶为Taq聚合酶。在本发明更优选的实施方案中，聚合酶是大肠杆菌(E.Coli)III型聚合酶全酶(McHenry，生物化学综述年刊(Ann.Rev.Biochem.)1988；57：519)、T7聚合酶(Schwager等人，分子与细胞生物学方法(Methods in Molecular and Celular Biology)(1989/90)；卷1(4)：155-159)、或复合有大肠杆菌硫氧还蛋白的T7噬菌体基因5聚合酶(Tabor等人，生物化学杂志(J.Biol.Chem.)(1987)；262：1612-1623)。每种聚合酶都能以高忠实度与靶多聚核苷酸发生结合，并因此甚至在未发生有效聚合的时候维持聚合酶-多聚核苷酸复合物。
III型聚合酶全酶包括可产生加工酶的三个亚组：(I)核心聚合酶，包括聚合酶α亚基；(II)β-二聚体亚基，其作为围绕DNA的手镯样结构；和(III)含有τ和γ亚基的亚组，可结合并水解ATP以形成围绕DNA的β-二聚体。
作为测序过程的第一步，可使靶多聚核苷酸与合适的引物在杂交/聚合缓冲液进行作用。一般地，缓冲液的温度应足够高以破坏(或融解)靶多聚核苷酸中存在的任何二级结构。冷却时，引物与靶互补序列退火。再将该样品与固定化的聚合酶进行作用，以形成靶多聚核苷酸/聚合酶复合物。
在本发明的一个实施方案中，控制核苷酸的添加，使不同的核苷酸顺序加入聚合酶/靶复合物。例如，添加dGTP使之流过聚合酶/多聚核苷酸复合物；然后检测任何掺入情况。未结合的dGTP流出反应位点，再导入另一种核苷酸。以这种方式，动力学相互作用的检测可与其时出现的特定核苷酸有关，然后可以确定多聚核苷酸的序列。
不同的核苷酸同时存在时也可以实施本方法。为成功实施之，需要至少在核苷酸的3’位置导入一个封闭基团，但优选在3’和5’端均导入。封闭基团可以是光敏的，能用一定波长的光去除从而得到活性分子。若核苷酸的3’和5’端都掺入了封闭基团，则应能基于光谱吸收度对封闭基团加以区分，即应可通过使用特定波长的光选择性去除一种封闭基团而不影响其他封闭基团。也希望去除3’端封闭基团所需的光照时间比去除5’端封闭基团长。这样，封闭基团可通过光谱和时间的方式得以区分。
一般地，光敏性封闭基团在200nm到450nm波长范围发生光裂解。封闭基团一般衍生自分子式为R1-[O-CO-]X的化合物，其中R1是光不稳定基团，X是离去基团。例如R1是邻硝基苄基。特别优选的封闭基团包括WO-A-92/10092与WO-A-97/39151中描述的邻硝基苄基保护基。这些基团包括硝基藜芦基氧羰基(NVOC)、硝基胡椒基氧羰基(NPOC)、α-甲基-硝基藜芦基氧羰基(MeNVOC)、α-甲基-硝基胡椒基氧羰基(MeNPOC)和1-芘基甲基氧羰基(PYMOC)。
合适的3’封闭基团为(4，5-二甲氧基-2-硝基苄基)氧羰基，其可通过核苷酸与式(I)的化合物反应形成：
Figure C9880762300091
其中R是任何合适的酯化基，如甲基。可用360nm波长的光脉冲选择性去除该封闭基团。
5’端合适的封闭基团是1-(2-硝基苯)乙基(II)：
其中是R是任何合适的官能团，如卤素。可于260nm波长选择性去除该封闭基团。
例如，将双封闭的核苷酸注射到含有引物的多聚核苷酸上(已与高忠实度聚合酶复合物相结合)，在足以从每一核苷酸末端磷酸释放封闭基团的波长下使单色光聚焦到聚合酶的上游。将核苷酸流至结合的聚合酶上，掺入到新生的多聚核苷酸链中。然而，由于3’端的封闭基团仍处于结合状态，只能掺入一种核苷酸。对这种动力学相互作用进行测量可得知掺入到新生链中的特定核苷酸的信息。使用的聚合酶应为在反应终止时不易从靶上解离的高忠实度聚合酶。另外，可使用竞争性抑制剂防止聚合酶从靶上解离。
测量完掺入的核苷酸后，将单色光脉冲聚焦到聚合酶催化位点内的封闭基团上，以去除3’端的封闭基团。单色光脉冲时间可比去除5’端所需时间长，这样仅仅与聚合酶复合物中的核苷酸连接的封闭基团得以去除。这就减少了未与聚合酶复合物连接的核苷酸掺入的可能性。
一旦释放3’端的封闭基团，另一核苷酸到达聚合酶反应位点时可以继续聚合酶反应。通过改变去除封闭基团所需的光脉冲防止自由聚合。
尽管优选使用双封闭核苷酸，如上所述，也可使用仅在3’端带有封闭基团的核苷酸实施本方法。这种情况下，要用聚合酶的竞争性抑制剂来减少3’端没有封闭基团的核苷酸掺入到新生链中的可能性。合适的聚合酶的竞争性抑制剂是羰基二膦酸酯(COMDP)。
参照附图，用下述实施例说明本发明。
实施例
在改进的BIAcore2000系统(BIAcoreAB，Uppsala，瑞典)上进行以下分析，该系统以CM5传感器芯片(研究级，BIAcoreAB)作为光学传感器表面。该仪器带有完整的μm-流体筒(IFC)，可以通过一次样品注射在4个槽内进行分析。
聚合酶的制备
按照如所述方法(Millard等人，酶学方法(Methods Enzymol.)(1995)； 262：22)在缬氨酰-琼脂糖上通过疏水相互作用色谱法制备大肠杆菌III型聚合酶全酶，并在高盐浓度下纯化。纯化后，用Kirkegaard等人在生物化学分析(Anal.Biochem.)(1972)；50：122中所述的离子-过滤技术浓缩该酶。
聚合酶的固定
按照(Jonsson等人，生物技术(Biotechniques)(1991)； 11：620-627)所述，将聚合酶固定到传感器芯片表面。简言之，用Hepes缓冲液(10mMHepes、150mM氯化钠、0.05％表面活性剂P20(BIAcoreAB，Uppsala，瑞典)、pH7.4)使传感器芯片的环境平衡。将等体积的N-羟基琥珀酰亚胺(0.1M水溶液)和N-乙基-n’-(二甲基氨基丙基)碳二亚胺(EDC)(0.1M水溶液)混合，注射入芯片表面(CM5)使羧甲基化葡聚糖活化。将III型聚合酶核心亚组(160μl，500U)与10mM乙酸钠(100μl，pH5)混合，注射入活化表面。最后，使传感器芯片表面残余的N-羟基琥珀酰亚胺酯与乙醇胺(35μl，1M水溶液，pH8.5)反应，洗去表面上未结合的聚合酶。在25℃下用连续流动的Hepes缓冲液(5μl/分钟)进行固定。
寡聚核苷酸
采用标准的氨基亚磷酸酯(Phosphoramidity)化学法合成寡聚核苷酸。把序列为SEQ ID No.1的寡聚核苷酸用作靶多聚核苷酸，序列为SEQ ID No.2的寡聚核苷酸用作引物。
CAAGGAGAGGACGCTGTCTGTCGAAGGTAAGGAACGGAC
GAGAGAAGGGAGAG       SEQ ID No.1
CTCTCCCTTCTCTCGTC SEQ ID No.2
在杂交条件下使两寡聚核苷酸反应形成靶-引物复合物。
再将带有引物的DNA悬浮于缓冲液(20mM Tris-HCl，pH7.5、8mM氯化镁、4％(v/v)甘油、5mM二硫苏糖醇(DDT)、40μg牛血清白蛋白)中，该缓冲液含21μg ssDNA结合蛋白和形成手镯样结构所需的DNA聚合酶III亚组(1.6pmol-β-二聚体和195fmolγ亚基)。还有0.5mMATP和60μM羰基二膦酸酯(COMDP)。在此反应中，γ亚基作为分子媒体水解ATP，使β-二聚体亚基移到DNA上形成聚合酶亚组(Studwell等人，UCLA Symp.Mol.Cell.Biol.New Ser.1990；127：153)。
将带引物的DNA/亚组复合物以5μm/分钟的流速注射到传感器芯片表面的III型聚合酶上，并通过γ亚基的作用与聚合酶结合。
在本实验中，需要镁和ATP使聚合酶III与带引物的DNA结合。但是镁也可通过核验性3’→5’核酸外切酶活性促使引物被切除。通过纳入聚合酶活性的竞争性抑制剂羰基二膦酸酯可克服此问题(也可使用缺乏3’→5’核酸外切酶活性的III型聚合酶避免此具体问题)。
在芯片表面保持60μM羰基二膦酸酯的连续流动，防止核酸外切酶活性将引物从靶DNA上切除。
掺入两个封闭基团的核苷酸
如图3所示，每个核苷酸(dCTP、dTTP、dGTP和dATP)在5’端含有1-(2-硝基苯基)乙基封闭基团，3’端含有(4，5-二甲氧基-2-硝基苄基)氧羰基封闭基团。双封闭核苷酸的合成如下：
阶段1：(4，5-二甲氧基-硝基苄基)氧羰基核苷三磷酸的合成。
全部同样的方法应用于dGTP、dCTP、dTTP。室温下在15mlDMSO中将dATP二水合物(0.4mmol)与约3 mmol的4，5-二甲氧基-2-硝基苯基重氮甲烷混合液避光搅拌40小时，后者由900mg(4mmol)的4，5-二甲氧基-2-硝基苯腙新鲜制备而来(通过按照Wootton与Trentham，生物化学中的光化学探针(Photochemical Probes inBiochemistry)(Nielsen，P.E.，编)NATO ASI Ser.C，272卷，277-296页(1989)中的方法通过用肼单水合物氯仿溶液处理藜芦醛而合成)。通过氯仿/甲醇(5∶1v/v)溶剂系统中的TLC来监视反应，发现有Rf为0.54的点与笼闭核苷酸对应。通过60ml乙醚反复提取来去除DMSO、未反应的重氮化合物和低极性的反应产物。将含有未反应的核苷酸和所需产物及其他物质的剩余物质溶于最小量的氯仿中，并在硅胶柱(3×30cm)上用快速色谱法分离。用100％氯仿和甲醇/氯仿(95∶5v/v)洗脱去掉柱上的4，5-二甲氧基-2-硝基苯基重氮甲烷疏水性副产物。在旋转蒸发器上干燥级分。再冻干78mg笼闭产物。总产率为45％。用制备性反相HPLC从粗品中直接分离出纯度更高的3’封闭的4，5-二甲氧基-2-硝基苄基氧羰基dATP。
阶段2：5’1-(2-硝基苯基)乙基基团至3’4，5-二甲氧基-2-硝基苄基氧羰基封闭的dATP的添加。
室温下在15ml DMSO中将4，5-二甲氧基-2-硝基苄基氧羰基5’dATP(0.4mmol)与约3mmol的1-(2-硝基苯基)重氮乙烷混合液避光搅拌40小时，后者由716.7mg(4mmol)2-硝基苯乙酮的腙与2.9g(30mmol)MnO2(90％)在20ml氯仿中按照Walker等人的方法新鲜制备而来(Walker等人，酶学方法1989；172：288-301)，其中所述的腙通过用水合肼乙醇溶液处理2-硝基苯乙酮而合成。通过氯仿/甲醇(5∶1 v/v)溶剂系统中的TLC来监视反应，发现Rf为0.68和0.58的一对点，分别与4，5-二甲氧基-2-硝基苄基氧羰基5’dATP的1-(2-硝基苯基)乙酯平伏式的两个非对映异构体和轴向非对映异构体相对应。用50ml乙醚反复提取来去除DMSO、未反应的重氮化合物和低极性的反应产物。
将含有未反应的4，5-二甲氧基-2-硝基苄基氧羰基5’dATP和所需的双封闭dATP及其他物质的剩余物质溶于最小量的氯仿中，并在3×30cm硅胶柱上用快速色谱法分离。用100％氯仿洗脱去掉柱上的1-(2-硝基苯基)重氮乙烷疏水性副产物。在旋转蒸发器上干燥产物。冻干得到74mg笼闭化合物。总产率为57％。
聚合酶反应缓冲液(1mMTris-HCl pH8.8，5mM氯化钾，0.15mM氯化镁，0.01％(w/v)明胶)中每种核苷酸为0.2mM。
DNA测序
图1表示SPR传感系统和流体槽(7)，带有施加电磁辐射(1)至在传感器表面带有固定化的聚合酶(3)的传感器芯片(2)上装置，并带有使不同的核苷酸进入槽内的入口(4)和使脉冲单色光进入槽内的两个聚焦部件(5)和(6)。
在25℃和10Hz的数据收集速度下，使不同的核苷酸以30μl/分钟的流速进入流体槽(7)。当核苷酸经过聚焦部件(5)时，发射波长为260nm的单色光脉冲去除5’端的封闭基团。核苷酸然后流到传感器芯片(2)上，与该处被β-二聚体亚基固定的靶多聚核苷酸/聚合酶复合物接触。由于引物序列的3’端可以自由反应，核苷酸掺入到靶多聚核苷酸上的互补体时可发生聚合。再通过SPR设备中的p-极化的单色光检测该掺入。因为已掺入的核苷酸在3’端带有封闭基团，不会发生进一步的聚合。再在聚合位点通过聚焦部件(6)发射波长为360nm的单色光脉冲。流体槽内的高流速可确保未结合聚合酶的核苷酸在吸收足够能量以释放其3’端封闭基团之前从槽内流出。
一旦3’端的封闭基团从聚合的核苷酸中释放出来，可以发生进一步聚合。
图2表示测序实验中掺入到待测新生链中的每一核苷酸的结果。结果表明其顺序与SEQ ID No.1中的序列互补。
              序列表
(1)一般信息：
(i)申请人：
(A)姓名：Medical Biosystems Ltd.
(B)街道：The Old Mill，Beaston Cross
(C)城市：Broadhempston，Nr.Totnes
(D)州：Devon
(E)国家：英国
(F)邮政编码：TQ9 6BX
(ii)发明名称：核酸序列分析
(iii)序列数目：2
(v)计算机可读形式：
(A)介质类型：磁盘
(B)计算机：IBM兼容机
(C)操作系统：PC-DOS/MS-DOS
(D)软件：PatnetIn Release#1.0 Version#1.30(EPO)
(2)SEQ.ID.NO.1的信息：
(i)序列特征：
(A)长度：53个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑结构：线性
(ii)分子类型：其它核酸
(A)描述：/desc＝“寡核苷酸”
(xi)SEQ.ID.NO.1的序列描述：
CAAGGAGAGG ACGCTGTCTG TCGAAGGTAA GGAACGGACG AGAGAAGGGA GAG         53
(2)SEQ.ID.NO.2的信息：
(i)序列特征：
(A)长度：17个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑结构：线性
(ii)分子类型：其它核酸
(A)描述：/desc＝“寡核苷酸”
(xi)SEQ.ID.NO.2的序列描述：
CTCTCCCTTC TCTCGTC                                                 17

Claims (28)

1.一种多聚核苷酸的测序方法，包括步骤：(i)在聚合酶反应足以进行的条件下，使靶多聚核苷酸与固定在固体支持物上的聚合酶，以及不同的核苷酸进行反应；和(ii)检测与靶多聚核苷酸互补的特定核苷酸掺入后的作用结果。
2.权利要求1的方法，其中通过测量辐射来检测步骤(ii)的作用。
3.权利要求1或2的方法，其中轮流使用每种不同的核苷酸来进行步骤(i)和(ii)，直到掺入得以检测，然后重复上述步骤。
4.权利要求1或2的方法，其中在所有核苷酸均存在的情况下进行步骤(i)。
5.权利要求1的方法，其中核苷酸包括聚合酶反应后被去除的3’封闭基团。
6.权利要求5的方法，其中可通过脉冲单色光选择性去除封闭基团。
7.权利要求5或6的方法，其中核苷酸包括三磷酸链的末端磷酸基上的另一封闭基团，该封闭基团在3’封闭基团去除前被去除。
8.权利要求7的方法，其中上述另一封闭基团可在与去除3’封闭基团所需条件不同的条件下通过脉冲单色光选择性去除。
9.权利要求8的方法，其中上述另一封闭基团通过使用与去除3’封闭基团所需持续时间不同的脉冲单色光去除。
10.权利要求1的方法，其中步骤(i)还包括导入聚合酶的竞争性抑制剂。
11.权利要求1的方法，其中步骤(i)的靶多聚核苷酸通过β2二聚体复合物结合到聚合酶上。
12.权利要求1的方法，其中聚合酶为大肠杆菌DNA聚合酶III或T7聚合酶。
13.权利要求1的方法，其中聚合酶为Taq聚合酶。
14.权利要求1的方法，其中聚合酶为逆转录酶。
15.权利要求1的方法，其中步骤(ii)包括在一段时间内对共振信号变化的检测。
16.权利要求1的方法，其中辐射为电磁辐射。
17.权利要求16的方法，其中电磁辐射在红外光谱范围内。
18.权利要求1的方法，其中步骤(ii)包括使用表面等离子体共振。
19.权利要求16的方法，其中电磁辐射在射频光谱范围内。
20.权利要求19的方法，其中采用NMR检测核苷酸的掺入。
21.权利要求1的方法，其中多聚核苷酸为DNA。
22.权利要求1的方法，其中所述核苷酸为一种3’端和三磷酸链的末端磷酸基带有封闭基团的核苷酸，其中可通过不同波长的单色光去除这两种封闭基团。
23.权利要求22的方法，其中封闭基团衍生自式R1-[O-CO-]X的化合物，其中R1是光不稳定基团，X是离去基团。
24.权利要求22或23的方法，其中3’端的封闭基团是邻硝基苄基氧羰基。
25.权利要求22的方法，其中末端磷酸的封闭基团是邻硝基苄基。
26.权利要求22的方法，其中3’端的封闭基团是(4，5-二甲氧基-2-硝基苄基)氧羰基。
27.权利要求22的方法，其中末端磷酸的封闭基团是1-(2-硝基苯基)乙基。
28.一种用于权利要求1所述方法的多聚核苷酸的测序装置，包括光学传感器芯片、光源、成像设备和光检测器，其中传感器芯片含有固定于其上的聚合酶。
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