Measurement of incorporation kinetics of non-fluorescent native nucleotides by DNA polymerases using fluorescence microscopy.
Measurement of incorporation kinetics of non-fluorescent native nucleotides by DNA polymerases using fluorescence microscopy.
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DOI：
10.1093/nar/gkx833
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发表时间：
2017-12-01
影响因子：
14.9
通讯作者：
Huang X
中科院分区：
文献类型：
--
作者：
Walsh MT;Huang X
我们描述了一种通过DNA聚合酶测量非荧光天然核苷酸的单周转掺入动力学的方法。延时全内反射荧光（TIRF）显微镜用于直接测量单碱基核苷酸掺入到引发的DNA模板的动力学共价连接到盖玻片的表面，使用固定比例的天然核苷酸和相应的荧光标记的核苷酸在一系列的总核苷酸浓度。标记的核苷酸的存在允许通过待监测的DNA聚合酶的竞争性掺入反应的动力学。单周转催化速率常数和米氏常数的掺入的天然核苷酸可以通过建模的平行竞争反应的动力学来确定。我们的方法能够测量掺入天然或其他非荧光核苷酸的动力学参数，而不使用快速停流或猝灭流仪器，以及通常更多涉及和较少定量反应产物的反应后分析。作为我们的方法的演示，我们系统地确定了所有四个天然核苷酸和一组Cy3标记的核苷酸的大肠杆菌DNA聚合酶I的Klenow片段的单周转掺入动力学。
We describe a method for measuring the single-turnover incorporation kinetics of non-fluorescent native nucleotides by DNA polymerases. Time-lapse total internal reflection fluorescence (TIRF) microscopy is used to directly measure the kinetics of single-base nucleotide incorporation into primed DNA templates covalently attached to the surface of a glass coverslip using a fixed ratio of a native nucleotide and a corresponding fluorescently labeled nucleotide over a series of total nucleotide concentrations. The presence of a labeled nucleotide allows for the kinetics of competitive incorporation reactions by DNA polymerase to be monitored. The single-turnover catalytic rate constants and Michaelis constants of the incorporation of the native nucleotides can be determined by modeling the kinetics of the parallel competitive reactions. Our method enables the measurements of the kinetics parameters of incorporation of native or other non-fluorescent nucleotides without using a rapid stopped-flow or quench-flow instrument and the generally more involved and less quantitative post-reaction analysis of the reaction products. As a demonstration of our method, we systematically determined the single-turnover incorporation kinetics of all four native nucleotides and a set of Cy3-labeled nucleotides by the Klenow fragment of Escherichia coli DNA polymerase I.
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影响因子：
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