DNA聚合酶单核苷酸掺入产物的稳态动力学分析。
Steady-state kinetic analysis of DNA polymerase single-nucleotide incorporation products.
作者信息
O'Flaherty Derek K, Guengerich F Peter
机构信息
Department of Chemistry and Biochemistry, Concordia University, Montréal, Québec, Canada.
出版信息
Curr Protoc Nucleic Acid Chem. 2014 Dec 12;59:7.21.1-13. doi: 10.1002/0471142700.nc0721s59.
Abstract
This unit describes the experimental procedures for the steady-state kinetic analysis of DNA synthesis across DNA nucleotides (native or modified) by DNA polymerases. In vitro primer extension experiments with a single nucleoside triphosphate species followed by denaturing polyacrylamide gel electrophoresis of the extended products is described. Data analysis procedures and fitting to steady-state kinetic models is presented to highlight the kinetic differences involved in the bypass of damaged versus undamaged DNA. Moreover, explanations concerning problems encountered in these experiments are addressed. This approach provides useful quantitative parameters for the processing of damaged DNA by DNA polymerases.
摘要
本单元描述了通过DNA聚合酶对跨DNA核苷酸（天然或修饰的）的DNA合成进行稳态动力学分析的实验程序。描述了使用单一核苷三磷酸物种进行体外引物延伸实验，随后对延伸产物进行变性聚丙烯酰胺凝胶电泳。介绍了数据分析程序以及对稳态动力学模型的拟合，以突出损伤DNA与未损伤DNA绕过过程中涉及的动力学差异。此外，还讨论了这些实验中遇到的问题的解释。这种方法为DNA聚合酶处理损伤DNA提供了有用的定量参数。
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