DNA聚合酶反应产物的液相色谱-质谱分析
Liquid chromatography-mass spectrometry analysis of DNA polymerase reaction products.
作者信息
Chowdhury Goutam, Guengerich F Peter
机构信息
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
出版信息
Curr Protoc Nucleic Acid Chem. 2011 Dec;Chapter 7:Unit 7.16.1-11. doi: 10.1002/0471142700.nc0716s47.
Abstract
This unit describes experimental and analytical procedures for characterizing the efficiency and fidelity of translesion DNA synthesis across various DNA damages by DNA polymerases in vitro. This procedure utilizes primer extension assays followed by LC-MS and LC-MS/MS analysis of the extension products. Detailed explanations for the analysis of the LC-MS/MS data for deciphering the nucleotide sequences of the DNA fragments are also presented. This approach provides a significant improvement over conventional methods, as it allows detection of misincorporation, as well as frameshift products.
摘要
本单元描述了用于在体外表征DNA聚合酶跨各种DNA损伤进行跨损伤DNA合成的效率和保真度的实验和分析程序。该程序利用引物延伸测定法，随后对延伸产物进行液相色谱-质谱（LC-MS）和液相色谱-串联质谱（LC-MS/MS）分析。还介绍了用于解读DNA片段核苷酸序列的LC-MS/MS数据分析的详细解释。这种方法比传统方法有显著改进，因为它能够检测错配掺入以及移码产物。
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