使用琼脂糖包埋免疫染色(TARDIS)分析法对拓扑异构酶-DNA共价复合物进行可视化和定量分析。

Visualization and Quantification of Topoisomerase-DNA Covalent Complexes Using the Trapped in Agarose Immunostaining (TARDIS) Assay.

作者信息

Cowell Ian G, Austin Caroline A

机构信息

Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.

出版信息

Methods Mol Biol. 2018;1703:301-316. doi: 10.1007/978-1-4939-7459-7_21.

Abstract

The TARDIS assay was originally developed as a means of detecting and quantifying melphalan and cisplatin DNA adducts at the single cell level, but it has since been adapted to quantify topoisomerase DNA complexes that result from the actions of topoisomerase poisons and this is currently the main use of the assay. The method employs sensitive immunofluorescent detection to quantify topoisomerase molecules covalently coupled to DNA in what are often referred to as cleavage complexes. Free topoisomerase molecules, and other cellular constituents are first removed using salt-detergent extraction of agarose-embedded, unfixed cells. Using these stringent extraction conditions, genomic DNA remains in place in the agarose as "nuclear ghosts," and any covalent attached molecules can be detected and quantified by immunofluorescence with a low background.

摘要

TARDIS分析最初是作为一种在单细胞水平检测和定量美法仑和顺铂DNA加合物的方法而开发的,但后来它被用于定量由拓扑异构酶毒物作用产生的拓扑异构酶DNA复合物,这也是目前该分析的主要用途。该方法采用灵敏的免疫荧光检测来定量与DNA共价偶联的拓扑异构酶分子,这些分子通常存在于所谓的裂解复合物中。首先使用盐-去污剂对琼脂糖包埋的未固定细胞进行提取,去除游离的拓扑异构酶分子和其他细胞成分。在这些严格的提取条件下,基因组DNA作为“核幽灵”保留在琼脂糖中,任何共价连接的分子都可以通过免疫荧光检测并定量,背景较低。

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