利用轮廓钳位均匀电场（CHEF）系统检测哺乳动物细胞中DNA损伤诱导的双链断裂

Detection of DNA Damage-Induced DSBs by the Contour-Clamped Homogeneous Electric Field (CHEF) System in Mammalian Cells.

作者信息

Takiguchi Yuri, Kariyazono Ryo, Ohta Kunihiro

机构信息

Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.

出版信息

Methods Mol Biol. 2020;2119:101-109. doi: 10.1007/978-1-0716-0323-9_9.

DOI:10.1007/978-1-0716-0323-9_9

PMID:31989518

Abstract

Double-strand breaks (DSBs) and their repair mechanisms are essential for normal cell life. However, quantitative analysis of DSBs on mammalian whole chromosomes remains difficult. The method described here enables the quantitative detection of mammalian chromosomal DSBs by pulsed-field gel electrophoresis (PFGE) using a contour-clamped homogeneous electric field (CHEF). We illustrate this method by measuring DNA damage-induced DSBs in mammalian cells. The electrophoresis conditions presented here enabled the visualization of fragmented DNA (several mega-base pairs down to 500 kbp) as a single band. Using this protocol, about 10-45 samples can be analyzed on a single gel, depending on the direction of electrophoresis.

摘要

双链断裂（DSB）及其修复机制对于正常细胞生命至关重要。然而，对哺乳动物整条染色体上的DSB进行定量分析仍然困难重重。本文所述方法能够通过使用轮廓夹钳均匀电场（CHEF）的脉冲场凝胶电泳（PFGE）对哺乳动物染色体DSB进行定量检测。我们通过测量哺乳动物细胞中DNA损伤诱导的DSB来说明该方法。此处给出的电泳条件能够将片段化DNA（从几兆碱基对到500千碱基对）以单一条带的形式可视化。使用该方案，根据电泳方向，在一块凝胶上大约可以分析10 - 45个样品。