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

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.