Application of Programmable, Autonomously Controlled Electrode (PACE) Technology to the Development of an Improved Pulsed Field Gel Electrophoresis Assay for DNA Double-strand Breaks in Mammalian Cells

Abstract

PACE (programmable, autonomously controlled electrodes) pulsed field gel electrophoresis technology was used to develop a DNA double-strand break assay that simultaneously combined (1) resolution across a broad range of DNA sizes, (2) high sensitivity (in terms of detecting dsb at low doses) and (3) speed. A 48 h PACE/dsb assay resolves DNA fragments ranging in size from 0·2 to 6 Mb, and detects damage induced by as little as 2 Gy of γ-radiation. A different set of electrophoretic conditions resolves DNA between 1·5 and 6 megabases in 23 h, with a detection limit of about 5 Gy. A third electrophoretic protocol, while not resolving DNA fragments greater than 50 kb in length, detects DNA dsb in CHO cells induced by 15 Gy or more of γ-rays after only a 1 h run. In particular, the 48 h PACE/dsb assay should prove useful in studies aimed at understanding the mechanisms whereby a variety of biological, chemical and physical agents induce DNA dsb in eukaryotes.