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

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 gamma-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 gamma-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.