Measurement of DNA Double-Strand Breaks in Mammalian Cells by Pulsed-Field Gel Electrophoresis: A New Approach Using Rarely Cutting Restriction Enzymes

A new method is presented for measuring DNA double-strand breaks (DSBs) which combines the technique of pulsed-field gel electrophoresis (PFGE) with the idea of velocity sedimentation. Purified DNA samples were treated with restriction enzymes, which results in DNA of sizes which can be separated by PFGE. After electrophoresis, unirradiated DNA shows a size distribution (obtained with the help of a specially developed software program) similar to that obtained with the sedimentation technique; with X irradiation, this distribution is shifted to lower molecular weight with increasing dose. The rate of DSB induction was calculated by comparing the curves obtained experimentally with theoretical distributions (based on the assumption that breaks are formed according to Poisson statistics). The method was tested by measuring X-ray-induced DSBs in P3 (derived from human epithelial teratoma) cells. The induction of DSBs was found to be linear with dose and a rate of 5.4 x 10(-3)/Mbp/Gy was obtained.