Comparative measurements of radiation-induced DNA double-strand breaks by graded voltage and pulsed-field gel electrophoresis.

Abstract

We compared the ability of graded-voltage gel electrophoresis (GVGE) and pulsed-field gel electrophoresis (PFGE), to resolve DNA containing double-strand breaks (dsb) induced in human MS751 cells after exposure to gamma-radiation. For quantitation, prelabelling of the cells with [2-14C]-thymidine prior to electrophoresis and subsequent scintillation counting of excised bands was found to give closely similar results to Southern blotting and radiolabelled probe hybridization followed by phosphorimager quantitation. Compared with PFGE, DNA subjected to GVGE migrated further and generated distinct DNA bands. Dsb were detected and quantifiable with GVGE at much lower doses than with PFGE the radiation dose limits were approximately 2 and 10 Gy respectively. At all doses used, the amounts of dsb detected by GVGF, were higher than those by PFGE. GVGE coupled with Southern hybridization and phosphorimager analysis is thus a more sensitive approach to assessing dsb and their relationship with radiation sensitivity. The approach is also convenient when processing large numbers of samples and has the additional advantage of avoiding cell prelabelling such as in the case of cells extracted directly from human tumour biopsies and normal tissues.