A FILTER ELUTION ASSAY FOR THE SIMULTANEOUS DETECTION OF DNA DOUBLE AND SINGLE-STRAND BREAKS

This paper reports a gravity-flow filter elution assay that permits th e simultaneous detection of DNA double strand breaks (DSBs, as neutral elutable DNA) and DNA single strand breaks (SSBs, as alkaline elutabl e DNA). This assay is based on a modification of a conventional gravit y-flow alkaline elution procedure. The elution fraction resulting from cell lysis before the application of the alkaline elution buffer prov ides an estimate of DNA DSBs, while the fraction eluted by the alkalin e buffer (pH 12.3) measures the extent of DNA SSBs. Data show that, in addition to its good detection sensitivity for SSBs, this method prov ides a sensitive measure of DNA DSBs. As an illustration of its specif icity, this method unambiguously distinguished DNA SSBs induced by X-i rradiation of mouse leukemia L1210 cells from DNA DSBs occurring in ce lls undergoing nucleosomal DNA fragmentation and apoptosis. The neutra l elution fraction is a good predictor of loss of cell viability induc ed either by selenite at pharmacological levels or by growth factor de pletion. The filter elution method reported is more specific and infor mative than a nick translation assay for the detection of DNA strand b reaks. (C) 1996 Academic Press, Inc.