This paper reports a gravity-flow filter elution assay that permits the simultaneous detection of DNA double strand breaks (DSBs, as neutral elutable DNA) and DNA single strand breaks (SSBs, as alkaline elutable DNA). This assay is based on a modification of a conventional gravity-flow alkaline elution procedure. The elution fraction resulting from cell lysis before the application of the alkaline elution buffer provides an estimate of DNA DSBs, while the fraction eluted by the alkaline buffer (pH 12.3) measures the extent of DNA SSBs. Data show that, in addition to its good detection sensitivity for SSBs, this method provides a sensitive measure of DNA DSBs. As an illustration of its specificity, this method unambiguously distinguished DNA SSBs induced by X-irradiation of mouse leukemia L1210 cells from DNA DSBs occurring in cells undergoing nucleosomal DNA fragmentation and apoptosis. The neutral elution fraction is a good predictor of loss of cell viability induced either by selenite at pharmacological levels or by growth factor depletion. The filter elution method reported is more specific and informative than a nick translation assay for the detection of DNA strand breaks.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology