Visualizing DNA single- and double-strand breaks in the Flash comet assay by DNA polymerase-assisted end-labelling.

In the comet assay, tails are formed after single-cell gel electrophoresis if the cells have been exposed to genotoxic agents. These tails include a mixture of both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). However, these two types of strand breaks cannot be distinguished using comet assay protocols with conventional DNA stains. Since DSBs are more problematic for the cells, it would be useful if the SSBs and DSBs could be differentially identified in the same comet. In order to be able to distinguish between SSBs and DSBs, we designed a protocol for polymerase-assisted DNA damage analysis (PADDA) to be used in combination with the Flash comet protocol, or on fixed cells. By using DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides. Herein, TK6-cells or HaCat cells were exposed to either hydrogen peroxide (H2O2), ionising radiation (X-rays) or DNA cutting enzymes, and then subjected to a comet protocol followed by PADDA. PADDA offers a wider detection range, unveiling previously undetected DNA strand breaks.