DNA integrity under alkaline conditions: An investigation of factors affecting the comet assay

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

“…Even if other strategies have been described to monitor both SSBs and DSBs in modified protocols for the comet assay, including the so called two-tailed comet assay ( 18–21 ). However, not only our own work ( 14 ), but also findings reported by other research groups ( 22–24 ) have shown that SSBs and DSBs cannot be clearly distinguished by using the comet assay, neither under neutral, nor under alkaline conditions, when used in combination with conventional staining techniques. Therefore, the herein described approach has, to the best of our knowledge, never been used before to distinguish between SSBs and DSBs in comets after alkaline single-cell gel electrophoresis.…”

“…It would therefore be beneficial if it was possible to distinguish between SSBs and DSBs in comet tails when evaluating an induced increase in the level of DNA damage. It is often assumed that a comet assay performed under more neutral conditions (pH 10) will capture DSBs selectively, but as we recently have shown when investigating the DNA integrity at different pH (10, 12.5 or >13), this is not the case ( 14 ). Also, the so-called neutral version of the comet assay will show a mixture of SSBs and DSBs, even if the DSBs will dominate.…”