Assessment of DNA damage in filamentous fungi by single cell gel electrophoresis, comet assay

The single cell gel electrophoresis (SCGE) assay, or comet assay, is a highly sensitive method to measure DNA damage. The principle of the assay is based on the conversion of single-strand breaks and alkali-labile sites to double-strand breaks via alkaline lysis. The cells are subjected to electrophoresis, stained, and microscopically evaluated. Increasing damage leads to increasing migration of DNA from the nuclei. Experiments were conducted with Sordaria macrospora grown for 3 d directly on agarose minigels in order to examine the utility of the SCGE assay for the detection of effects caused by a variety of DNA-damaging agents. When the mycelia were exposed to a variety of genotoxic conditions and analyzed by SCGE, DNA strand breaks were detected by an increase in the DNA migration distance from the nucleus. The results show that this assay allows for a fast and sensitive detection of a wide spectrum of DNA-damaging effects.