The Comet Assay: A Sensitive Genotoxicity Test for the Detection of DNA Damage

The comet assay or single-cell gel (SCG) test is a microgel electrophoresis technique that measures DNA damage at the level of single cells. A small number of cells suspended in a thin agarose gel on a microscope slide is lysed, electrophoresed, and stained with a fluorescent DNA binding dye. Cells with increased DNA damage display increased migration of chromosomal DNA from the nucleus toward the anode, which resembles the shape of a comet. In its alkaline version, which is mainly used, DNA single-strand breaks, DNA double-strand breaks, alkali-labile sites, and single-strand breaks associated with incomplete excision repair sites cause increased DNA migration. On the other hand, crosslinks (DNA-DNA or DNA-protein) can lead to decreased DNA migration. Variations of the comet assay have been established for the detection of specific DNA base modifications. Here we describe the basic methodology of the alkaline comet assay, establishing a sensitive protocol for obtaining reproducible and reliable data. Applications of the comet assay for detecting DNA damage in individual cells are briefly reviewed.