Evaluation of DNA damage using single-cell gel electrophoresis (Comet Assay)

Detection of deoxyribonucleic acid (DNA) damage at the level of an individual eukaryotic cell warrants high significance in the fields of toxicology, pharmaceuticals, genotoxicity testing, environmental/ human bio-monitoring, diagnosis of genetic disorders etc. Single cell gel electrophoresis (SCGE) or the comet assay is a versatile, sensitive yet simple and economical technique used to measure DNA damage and repair in individual cells. The comet assay helps to measure the single/ double-strand DNA breaks, alkali labile sites (apurinic/ apyrimidinic sites), DNA cross-links, base/ base-pair damages and apoptotic nuclei in the cells. In 1984, Ostling and Johnson demonstrated migration of DNA strands from nuclei which were exposed to an electric field under neutral conditions. [1] Later, in 1988, Singh and his co-workers modified and optimized this procedure using alkaline conditions which substantially increased its specificity and reproducibility.[2] Since then SCGE has gained huge popularity and evolved as a standard technique for evaluation of DNA damage/repair. There has been constant modification and innovations in the protocol which has led to an array of comet assay variants [Table 1].[3] A wide range of samples including peripheral blood, cultured cells, buccal mucosal cells, solid tumor, cancer cells, sperm, yeast cells, bacteria etc., can be subjected to SCGE which makes the assay more versatile. The most widely used method for assessment of DNA damage is the alkaline comet assay; hence this article describes the various steps involved in the alkaline comet assay using peripheral blood mononuclear cells.