Single-Cell Gel Electrophoresis (Comet) Assay: Foundations, Refinements and Applications in Genetic Toxicology

The single-cell gel electrophoresis (SCGE) assay, universally designated as the comet assay, has established itself as a foundational methodology across contemporary molecular toxicology, environmental biomonitoring, and regulatory genetic toxicology. By micro-electrophoretically resolving DNA damage within individual eukaryotic nuclei, the assay delivers exceptional analytical sensitivity, broad compatibility across proliferating and quiescent tissues, and direct visual assessment of cell-to-cell genomic heterogeneity. The alkaline adaptation (pH > 13) provides simultaneous detection of primary DNA single-strand breaks (SSBs), double-strand breaks (DSBs), alkali-labile sites (ALS), and transient enzymatic repair scission intermediates. Furthermore, incorporating lesion-specific repair endonucleases (such as Fpg and hOGG1) enables high-precision quantification of oxidative and alkylative purine/pyrimidine base damage. This comprehensive article details the fundamental biophysical mechanisms governing nucleoid unwinding and electrophoretic fragment migration, provides exhaustive step-by-step experimental optimization criteria, evaluates quantitative scoring metrics (% tail DNA, tail moment, tail length), addresses critical analytical confounders including cytotoxicity-induced hedgehogs, surveys high-throughput miniaturization platforms (12-gel arrays and microfabricated CometChips), and delineates regulatory validation under OECD Test Guideline 489.