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The comet assay (single‐cell gel electrophoresis) is a simple method for measuring DNA strand breaks in cells that are embedded in agarose and lysed to remove membranes and soluble cell constituents (including most histones). The assay depends on the ability of breaks to relax supercoiling, which allows DNA still attached to a nuclear matrix to move toward the anode under electrophoresis, forming a ‘comet tail’ when viewed by fluorescence microscopy. The percentage of DNA in the tail indicates the frequency of DNA breaks. An additional step — digestion with lesion‐specific endonucleases — is introduced after lysis in order to detect different kinds of DNA damage. The assay can be calibrated to give quantitative measures of DNA damage. It has been widely used in genotoxicity testing and human biomonitoring, and also in ecogenotoxicology, as well as in basic research. We here discuss the development of the method, its principles, applications, and limitations, and attempt to dispel some fallacies that trouble the assay. We provide a detailed protocol, including a recent modification that increases the assay's throughput.

DNA repair pathways provide a critically important cellular defence system, effectively protecting us from mutations and cancer. Distinct pathways deal with various classes of damage: single-and double-strand breaks, oxidized and alkylated bases, bulky adducts, intra-and inter-strand cross-links. A simple approach to measuring the DNA repair capacity of cell lines, or samples of blood cells, for instance, is the cellular repair assay, or challenge assay, in which cells are treated with a specifi c DNA-damaging agent, and incubated; at intervals, samples are taken and the residual damage is measured. The comet assay is well suited for measuring strand break rejoining, excision repair of oxidized or alkylated bases (with a lesion-specifi c endonuclease to convert the altered bases into breaks), and nucleotide excision repair of UV-induced lesions (again, using an appropriate enzyme to detect the damage). An alternative way to assess nucleotide excision repair capability is the incision assay: after UV irradiation, cells are incubated with inhibitors of repair DNA synthesis, so that incomplete repair sites accumulate as DNA breaks.We provide protocols for these DNA repair assays, and discuss their applications-and their limitations. We also raise some important, so far unanswered questions concerning the regulation of repair, and the factors that might account for the wide variations seen in individual repair capacities.