The comet assay, or single-cell gel electrophoresis (SCGE), is a simple and sensitive method for detecting DNA strand breaks in eukaryotic cells. Cells are embedded in agarose, lysed with detergent and high salt, and then subjected to electrophoresis. The resulting structures, resembling comets, are observed under fluorescence microscopy, with the intensity of the comet tail reflecting the number of DNA breaks. The assay can be enhanced by incubating nucleoids with bacterial repair endonucleases that recognize specific DNA lesions, increasing the amount of DNA in the tail. The comet assay has applications in genotoxicity testing, human biomonitoring, molecular epidemiology, and DNA damage and repair research. It can detect various types of DNA damage, including UV-induced pyrimidine dimers, oxidized bases, and alkylation damage. The assay can also monitor DNA repair by measuring damage remaining after treatment with a damaging agent or by measuring repair activity in cell extracts. The comet assay is commonly used for animal cells but has also been adapted for plant cells. Variants include alkaline and neutral SCGE, as well as the use of lesion-specific enzymes to detect specific DNA lesions. The comet assay can also be used to detect replicating DNA and intermediates in DNA repair. It is not a reliable indicator of apoptosis, as cells with severe DNA damage can repair and no longer show comet-like structures. The comet assay can also be used to assess DNA repair capacity and to detect DNA damage in human studies, including biomonitoring, nutritional studies, and diagnosis. The assay is sensitive and can detect DNA damage at low levels, but it is important to ensure proper calibration and standardization for accurate results. The comet assay is a valuable tool for studying DNA damage and repair in various contexts, including environmental monitoring, human health, and cancer research.The comet assay, or single-cell gel electrophoresis (SCGE), is a simple and sensitive method for detecting DNA strand breaks in eukaryotic cells. Cells are embedded in agarose, lysed with detergent and high salt, and then subjected to electrophoresis. The resulting structures, resembling comets, are observed under fluorescence microscopy, with the intensity of the comet tail reflecting the number of DNA breaks. The assay can be enhanced by incubating nucleoids with bacterial repair endonucleases that recognize specific DNA lesions, increasing the amount of DNA in the tail. The comet assay has applications in genotoxicity testing, human biomonitoring, molecular epidemiology, and DNA damage and repair research. It can detect various types of DNA damage, including UV-induced pyrimidine dimers, oxidized bases, and alkylation damage. The assay can also monitor DNA repair by measuring damage remaining after treatment with a damaging agent or by measuring repair activity in cell extracts. The comet assay is commonly used for animal cells but has also been adapted for plant cells. Variants include alkaline and neutral SCGE, as well as the use of lesion-specific enzymes to detect specific DNA lesions. The comet assay can also be used to detect replicating DNA and intermediates in DNA repair. It is not a reliable indicator of apoptosis, as cells with severe DNA damage can repair and no longer show comet-like structures. The comet assay can also be used to assess DNA repair capacity and to detect DNA damage in human studies, including biomonitoring, nutritional studies, and diagnosis. The assay is sensitive and can detect DNA damage at low levels, but it is important to ensure proper calibration and standardization for accurate results. The comet assay is a valuable tool for studying DNA damage and repair in various contexts, including environmental monitoring, human health, and cancer research.