Comet-FISH for Ultrasensitive Strand-Specific Detection of DNA Damage in Single Cells

The genome integrity of living organisms is constantly threatened by endogenous cellular metabolic processes and environmental agents. To quantify these low, physiologically relevant levels of DNA damage, a single-cell gel electrophoresis (comet) combined with strand-specific fluorescence in situ hybridization (FISH)-based approach has been developed. This approach enables the quantification of low levels of specific DNA lesions in each strand of the selected sequence at the single-molecule sensitivity, as well as in the genome overall in single cells. In this method, the percentage of DNA in the comet tail is used to quantify lesions in the genome overall. Lesions in the respective strands of the designated sequence are analyzed using strand-specific FISH probes. These probes targeting the 3' and 5' termini of the selected sequence are conjugated with two distinct fluorophores. Following the comet-FISH assay, the two termini of the designated sequence are visualized as two spots with different colors, under a fluorescence microscope. Separated spots indicate a damage strand, while adjacent or colocalized spots imply an intact strand. Any DNA lesions or DNA modifications, which can be converted into strand breaks enzymatically or chemically, can be quantified by this method. The comet-FISH approach described here can be applied to the study of the molecular mechanisms of various repair pathways, as well as in drug screening to develop inhibitors for specific repair pathways.