The Comet-FISH Assay for the Analysis of DNA Damage and Repair
In this chapter, I describe the alkaline single-cell gel electrophoresis (Comet assay) combined with fluorescence in situ hybridization (FISH) technology, used in our laboratory, to study the incidence and repair of lesions induced in human cells by ultraviolet light. The Comet-FISH method permits the simultaneous and comparative analysis of DNA damage and its repair throughout the genome and in defined chromosomal regions. This very sensitive approach can be applied to any lesion, such as those induced by chemical carcinogens and products of cellular metabolism that can be converted to DNA single- or double-strand breaks. The unique advantages and limitations of the method for particular applications are discussed.
Key wordsDNA damage DNA repair Transcription-coupled repair Comet FISH Fluorescence Ultraviolet Cyclobutane pyrimidine dimers Single-cell gel electrophoresis Human fibroblasts
The author would like to thank Alexia Chollat-Namy and Rachel A. Cox for their patience and perseverance in developing the assay, and Phil Hanawalt for believing in the project. This work was supported by a grant CA91456 from NIH.
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