Combining fluorescent in situ hybridization with the comet assay for targeted examination of DNA damage and repair.
The comet assay is a simple and sensitive method for measuring DNA damage. Cells are embedded in agarose on a microscope slide, lysed, and electrophoresed; the presence of strand breaks allows the DNA to migrate, giving the appearance of a comet tail, the percentage of DNA in the tail reflecting the break frequency. Lesion-specific endonucleases extend the usefulness of the method to investigate different kinds of damage. DNA repair can be studied by treating cells with damaging agent and monitoring the damage remaining at intervals during incubation. An important feature of the assay is that damage is detected at the level of individual cells. By combining the comet assay with fluorescent in situ hybridization (FISH), using labeled probes to particular DNA sequences, we can examine DNA damage and repair at the level of single genes or DNA sequences. Here we provide protocols for the comet assay and the FISH modification, answer some technical questions, and give examples of applications of the technique.
Department of Nutrition, Faculty of Medicine, University of Oslo, Oslo, Norway.
Sergey
S
Shaposhnikov
Preben D
PD
Thomsen
Andrew R
AR
Collins
D000906:Antibodies; D001710:Biotin / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage; D015342:DNA Probes / Q000378:metabolism; D004260:DNA Repair; D004076:Digoxigenin / Q000378:metabolism; D004586:Electrophoresis; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009691:Nucleic Acid Denaturation; D012685:Sepharose; D012996:Solutions; D013194:Staining and Labeling
D000906:Antibodies; D015342:DNA Probes; D012996:Solutions; D001710:Biotin; D012685:Sepharose; D004076:Digoxigenin