title: Measurement of oxidatively-induced clustered DNA lesions using a novel adaptation of single cell gel electrophoresis (comet assay).
abstract: The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results.
authors.affiliation: Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, North Carolina, USA.
authors.forename: Alexandros G
authors.initials: AG
authors.lastname: Georgakilas
authors.forename: Stewart M
authors.initials: SM
authors.lastname: Holt
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Hair
authors.forename: Charles W
authors.initials: CW
authors.lastname: Loftin
mesh_terms: D000818:Animals; D002460:Cell Line; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage / Q000528:radiation effects; D006801:Humans; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011839:Radiation, Ionizing