Single-cell gel electrophoresis (the comet assay): loops or fragments?
Single-cell gel electrophoresis, or the comet assay, is widely used to measure DNA damage and repair. Upon electrophoresis, the DNA of lysed, agarose-embedded cells known as nucleoids, extends towards the anode in a structure resembling a comet, the relative intensity of the tail reflecting the frequency of DNA breaks. The structural organization of the DNA within comet preparations is not fully understood. We have used fluorescent in situ hybridization with large-insert genomic probes and human Cot-I DNA to investigate whether the production of the comet tail is simply explained by the relaxation of supercoiled DNA loops. We find that, under neutral electrophoresis conditions, when the tail and head DNA are double-stranded, the probed sequence of DNA is seen as a linear array, consistent with extension from a fixed point on the nuclear core or matrix. After alkaline electrophoresis, the appearance of the fluorescent probes suggests that linear DNA has coalesced into a granular form.
Shaposhnikov
Sergey A
SA
Department of Nutrition, Faculty of Medicine, University of Oslo, Oslo, Norway. sergey.shaposhnikov@medisin.uio.no
Salenko
Veniamin B
VB
Brunborg
Gunnar
G
Nygren
Jonas
J
Collins
Andrew R
AR
D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage; D015342:DNA Probes; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017404:In Situ Hybridization, Fluorescence; D014466:Ultraviolet Rays
D015342:DNA Probes; D006861:Hydrogen Peroxide