Single cell gel electrophoresis: detection of DNA damage at different levels of sensitivity.
Single cell gel electrophoresis, also known as the comet assay, is widely used for the detection and measurement of DNA strand breaks. With the addition of a step in which DNA is incubated with specific endonucleases recognising damaged bases, these lesions can be measured, too. In the standard protocol, electrophoresis is carried out at high pH. If, instead, electrophoresis is in neutral buffer, the effect of DNA damage seems to be much reduced--either because alkaline conditions are needed to reveal certain lesions, or because the effect of the same number of breaks on DNA migration is greater at high pH. A lower sensitivity can be useful in some circumstances, as it extends the range of DNA damage levels over which the assay can be used. Here we compare the performance of standard and modified techniques with a variety of DNA-damaging agents and offer possible explanations for the differences in behaviour of DNA under alternative electrophoretic conditions.
Angelis
K J
KJ
Institute of Experimental Botany of Czech Academy of Sciences, Praha, Czech Republic.
Dusinská
M
M
Collins
A R
AR
D002021:Buffers; D004249:DNA Damage / Q000187:drug effects; D043382:DNA-Formamidopyrimidine Glycosylase; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007887:Fabaceae / Q000235:genetics; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D010946:Plants, Medicinal; D011740:Pyrimidine Dimers / Q000032:analysis; D011759:Pyrrolidines / Q000494:pharmacology; D011807:Quinolizines / Q000494:pharmacology; D012680:Sensitivity and Specificity; D014812:Vitamin K / Q000494:pharmacology
D002021:Buffers; D011740:Pyrimidine Dimers; D011759:Pyrrolidines; D011807:Quinolizines; C076809:Ro 19-8022; D014812:Vitamin K; D008741:Methyl Methanesulfonate; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase