The comet assay: topical issues.
The comet assay is a versatile and sensitive method for measuring single- and double-strand breaks in DNA. The mechanism of formation of comets (under neutral or alkaline conditions) is best understood by analogy with nucleoids, in which relaxation of DNA supercoiling in a structural loop of DNA by a single DNA break releases that loop to extend into a halo-or, in the case of the comet assay, to be pulled towards the anode under the electrophoretic field. A consideration of the simple physics underlying electrophoresis leads to a better understanding of the assay. The sensitivity of the assay is only fully appreciated when it is calibrated: between one hundred and several thousand breaks per cell can be determined. By including lesion-specific enzymes in the assay, its range and sensitivity are greatly increased, but it is important to bear in mind that their specificity is not absolute. Different approaches to quantitation of the comet assay are discussed. Arguments are presented against trying to apply the comet assay to the study of apoptosis. Finally, some of the advantages and disadvantages of using the comet assay on lymphocyte samples collected in human studies are rehearsed.
Collins
Andrew R
AR
Department of Nutrition, Faculty of Medicine, University of Oslo, Oslo, Norway. a.r.collins@medisin.uio.no
Oscoz
Amaia Azqueta
AA
Brunborg
Gunnar
G
Gaivão
Isabel
I
Giovannelli
Lisa
L
Kruszewski
Marcin
M
Smith
Catherine C
CC
Stetina
Rudolf
R
D002138:Calibration; D002470:Cell Survival; D020552:Comet Assay / Q000379:methods* / Q000592:standards*; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D004272:DNA, Mitochondrial / Q000032:analysis; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry; D012680:Sensitivity and Specificity
D004272:DNA, Mitochondrial