The Comet Assay for DNA Damage and Repair: Principles, Applications, and Limitations

Abstract

The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.

Modulation of DNA damage/DNA repair capacity by XPC polymorphisms

“…During this 24-hour period, cells were continuously removing and repairing BPDE-induced DNA damages. Therefore, the level of DNA breaks in our study after 24-hour incubation should reflect the overall host NER capacity [21,25]. In support of this notion, we previously performed experiments using various XP cells deficient in XPC, XPA, or XPD genes.…”

“…It has been widely use to quantify DNA damages in peripheral blood lymphocytes (PBLs) challenged by mutagens such as BPDE, NNK, H 2 O 2 , and gamma-radiation [20]. Comet assay is also used to evaluate the in vitro host DRC [21]. In this study, to test the hypothesis that three common polymorphisms in XPC gene, Ala499Val (C→T), PAT (-/+), and Lys939Gln (A→C), modulate DRC, we determined the DNA damage/repair capacity in PBLs challenged by BPDE and gamma-radiation using alkaline comet assay in 476 healthy subjects.…”

Oxidative stress status of highly prolific sows during gestation and lactation

“…It has been reported that freezing of lymphocytes does not increase DNA strand breaks as compared to those of freshly isolated lymphocytes, and that fresh and frozen lymphocytes responded almost identically to hydrogen peroxide (Duthie et al, 2002). Visual scoring system used in this study reported to be accurately corresponding to the data that are measured by computer image analysis in human lymphocytes (Collins, 2004), in equine lymphocyte (Marlin et al, 2004) and in canine and feline leukocytes (Heaton et al, 2002). The method used for visual scoring involves classifying comets into four categories on the basis of the perceived length of migration and relative proportion of DNA in the tail.…”

Dose-Dependent Protective and Inductive Effects of Xanthohumol on Oxidative DNA Damage in Saccharomyces cerevisiae

“…The genome-protective eff ect of xanthohumol against oxidative DNA damage induced by H 2 O 2 was evaluated using the yeast comet assay. The yeast comet assay is a well-established, simple, versatile, sensitive and inexpensive technique used to assess DNA oxidative damage quantitatively and qualitatively in eukaryotic cell populations (30)(31)(32)(33). Yeast spheroplasts were incubated with diff erent xanthohumol concentrations and 10 mM H 2 O 2 in S buff er (containing 1 M sorbitol) in order to maintain osmotic protection.…”