Comet Assay for the Detection of Single and Double-Strand DNA Breaks

Abstract

DNA damage is a common event that occurs during the life span of the majority of cells, as a result of aberrant energy metabolism, exposure to radiation, DNA damaging chemicals, and viral infections. Rapid, sensitive, and economical methods for the detection of DNA damage offer valuable insights into DNA repair, drug genotoxicity, and biomonitoring. The comet assay, or single cell electrophoresis, has emerged as a popular method for detecting single-and double-stranded DNA breaks in single cells. This experimental technique is based on the fact that, when exposed to an electric current, damaged DNA fragments in a gel, migrate farther from the nucleus toward the anode resulting in the shape of a comet. This assay is versatile, quantitative, simple to perform and exhibits high sensitivity; however, consistent experimental conditions must be maintained to ensure assay reproducibility. Electrophoresis can be performed under neutral conditions to detect solely double stranded breaks, or under alkaline conditions to detect both single-and double-stranded breaks. This chapter describes the protocol for both alkaline and neutral comet assays.

DNA damage in prepared semen is associated with fertilisation rate in assisted reproduction technology (ART) treatment

“…The differences between these studies might re ect analytical and population differences but it is unclear to what extent (if any) the differences might also re ect the neutral and alkaline Comet assays measuring different types of DNA damage. The type of DNA damage detected by either the neutral or the alkaline comet assay has not been fully characterised, but it has been suggested that the neutral Comet assay detects double strand DNA breaks better than the alkaline Comet assay [3,13]. This, though, is controversial with other authors reporting that the neutral comet assay measures single strand nicks when the DNA remains attached to the nuclear matrix [36].…”

“…The contribution then that each type of damage makes to the overall response is unclear. Other variants of this assay have been described such as the enzyme modi ed Comet assay [12] which detects strand breaks induced by enzymatic removal of DNA base damage and the neutral comet assay which has recently been described as detecting double strand breaks better than the alkaline Comet assay [3,13].…”

The relationship between sperm nuclear DNA fragmentation, mitochondrial DNA fragmentation, and copy number in normal and abnormal human ejaculates

“…4,13 These assays do not distinguish if the DNA fragmentation of an individual sperm cell is only due to SSBs (SDF-SSBs) or DSBs (SDF-DSBs). 3,4,14 The presence and differentiation of DSBs, may however, be detected using the non-denaturing comet assay, 15 or a more recently reported variant of the SCD test (DSBs-SCD), where DNA fragments spread through passive diffusion. 16 The subtraction of SDF-DSBs value from that of global SDF obtained with the standard SCD test can thereby provide an estimate of the SSBs' sub-population.…”