Single‐cell gel electrophoresis (the comet assay): Loops or fragments?

Abstract

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.

DNA Repair Measured by the Comet Assay

“…Based on our hypothesis that DNA organisation in comets reflects the DNA loop organization in living cells, it is reasonable to expect that the number of signals detected in comets will be related to the number of signals observed on chromosome spread preparations. Our results with chromosome 16 probes confirmed this hypothesis: twice as many signals were observed in the alkaline version of the assay relative to the number seen under neutral conditions (Shaposhnikov et al, 2008). This can be explained by DNA denaturation in alkaline comets since each strand of DNA will act as a target for the FISH probe.…”

SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

“…To further examine the cause of MVC infection-induced DDR, we asked whether virus infection is able to cause cellular DNA damage. To this end, we performed a Comet assay, which is commonly used for the detection of both DSBs and SSBs of chromosome DNA (65)(66)(67)(68). H 2 O 2 treatment, as a control, was able to cause severe damage to cellular DNA as shown by the fact that nearly all the cells were Comet positive (Comet ϩ ; DNA damaged); however, neither mock-nor MVC-infected cells contained Comet ϩ cells at a level of over 1% (Fig.…”

Genotoxic potential of dental bulk-fill resin composites

“…To quantify DNA damage by means of the comet assay, the parameters tail length (µm) and tail intensity (% DNA) are most frequently used. Tail length determines the length of DNA migration and is directly related to DNA fragment size and the extent of DNA damage, whereas tail intensity denotes the number of DNA fragments, which directly indicates the proportion of the genome affected by the damage [25].…”