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Why do I sometimes see images resembling halos, rather than comets, even when there is sufficient damage to produce tails?
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Do I need to buy image analysis software?
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Can the comet assay be used to measure DNA repair?
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icaworkgroup
Core Group
May 11, 2020
Yes. There are several approaches. The simplest is to treat cells with damaging agent and measure the damage remaining (as strand breaks or enzyme-sensitive sites) at intervals during incubation. Thus you can measure the kinetics of cellular repair after very low (‘physiological’) doses of DNA damage. Another method is the in vitro repair assay – a biochemical approach which measures the repair capacity. A substrate is prepared, of cells treated with an agent delivering an excess of damage of the relevant sort for the enzyme of interest. An extract is prepared from the cells whose repair capacity is to be investigated, and this extract is incubated with the substrate, for 10 or 20 min. Breaks accumulate as the incision events take place. Rates of repair vary between individuals. Also, inhibitors of DNA synthesis (hydroxyurea plus cytosine arabinoside or aphidicolin) may be used to block the resynthesis step of nucleotide excision repair, causing breaks to accumulate; these give a measure of repair capacity, since incision is the rate-limiting step. Some hints: Pre-incubate cells for 15-30 min before giving damage; do not incubate for longer than 20-30 min after treatment (as breaks do not accumulate linearly after that); beware of the rapid rejoining of breaks when inhibitors are removed (so scraping may be better than trypsinisation to harvest the cells). "FAQ by Andrew Collins, Gunnar Brunborg and Jonas Nygren, 2006, NewGeneris FP7-project"
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Do you need to use alkaline conditions to see single strand breaks?
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icaworkgroup
Core Group
May 11, 2020
No. Other methods, such as alkaline unwinding, depend on the high pH to unravel the DNA molecules, starting at DNA breaks; the rate of unravelling depends on the frequency of breaks, and is assessed as the proportion of single-stranded DNA present after a certain unwinding time. But the comet assay is different. The original comet paper (Östling and Johanson, 1984; BBRC 123, 291-298) used pH 10 (effectively neutral – certainly not high enough to unwind DNA), and reported the effect of low doses of ionising radiation. The dose range giving detectable damage was almost the same as that reported for the alkaline version by Singh et al. (1988; Exp. Cell Res. 175, 184-191). The most likely explanation is in terms of supercoiling. The DNA in the nucleoids (the structures remaining after lysis with Triton X-100 and 2.5 M salt) is supercoiled in a negative sense. Imagine the DNA as a series of supercoiled loops, associated at their bases to the nuclear matrix (a scaffold of protein and RNA which supports the DNA). Comets from undamaged cells have tightly packed, supercoiled DNA and no tail. Damage that leads to DNA breaks relaxes the supercoiling in the loops with breaks; these loops relax, and are pulled into a tail under electrophoresis. A single-strand break is sufficient to relax supercoiling. So the neutral and the alkaline comet assays detect both single- and double-strand breaks, and it is impossible to distinguish between them. "FAQ by Andrew Collins, Gunnar Brunborg and Jonas Nygren, 2006, NewGeneris FP7-project"
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