I would be studying the cell line and in one experiment the correlation of the damage to the cell ( DNA treatment) should be correlated to the concentration of the dose. I do not have western blotting available. Can anyone suggest a unique marker of damage that can be visualised by microscopy?
I am thinking of using gamma h2a.x but are any other alternatives available?
yH2AX and TUNEL stain double strand breaks. 53BP1 is a late damage marker or look at the MRE11 complex.
You can also try the micronucleus assay or the comet assay. Micronuclei are chromosomal fragments lacking a centromere unit and can be detected as small, DNA containing particles after staining with Giemsa or DAPI. In the comet assay, chromosomal DNA from single cells is used in electrophoresis. DNA with breaks loose their supercoiling and migrate faster and form a tail.
The comet assay for double-stranded breaks is based on migration pattern of DNA and doesn't use any protein markers. But it will ONLY detect double-stranded breaks and not single-stranded.
Hi Joyita,
I think you are trying to find what most studies have tryed to avoid: most studies have focused in finding markers which are specific for DSB or SSB. DSB and SSB are different types of DNA damage, with different consequences for the cell. Usually DSB are much more lethal. Measuring one, or both types of damage would depend on your goals, but I would not recommend to mix them.
Regarding gamma-H2AX foci, they are typicaly associated with DBS, but there are some reports of foci in SSB and stalled replication forks, so it seems to be influenced by the cell cycle stage of your cells.
I think I did not answer your primary question, but I hope my comments help you a bit with your research.
Good luck.
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