I want to know the assay for DNA damage in response to pathogen and heat stress and also due to reactive oxygen species produced in stress response
Hi Sudhir,
The assay for the DNA damage depends on what particular kind of damage you're interested in - for example, double strand breaks (DSBs), bulky base adducts, DNA mismatches, etc.. Though my knowledge is more mammalian- than plant-based, I believe that plants (like mammals) repair each by a different pathway, so different assays/markers may be needed to assess different types of damage.
For DSBs, in mammals we tend to use immunofluorescence to stain for 53BP1, a protein that localises to DSBs, forming bright foci. A brief perusal of a review of DSB repair in Arabidopsis (PMC3797388) suggests that, provided you can purchase antibodies (for example against RAD51 - I don't think 53BP1 has a reported Arabidopsis homolog) and can fix/permeabilise your cells, the same approach may work. You may also be able to do the same for other types of DNA damage (e.g. XRCC1 for SSBs, I think?) but that may require some further reading on possible markers and antibodies available - outside my range of knowledge I'm afraid!
A couple of papers in PubMed also mention using TUNEL technology to label DSBs in Arabidopsis, which may represent an alternative approach. For a more low-tech (and maybe cheaper!) alternative, you may be able to adapt Comet assays to look at genome fragmentation - I've not heard of this being done in plants, but then I don't know if I would have heard even if it had been done!
In terms of assessing oxidative DNA damage, we have had some success with a "Southwestern" approach - spotting total cellular DNA onto a membrane, blocking with salmon sperm DNA, and hybridising with an antibody against 8-hydroxydeoxyguanosine (8-OHdG), a common product of DNA oxidation. While we've not tried it in plants, I don't off the top of my head foresee a reason why a similar approach wouldn't work. The antibody we use is from Millipore (AB5830) but antibodies against 8-OHdG are fairly widely available (AbCam etc.).
I hope that all helps! Let me know if you have any other questions.
Regards,
Dan
Thanks Danial
I came through Comet assay for the same the same I want to know more about it like sample preparation, slide preparation for comet single cell gel electrophoresis. Plz if you could put some light on this
Hi,
Comet assay detects single strand breaks, please see my profile to get all the information that you need.
You may also find this reference useful, since it touches on both plant cells and non-SSB damage types:
doi:10.1038/nprot.2006.5
Cheers,
Dan
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