Hello everyone. I want to know very simple method to check the damage introduced to DNA by exposure to UV light or some chemicals for example hydrogen peroxide.
This may be useful: http://pubs.rsc.org/en/Content/ArticleLanding/2013/CC/c3cc38884k#!divAbstract
Dear Dr. Amina.
Please find the article related to your question.
Regards.
Dr. Nasir Mahmood
Dear Amina,
You can do one thing, give direct exposure to blood lymphocytes under UV light for some time and then isolate DNA to check its damage.
I would do a DNA dot blot on nylon+ membrane and use anti-pyrimidine dimer (CPD) (from cosmo bio or trevigen) or 8-oxodG antibody. There are plenty of other techniques (Comet, ELISA, denaturing gels following T4 endo V tx) but the dot blot is the easiest.
You must evaluate firstly the level of accuracy you need in order to assess the UV or chemical damage. Then, you must classify the chemical under evaluation according to its action mechanism. If it is an alkilating agent you may consider the use of chromosomal aberration (at higher doses than those that yield positive results with techniques as comet or alkaline elusion assay)
If the chemical you are scrutinizing is able to produce adducts, If those adducts are characterized, you may consider inmunodetection (is more time consuming and need an skilled operator).
DNA electrophoresis is versatile and relatively easy to do but if the damage does not cause fragmentation or a sensible hampering in migration, it is a better option to use comet assay. Comet assay may be adapted to detect specific modifications as (OH dG ) by using enzymatic treatment of the samples or variation in the pH of cells pretreatment or electrophoresis run.
I think that as a beginning technique comet assay will allow you to screen your substances to evaluate if it is able to produce single strand breaks (pH 13.1), double strand (neutral pH).
It is easy and extremely sensitive, it means that you will get results if even an slight effect of that chemical is present, but also that false positive are the rule if you don´t take all the precautions. See Tice and Vasquez: Protocol for Comet Assay, it is easy to find in the web.
Then you may scale and direct your technical options according to your results.
When you evaluate UV radiation, you should take in account the wave length of that UV, There is not the same the pro oxidant UVA than UVB with a greater capacity to produce cyclobutane dimers or pyrimidino pyrimidone photoproducts.
Any way you may evaluate Sister Chromatid Exchange that is very sensitive to UV and also comet assay. Inmuno detection is also possible if it is justifiable for your objectives.
Dear Amina,
You can use the sister chromatid exchange test and comet assay for detect chemical and UV damage. Best regards
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