We want to determine/understand DNA damage/mutation caused by different doses of gamma irradiation on different organisms. However, although I have performed DNA sequencing using Genetic Analyzer 3500, but I don't have any experience on determining DNA damage/mutation caused by ionizing radiation. Those who have experience on this field, please provide your insightful guidelines on this issue.
Thank you very much.
You can try comet assay for determination of DNA damage in single cell level otherwise you can study the metaphase chromosome from mouse bone marrow cells, human blood lymphocytes etc for to ascertain the extent of DNA damage.......
The best biomarker of DNA damage by gamma-irradiation is the detection of the phosphorylated form of variant histone H2AX (γ-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs). There are many protocol that can be used. Check for example this paper:
γ-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin.
If you want to compare DNA damage after different doses, gamma-H2AX is probably the easist way (do immunodetection within couple of minutes after irradiation). If you want to check for futher time intervals and be more quantitative thae you should perform analysis of dicentric chromosomes, but you will need more time and a skilled lab-technician, who can read out the preparates under microscope (that is not a job for rookie). If you want to discuss more, send me an email..
Good luck!
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