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MEASURING DNA DAMAGE - the comet assay

The comet assay - single cell gel electrophoresis assay

Both DNA damage and DNA repair can be measured.
Oxidised bases such as 8-oxoguanine can be measured readily by chromatography (HPLC), or using the comet assay in combination with lesion-specific endonucleases. The bacterial repair endonucleases FPG and Endonuclease III are useful in combination with the comet assay for detecting oxidised bases.

http://meddev.uio.no/elaring/lcms13/ernaeringslaere/nutr-cancer-biology/illustrations/Comet-assay.jpg

Figure Cells are embedded in agarose on glass slides. After lysis of the cell membrane and nuclear membrane, the DNA is digested with lesion-specific endonuclease that cuts in one strand. Digested DNA loops out during electrophoresis to produce a tail. % of DNA in tail is related to DNA break frequency.

Flow scheme of the procedure:

http://meddev.uio.no/elaring/lcms13/ernaeringslaere/nutr-cancer-biology/illustrations/Comet-flow.jpg

ESCODD - European standards committee on oxidative DNA damage

What is the most reliable method for measuring the level of DNA oxidation?

Watch the video and find out:

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