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.
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:
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: