Diversity in the applications of the single cell gel electrophoresis (comet) assay

Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2005.

The development of the single cell gel electrophoresis assay (Comet assay) as a powerful method for measuring DNA strand breakage and repair, has lead to a broader understanding of the impact of certain internal and external factors on DNA damage. This study describes the establishment of the Comet assay in our laboratory and its application in a diversity of studies. These studies include the monitoring of the effect of exercise on DNA damage and repair with the purpose of establishing the optimal exercise program for middle aged men; in the analysis of DNA repair in a breast cancer patient after chemotherapy, we demonstrated that to get a really informative picture of baseline DNA damage, it is necessary to invoke some type of insult to the DNA; we also studied the antioxidant activity of plant extract and finally an industrial application of the Comet assay where we studied DNA damage and repair in the lymphocytes of people exposed to a variety of chemicals. The Comet assay was made even more informative by incorporating additional steps by digesting the DNA on the microscope slides with enzymes that recognize particular kinds of damage to the nucleic acid (formamidopyrimidine DNA glycosylase (Fpg) and Endonuclease lll (Endo lll). From these applications we can safely conclude that we have established the Comet assay as reproducible assay with proven diversity in its applications. The most significant contribution of this study is the quite novel modification of the Comet assay to monitor the methylation status of DNA. In this we applied the restriction endonuclease enzymes, Hpa ll and Msp I, which have different sensitivities to methylation of their common target site. Changes in the methylation levels of DNA after treatment of animals and isolated cells with chemicals with known effects on DNA, were convincingly demonstrated and indications of cell specificity in DNA methylation were observed. We believe that this modification opens a new field of applications for the Comet assay.

Masters