| Summary: | 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 Ill (Endo Ill).
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. === Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2005.
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