Modifying the comet assay for measuring global DNA methylation in a variety of tissue cells

Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

It is becoming abundantly clear that DNA methylation plays a crucial role in gene regulation and that aberrant regulation of DNA methylation influences the development of certain diseases such as cancer. Although a wide variety of methylation analysis techniques are available today, they are still relatively expensive and a large number of them is platform specific. The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive and simple technique which is traditionally used for analysing and quantifying DNA integrity in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. The alkaline comet assay was standardised to use the difference in methylation sensitivity of the isoschizomeric restriction endonucleases Hpall and Mspl to measure the global DNA methylation levels of individual cells. Cultured cells exposed to the demethylating agent 5-azacytidine and the accumulating tyrosinemia type I metabolite, succinylacetone, was used to investigate whether it was possible to detect differences in the degree of global DNA methylation with the comet assay. The methylation sensitive comet assay's results were then verified using the well established cytosine extension assay (CEA). The CEA is also based on the selective use of the methylation-sensitive restriction enzymes Hpall and Mspl, both of which leave a 5' guanine overhang after DNA cleavage followed by a single nucleotide extension with [3H]dCTP. The study concluded that the methylation sensitive comet assay is indeed able to show clear variations in DNA methylation after treatment of cultured cells. In conclusion, the comet assay was successfully modified to determine changes in the level of global and regional DNA methylation in single cells. This modification further expands the versatility of the comet assay by increasing the variety of observations that can be made in one experiment. Since DNA methylation was shown to be a tissue-specific event, this modification of the comet assay provides the opportunity to study the DNA methylation status of single cells that are prepared from different tissues under various physiological conditions.

Masters