Establishing a method for measuring the DNA methylation status of specific human genes
Van Heerden, Chrisna
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DNA methylation is an epigenetic alteration which occurs during the development and lifetime of mammalians. It has an essential gene transcription regulation function and has been shown to be a common alteration at the root of several malignancies. Regions with high concentrations of CpG sites are classified as CpG islands which usually lie in the promoter regions. Hypermethylation of these promoter regions represses transcription of tumour repressor genes, which causes gene silencing. Specific methylation profiles can be compiled for different types of cancers. These profiles are of great importance due to their diagnostic, risk assessment and therapeutic potential, in addition to increasing the knowledge and understanding of the pathogenesis of tumorigenesis and oncogenesis. The aim of this study was to establish and optimize a method to measure the DNA methylation status of specific human genes, i.e. cancer related genes. Methylationspecific PCR (MSP) was used to analyze the methylation status of the p76 and RASSFIA promoter CpG islands following the sodium bisulfite conversion of unmethylated cytosines to uracils since this is a sensitive, efficient and relatively fast method with high-throughput potential. The MSP assay was established and applied successfully to DNA isolated from whole blood, plasma (free-circulating DNA) and paraffin-embedded tumour tissue. The results obtained showed that for the p76 promoter sequence, the methylation status in all control individual DNA samples was unmethylated. The patient DNA samples had diverse p16 promoter methylation levels. The tumour DNA sample displayed both methylated- and unmethylated-specific products. This was also observed for this patient's free-circulating DNA sample, but not for the whole blood DNA sample.