Pyrosequencing assays to study promoter CpG site methylation of the O6-MGMT, hMLH1, p14ARF, p16INK4a, RASSF1A, and APC1A genes.

DNA methylation of CpG sites in promoter regions of several cancer related genes, such as O6-MGMT, hMLH1, p14ARF, p16INK4a, RASSF1A and APC1A, has been actively explored recently. Much of the data has been obtained using a variation of an allele-specific PCR assay known as methylation specific PCR. This technique is objectionable for a number of methodological limitations and drawbacks. We wanted to study the promoter regions of the above mentioned genes using bisulfite-treated genomic DNA amplified by PCR, using primers designed to bind only to CpG-free sequences. The methylated fraction (%) of each CpG site was measured by Pyrosequencing technology. For three of the genes, O6-MGMT, hMLH1 and p14ARF, two amplicons were designed to cover all relevant CpG sites. Several of the amplicons were analyzed by two different Pyrosequencing assays. In all, we designed nine optimized PCR protocols and 13 Pyrosequencing assays covering the promoters of all six studied genes. In all cases, standard PCR generated sufficient quantities of pure amplicons to be further analyzed by Pyrosequencing technology. Thus, a total of 119 CpG sites in six genes could be quantified. We conclude that standard PCR followed by Pyrosequencing is a workable, more specific and quantitative alternative to 'methylation specific' PCR. This approach provides a more comprehensive picture of the distribution of DNA methylation throughout the promoter regions of the studied set of six genes, which will be of benefit in oncological research.