Improved quantification of DNA methylation using methylation-sensitive restriction enzymes and real-time PCR.

Heterogeneity of cells with respect to the DNA methylation status at a specific CpG site is a problem when assessing methylation status. We have developed a simple two-step method for the quantification of the percent of cells that display methylation at a specific CpG site in the promoter of a specific gene. The first step is overnight digestion of genomic DNA (optimal conc. 20 ng/5 microl) with a relevant methylation-sensitive restriction enzyme (optimal 2 units). This is followed by real time PCR, using the SYBR Green method, with primers that bracket the site cleaved by the enzyme. By including fully methylated and fully non-methylated DNA in each PCR plate, the errors caused by non-specific digestion or incomplete digestion can be measured and used to adjust the raw results and thus increase specificity. The method can detect differences in methylation status if these are more than 10%. No specialized equipment is required beyond the real-time PCR system and the method can be adapted for any of the 53 commercially available methylation-sensitive restriction enzymes.