Enzymatic Regional Methylation Assay: A Novel Method to Quantify Regional CpG Methylation Density

Abstract

We have developed a novel quantitative method for rapidly assessing the CpG methylation density of a DNA region in mammalian cells. After bisulfite modification of genomic DNA, the region of interest is PCR amplified with primers containing two dam sites (GATC). The purified PCR products are then incubated with ¹⁴C-labeled S-adenosyl-L-methionine (SAM) and dam methyltransferase as an internal control to standardize DNA quantity. This is followed by an incubation with ³H-labeled SAM and SssI methyltransferase for methylation quantification. By use of standard mixtures of cell line DNA with a defined methylation status in every assay, the ratio (³H/¹⁴C signal) of each sample can be converted into percentage values to assess the methylation density of the amplified sequence. This methylation-sensitive technique, termed ERMA (Enzymatic Regional Methylation Assay) provides several advantages over existing methods used for methylation analysis as it determines an exact measurement of the methylation density of the region studied. We demonstrate a use of this technique in determining the methylation density of the promoter region of the tumor suppressor genep15^INK4B and changes that occur after treatment with demethylating agents.