Analysis of Locus-Specific Changes in Methylation Patterns Using a COBRA (Combined Bisulfite Restriction Analysis) Assay

DNA methylation is a major mechanism for the reversible control of gene expression, chromatin structure, and genome stability. Methylation analysis at a given locus allows one to evaluate levels of chromatin packaging, gene expression, and even homologous recombination. We have shown that the combined bisulfite restriction analysis (COBRA) assay makes it possible to analyze methylation levels at a defined locus. The major steps are: bisulfite conversion of nonmethylate cytosines to uracils, locus-specific PCR amplification of converted DNA, restriction digestion, and analysis of restriction patterns on the gel. Due to the availability of various restriction enzymes that have cytosines in the restriction recognition sequence, the assay allows analysis of various cytosines, including those potentially targeted for symmetrical and nonsymmetrical methylation.