The Combined Bisulfite Restriction Analysis (COBRA) Assay for the Analysis of Locus-Specific Changes in Methylation Patterns

DNA methylation is a heritable but reversible epigenetic mechanism of control over gene expression. The level of DNA methylation of specific genomic regions correlates with chromatin condensation, the level of gene expression, and in some cases genome stability and the frequency of homologous recombination. Here, we describe the combined bisulfite restriction analysis (COBRA) assay that allows analyzing the methylation status at a specific locus. The protocol consists of the following major steps: bisulfite conversion of non-methylated cytosines to uracils, the locus-specific PCR amplification of converted DNA, restriction digestion, the analysis of restriction patterns on the gel, and the quantification of these restriction patterns using ImageJ or a similar program.