DNA methylation analysis based on bisulfite conversion
Exposing DNA to bisulfite rapidly leads to the deamination of unmethylated cytosines which are converted to 6-sulfonyluracil. At high pH, 6-sulfonyluracil is desulfonated to uracil, which ultimately after amplification will translate into thymidine, while methylated cytosines will not be converted. Comparing this converted DNA to the original unconverted sequence enables detailed evaluation of the location and abundance of methylated sites in CpG islands. High-resolution melting (HRM) analysis provides a rapid screening tool to accurately detect changes in CpG methylation status of bisulfite converted DNA. Detailed quantitative measures of percent methylation at individual CpG sites are obtained by Pyrosequencing. Alternative methods for broad-scale methylation analysis include methylation-specific PCR (MSP) which is highly specific and sensitive. Commercial kits are available for all forms of analysis.
DNA methylation analysis based on restriction enzyme digest
This methodology enables the study CpG island methylation of individual genes and disease or pathway-focused gene panels without bisulfite modification. It relies on differential cleavage of target sequences by two different restriction endonucleases whose activities require either the presence or absence of methylated cytosines in their respective recognition sequences. The relative amount of DNA remaining after each enzyme digest is quantified by real-time PCR, delivering reliable calculation of the methylation status of individual genes and the methylation profile across a gene panel.