Methylation Analysis by Restriction Endonuclease Digestion and Real-Time PCR

DNA methylation is a highly characterized epigenetic modification of the human genome that is critical for normal cell processes, including tissue regulation, genetic expression development, genomic imprinting, X-chromosome inactivation, and DNA repair. DNA methylation in humans occurs almost exclusively at the C5 position of CpG dinucleotides in genomic promoter regions. Alterations in DNA methylation cause developmental diseases, such as genetic imprinting defects, cancer, and potentially a wide range of adult-onset chronic diseases. Therefore, sensitive techniques that detect methylation are critical for epigenetic research, clinical diagnostics, cancer prognosis, and therapeutics (1)(2). Methylation-specific PCR is a commonly used rapid technique for methylation screening that replaces the laborious Southern blot assay, the standard procedure on which most DNA analysis is based. Methylation-specific PCR and other PCR-based methylation methods that use bisulfite-treated DNA as a template are generally accepted as the most analytically sensitive and specific techniques for analyzing DNA methylation at a single locus. The protocol described by Frommer et al. has been widely used for sodium bisulfite modification, and a variety of commercial kits are available for this purpose (3). Sodium bisulfite deaminates unmethylated cytosine to uracil, and the methylation is accessed by the PCR with either methylation-specific primers or methylation-independent primers (MIPs).1 Methylation-specific primers amplify either methylated or unmethylated alleles. These primers are highly sensitive and able to detect the presence of a methylated allele at a frequency as low as 0.1% in an unmethylated population (4). PCR with methylation-specific primers is not quantitative, however. Alternatively, the MethyLight method is a sensitive, fluorescence-based real-time PCR technique capable of quantifying DNA methylation at a specific locus (5)(6). MethyLight can detect completely methylated or unmethylated alleles but is oblivious to partially methylated CpGs. Considering the random conversion of cytosine to uracil, it is possible that a subset …