Altered epigenetic patterns are a hallmark of cancer. Many genes are silenced in cancerous cells due to newly acquired de novo methylation of CpG islands (1). Currently, there are several methods that detect methylated CpGs. Methylation-specific PCR (MSP) is a sensitive technique used for the detection of gene methylation in the genome (2). MSP uses an initial bisulfite reaction to modify the DNA by converting unmethylated cytosines to uracils while 5-methylcytosines remain unaltered. This reaction is followed by PCR amplification with specific primers designed to distinguish methylated from unmethylated DNA. Since this is an extremely sensitive PCR-based assay, the use of control DNA is necessary to determine the quality of the bisulfite conversion and to identify artifacts such as primer-dimer pairing and mispriming that can cloud the interpretation of results.
To create a methylation-positive DNA control, all cytosine residues (C5) within the double-stranded dinucleotide recognition sequence 5´…CG…3´ in HeLa, NIH-3T3 and Jurkat genomic DNA have been enzymatically methylated with CpG Methylase (M.SssI) (NEB #M0226). These modified DNAs are extensively tested for complete methylation by an additional methyl group transfer assay and MSP. A partially demethylated DNA control has also been created by incubating Jurkat cells with a potent methyltransferase inhibitor, 5-aza-2-deoxycytidine (5-Azadc). Bisulfite conversion (3) and sequencing of a section of intergenic (IGS) repetitive DNA (rDNA) that is normally methylated revealed significant CpG demethylation.