MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome

Here we present MethylQuant, a novel method that allows accurate quantification of the methylation level of a specific cytosine within a complex genome. This method relies on the well-established treatment of genomic DNA with sodium bisulfite, which converts cytosine into uracil without modifying 5-methyl cytosine. The region of interest is then PCR-amplified and quantification of the methylation status of a specific cytosine is performed by methylation-specific real-time PCR with SYBR Green I using one of the primers whose 3′ end discriminates between the methylation states of this cytosine. The presence of a locked nucleic acid at the 3′ end of the discriminative primer provides the specificity necessary for accurate and sensitive quantification, even when one of the methylation states is present at a level as low as 1% of the overall population. We demonstrate that accurate quantification of the methylation status of specific cytosines can be achieved in biological samples. The method is high-throughput, cost-effective, relatively simple and does not require any specific equipment other than a real-time PCR instrument.