In order to verify that the above method can accurately detect differences between a population of differentially methylated DNAs, mixtures of in vitro synthesized oligonucleotides that are unmethylated (C), methylated (5-mC), or hydroxymethylated (5-hmC) were prepared. Two heterogeneous mixed sets of these sequences were assessed: set #1 contained a mixture of 20% C / 60% 5-mC / 20% 5-hmC, and set #2 contained a mixture of 20% C / 50% 5-mC / 30% 5-hmC.
Both sets were treated with beta-GT followed by digestion with MspI or HpaII. Undigested DNA served as a positive control for amplification, while the digestion of nonglucosylated DNA with MspI served as a negative control. Quantitative PCR of the synthetic DNA yielded results consistent with the input amounts of the differentially methylated DNAs in each sample (Table).
These results indicate that this method reliably differentiates between unmethylated, 5-mC, and 5-hmC modified DNAs, and allows the quantitation of the various levels of each.