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.
Quantification of locus-specific epigenetic modifications in the mammalian genome is an integral part of understanding epigenomic changes during development and disease. The EpiMark 5-hmC and 5-mC Analysis Kit from New England Biolabs offers a simple, reproducible, and robust method to examine the methylation status of a DNA population; addresses the need for detection and quantitation of 5-hmC in genomic DNAs; and opens up opportunities to determine whether 5-hmC modifications of DNA can be exploited in the identification of novel biomarkers.
Further, quantitative PCR can be replaced by end-point PCR, providing scientists with a simple “yes or no” answer to whether a given loci contains 5-hmC. Lastly, multiplexing of this protocol for end-point PCR may be performed for different amplicons using standard thermocyclers.
The EpiMark method followed by qPCR analysis is currently being applied to study the differences in hydroxymethylation patterns for a variety of biologically relevant modifications in mammalian DNAs during the different stages of development. In unpublished studies, researchers at NEB have shown variations in 5-hmC levels of genomic DNAs extracted from brain, heart and liver tissue samples from murine and human.