Enzymatic regional methylation assay for determination of CpG methylation density

DNA methylation is a critical mechanism for epigenetic gene regulation. In mammalian cells, CpG methylation density often influences the transcription level of related genes. Here, we report a short and accurate method to determine the CpG methylation density of any DNA region by sequentially applying bisulfite PCR and SssI (CpG) methylase. We introduced simple and efficient binding and washing steps that greatly improve the previous methodology and considerably enhance the accuracy of the assay. The accuracy of this method allows the detection of differences at the level of a single CpG methylation site when homogeneous PCR product was used as substrate. We validated our method by bisulfite sequencing multiple clones of samples with variable levels of CpG methylation. We envision that for its accuracy, simplicity, low-cost, flexibility, minimum instrumentation requirements, and high-throughput potential our method could greatly benefit research on DNA methylation.