Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces

Various technologies are currently available to quantify DNA methylation. However, rapid and simple methods for determining the DNA methylation status of CpG sites in genes still remain elusive. In this report, we describe a novel method for the rapid quantification of CpG methylation on the basis of direct bisulfite-PCR sequencing method. According to the principles of bisulfite-PCR, converting unmethylated cytosines to thymine while leaving methylated cytosines unchanged, we regard the CpG site as a SNP and estimate the methylation status of cytosines in the given CG dinucleotides by measuring the ratio of the cytosine peak height to the sum of cytosine and thymine peak heights in automated DNA sequencing traces. Furthermore, we take several effective measures to break through the 'bottleneck' problems that render the routine bisulfite sequencing method unsuitable for quantitative methylation. In comparison with pyrosequencing and bisulfite-cloning sequencing, our method is confirmed to be a simple, high-throughput and cost-effective technology for determining the methylation status of specific genes. Accordingly, this novel method is anticipated to be an efficient and economical alternative tool for rapid quantification of methylation patterns in screening large numbers of clinical samples across multiple genes.