Novel Assay for Quantitative Analysis of DNA Methylation at Single-Base Resolution

Abstract BACKGROUND The DNA methylation profile provides valuable biological information with potential clinical utility. Several methods, such as quantitative methylation-specific PCR (qMSP), have been developed to examine methylation of specific CpG sites. Existing qMSP-based techniques fail to examine the genomic methylation at a single-base resolution, particularly for loci in gene bodies or extensive CpG open seas lacking flanking CpGs. Therefore, we established a novel assay for quantitative analysis of single-base methylation. METHODS To achieve a robust single-base specificity, we developed a PCR-based method using paired probes following bisulfite treatment. The 6-carboxyfluorescein- and 2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein-labeled probes conjugated with minor groove binder were designed to specifically bind to the methylated and unmethylated allele of targeted single CpGs at their 3′ half regions, respectively. The methylation percentage was calculated by values of methylation / (methylation + unmethylation). RESULTS In the detection of single CpGs within promoters or bodies of 4 human genes, the quantitative analysis of the single-base methylation assay showed a detection capability in the 1 to 1:10000 dilution experiments with linearity over 4 orders of magnitude (R2 = 0.989–0.994; all P < 0.001). In a cohort of 10 colorectal cancer samples, the assay showed a comparable detection performance with bisulfite pyrosequencing (R2 = 0.875–0.990; all P < 0.001), which was better than conventional qMSP methods normalized by input control reaction (R2 = 0.841 vs 0.769; P = 0.002 vs 0.009). CONCLUSIONS This assay is highly specific and sensitive for determining single-base methylation and, thus, is potentially useful for methylation-based panels in diagnostic and prognostic applications.