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-7phenyl-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 1:10 000 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.