OBJECTIVETo setup a quantitative assay for detection of methylation of SNCG CpG island in human tissue samples.

METHODSMethylation status of the 16 tested CpG sites within the CpG island was analyzed by bisulfite-clone-sequencing for 2 gastric carcinoma cell lines, 2 normal gastric mucosa samples, and 2 pairs of primary gastric carcinomas and their corresponding non-neoplastic tissues, respectively.

RESULTSThe methylation of -88 and other four CpG sites was well correlated with the methylation of the overall CpG island. Thus, a combined bisulfite-restriction assay (COBRA) was developed based on the enzyme AciI, which digested the only one GCGG sequence in the PCR products of the methylated CpG island, but not the GTGG in the demethylated one. The digested fragments (144 bp and 85 bp) and undigested fragment (229 bp) could be completely separated by denaturing high performance liquid chromatography (DHPLC). According to the peak areas of these fragments, the proportion of the methylated copies of the SNCG CpG island was calculated easily. The result of the COBRA-DHPLC assay was reproducible and consistent with that of clone-sequencing.

CONCLUSIONA COBRA-DHPLC assay is setup successfully for quantification of methylation of the SNCG CpG island.