The semi-quantitative methylation analysis (A) and the cloning and sequencing (B) in the transitional-CpG sites. The methylation of transitional-CpG sites was evaluated by performing a semi-quantitative methylation analysis (A) and it was verified with cloning and sequencing of the common primer (B). (A) A total of 14 transitional-CpG sites were examined in the H. pylori-negative and H. pylori-positive normal gastric mucosa and cancer lesion of gastric cancer patients. The methylation density was calculated according to the proportion of the methylation (M) band intensity against the total unmethylation (U) and methylation band intensity. The methylation density of each CpG site was classified into 5 levels with 20%-methylation increment. The methylation status of each gene was categorized as undermethylation (under-) or overmethylation (over-) based on an intermediate methylation level (inter-) of the H. pylori-negative normal gastric mucosa. (B) Representative results of cloning and sequencing of common PCR. The transitional-CpG sites of the CDH1, CDKN2A and RUNX3 genes were analyzed by cloning and sequencing the common PCR product. The genomic DNAs represented in Figure 1A were used to compare the MSP results with the sequencing results. The open and closed circles indicate unmethylated and methylated cytosine residues, respectively. The rectangular boxes indicate the MSP primer sites.