Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography

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Oxford University Press

RESUMO

We report here a novel method to simultaneously detect CpG methylation and single nucleotide polymorphisms (SNPs) using denaturing high performance liquid chromatography (DHPLC). PCR products of bisulfite-modified CpG islands were separated using DHPLC. BstUI digestion and DNA sequencing were used in confirmation studies. Consistent with the BstUI digestion assay, the 294 bp PCR product of the modified hMLH1 promoter showed different retention times between the methylated cell lines (RKO and Cla, 6.7 min) and the unmethylated cell lines (PACM82 and MGC803, 6.2 min). No hMLH1 methylation was observed in 13 primary gastric carcinomas and their matched normal tissues. One hMLH1 SNP was detected in gastric cancer patients, in both cancer and normal tissues. DNA sequencing revealed that the SNP is a G→A variation at –93 nt of the hMLH1 promoter. A two-peak chromatogram was also obtained in the 605 bp PCR product of the Cox-2 promoter of the AGS, HEK293 and MKN45 cell lines by DHPLC. Another peak corresponding to methylated CpG islands was observed on the chromatogram of the Cox-2-methylated AGS cell line after bisulfite treatment. In conclusion, methylation in homoallelic and heteroallelic CpG islands could be detected rapidly and reliably by bisulfite–DHPLC. A SNP in the target sequence could also be detected at the same time.

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