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

Abstract

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.

Methylation of RASSF1A, RASSF2A, and HIN-1 Is Associated with Poor Outcome after Radiotherapy, but not Surgery, in Oral Squamous Cell Carcinoma

“…The methylation status of RASSF1A, RASSF2A, PTEN, and HIN-1 was determined by DHPLC on a WAVE DNA Fragment Analysis System (Transgenomic) as described previously (24,25). In brief, PCR products were denatured, renatured, and analyzed by DHPLC.…”

Methylation of p16 CpG Islands Associated with Malignant Transformation of Gastric Dysplasia in a Population-Based Study

“…To further confirm the specificity of MSP and the reliability of DHPLC analysis, the MSPs in which the methylated p16 fragments were observed by DHPLC analysis were subsequently purified and sequenced as described previously (25).…”

Cyclooxygenase Isozymes: The Biology of Prostaglandin Synthesis and Inhibition

“…In addition to variant COX mRNAs, which potentially produce COX or PCOX proteins with altered or expanded biological function, is the issue of mutations and epigenetic (e.g., CpG methylation; Deng et al, 2002) changes in COX genes or regulatory regions that may be involved in disease states. Numerous COX-1 and COX-2 single nucleotide polymorphisms (SNPs) have been identified, and a more complete discussion of them has been done by Ulrich et al (2002) and Cipollone and Patrono (2002).…”