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[Analysis of the methylation in CpG island by denaturing high-performance liquid chromatography]
1Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing 100034, China.
Objective:
To establish a new approach for analyzing methylation circumventing the limitations of the existing methods.
Methods:
A region containing CpG sites in mismatch repair gene hMLH1 promoter was amplified by strand-specific polymerase chain reaction (PCR) after sodium bisulfite treatment. The retention time of the PCR product at partially denaturing temperature was determined by denaturing high-performance liquid chromatography (DHPLC). The methylation status obtained by DHPLC was proved by comparing it with that from sodium bisulfite-restriction enzyme digestion.
Results:
Methylation of hMLH1 promoter from a colorectal cancer cell line RKO and a gastric cancer cell line PACM82 was analyzed by DHPLC. The retention time of the PCR product from RKO was obviously longer than that from PACM82 (6.7 min vs. 6.2 min). Thus, it was concluded that the hMLH1 promoter from RKO was methylated, while that from PACM82 was not, since the longer retaining time of RKO was due to the higher C/G content after sodium bisulfite treatment. The results from DHPLC were consistent with those from sodium bisulfite-restriction enzyme digestion.
Conclusion:
DHPLC method is a rapid and reliable approach for analyzing methylation in CpG island of the sequence of interest.