DETECTION OF CPG METHYLATIONS IN HUMAN MISMATCH REPAIR GENE HMLH1 PROMOTER BY DENATURING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (DHPLC)
Abstract
Objectives: To develop a novel method to detect CpG methylafion by DHPLC.
Methods: After DNA was treated with sodium bisulfite, mismatch repair gene hMLH1 promoter was amplified by polymerase chain reaction (PCR). DHPLC was used to separate the PCR products at their partially denaturing temperatures. BstUI digestion assay was also used for comparison study.
Results: A 294bp band was obtained by PCR after each DNA samples of colon cancer cell line RKO and gastric cancer cell line PACM82. These two bands could be separated completely by DHPLC at 53°C (retention time 6.7 min for RKO vs. 6.2 min for PACM82). We concluded that the hMLH1 promoter in RKO cells is methylated, while PACM82 is not methylated, since methylation can protect the conversion of C to T and keep higher C/G content after bisulfite treatment, leading to the delayed llme. These results consistent with those from BstUI digestion assay.
Conclusion: Methylation in CpG islands of hMLH1 could be detected conveniently by DHPLC after bisulfite modification.
Methods: After DNA was treated with sodium bisulfite, mismatch repair gene hMLH1 promoter was amplified by polymerase chain reaction (PCR). DHPLC was used to separate the PCR products at their partially denaturing temperatures. BstUI digestion assay was also used for comparison study.
Results: A 294bp band was obtained by PCR after each DNA samples of colon cancer cell line RKO and gastric cancer cell line PACM82. These two bands could be separated completely by DHPLC at 53°C (retention time 6.7 min for RKO vs. 6.2 min for PACM82). We concluded that the hMLH1 promoter in RKO cells is methylated, while PACM82 is not methylated, since methylation can protect the conversion of C to T and keep higher C/G content after bisulfite treatment, leading to the delayed llme. These results consistent with those from BstUI digestion assay.
Conclusion: Methylation in CpG islands of hMLH1 could be detected conveniently by DHPLC after bisulfite modification.