Methylation analysis of hMLH1 gene promoter by a bisulfite‐sensitive single‐strand conformation polymorphism–capillary electrophoresis method

DNA methylation is an important epigenetic modification that alters transcription in those genes containing CpG islands. In this report, a novel DNA methylation analysis method was developed employing bisulfite–single strand conformation polymorphism combined with capillary electrophoresis (bisulfite–SSCP–CE). During the bisulfite treatment of genomic DNA, a high concentration of sodium bisulfite (4.8 mol/L) was preferred in order to shorten reaction time and minimize template degradation. The methylated and unmethylated ssDNA of hMLH1 promoter were simultaneously separated under the optimized CE conditions, including 6% SLPA with 10% glycerol as sieving medium, 25°C as separation temperature and 12 kV as running voltage. The heterogeneous methylation of hMLH1 promoter was identified in 13 of 64 colorectal cancer patients. Moreover, hMLH1 promoter methylation had a significant relationship with protein expression loss and increased with the age of patients. Our results indicated that DNA methylation analysis for a large number of clinical samples would be facilitated by use of the bisulfite–SSCP–CE method. Copyright © 2005 John Wiley & Sons, Ltd.