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 degrees 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.
Shi
Xianzhe
X
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, People's Republic of China.
Li
Jianhua
J
Zhao
Chunxia
C
Lv
Shen
S
Xu
Guowang
G
D048868:Adaptor Proteins, Signal Transducing; D000328:Adult; D000368:Aged; D002352:Carrier Proteins / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects; D004273:DNA, Neoplasm / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013447:Sulfites / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004273:DNA, Neoplasm; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D013447:Sulfites; D000070957:MutL Protein Homolog 1; C042345:hydrogen sulfite