A quantitative DNA methylation assay using mismatch hybridization and chemiluminescence.
OBJECTIVE
To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence.


METHODS
Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes.


RESULTS
The percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines.


CONCLUSIONS
Compared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.
Yao
Qun-Feng
QF
Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.
Kang
Xin-Jiang
XJ
Hao
Qiao-Ling
QL
Zhou
Yi-Kai
YK
D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D019942:Genes, p16; D006801:Humans; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013447:Sulfites
D013447:Sulfites; C009279:sodium bisulfite