A quantitative DNA methylation assay using mismatch hybridization and chemiluminescence.
- Author:
Qun-Feng YAO
1
;
Xin-Jiang KANG
;
Qiao-Ling HAO
;
Yi-Kai ZHOU
Author Information
- Publication Type:Journal Article
- MeSH: Cell Line, Tumor; CpG Islands; DNA Methylation; Genes, p16; Humans; Luminescent Measurements; Nucleic Acid Hybridization; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Sulfites
- From: Biomedical and Environmental Sciences 2005;18(1):48-52
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence.
METHODSGenomic 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.
RESULTSThe 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.
CONCLUSIONCompared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.
