A methylation sensitive dot blot assay (MS-DBA) for the quantitative analysis of DNA methylation in clinical samples.
BACKGROUND
There is increasing interest in DNA methylation and in its implication in transcriptional gene silencing, a phenomenon commonly seen in human cancer.


OBJECTIVE
To develop a new method that would allow quantitative DNA methylation analysis in a large range of clinical samples, independently of the processing protocol.


METHODS
A methylation sensitive dot blot assay (MS-DBA) was developed, which is quantitative and combines bisulfite modification, PCR amplification using primers without CpG sites, and dot blot analysis with two probes specific for methylated and unmethylated DNA.


RESULTS
The established method was used to study methylation of the hTERT, APC, and p16 promoter regions in microdissected, formalin fixed and paraffin wax embedded tissues.


CONCLUSIONS
MS-DBA is a sensitive, specific, and quantitative approach to analyse DNA methylation in a variety of frozen or fixed tissues. Moreover, MS-DBA is rapid, easy to perform, and permits the screening of a large panel of samples in one experiment. Thus, MS-DBA can facilitate the routine analysis of DNA methylation in all types of clinical samples.
Clément
G
G
Institut de Pathologie, Centre Hospitalier Universitaire Vaudois, Bugnon 25, 1011 Lausanne, Switzerland.
Benhattar
J
J
D000230:Adenocarcinoma / Q000235:genetics; D019175:DNA Methylation; D015342:DNA Probes; D004268:DNA-Binding Proteins; D004938:Esophageal Neoplasms / Q000235:genetics; D019942:Genes, p16; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D019098:Telomerase / Q000378:metabolism
D015342:DNA Probes; D004268:DNA-Binding Proteins; D019098:Telomerase