This protocol describes a homogeneous, convenient and sensitive DNA methylation detection method, using an optically amplifying cationic conjugated
polymer (CCP, poly((1,4-phenylene)-2,7-[9,9-bis(6'-N,N,N-trimethyl
ammonium)-hexyl
fluorene] dibromide)). Genomic
DNA from
cancer cells is pretreated with a methylation-sensitive
restriction endonuclease, followed by PCR amplification in the presence of
fluorescein-labeled dNTP and
Taq polymerase. The PCR only occurs for methylated
DNA. DNA methylation of the gene sequence of interest is detected as a result of the fluorescence resonance energy transfer (FRET) between CCP and
fluorescein that is incorporated into
DNA. The methylated statuses of the p16, HPP1 and GALR2 promoters of five
cancer cell lines (HT29, HepG2, A498, HL60 and M17) were assayed to provide an association study between the
cancers and susceptibility genes, which shows great potential for early
cancer diagnosis. This protocol simplifies previously available procedures by avoiding the need for primer labeling, isolation or purification steps, and sophisticated instruments. The assay takes about 20 h to obtain the methylated statuses of
cancer cells.