Fluorescent conjugated polymer-based FRET technique for detection of DNA methylation of cancer cells.
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.
Feng
Fude
F
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China.
Liu
Libing
L
Wang
Shu
S
D015374:Biosensing Techniques / Q000379:methods*; D002412:Cations; D045744:Cell Line, Tumor; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D019175:DNA Methylation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005821:Genetic Techniques; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011108:Polymers / Q000032:analysis / Q000737:chemistry; D011401:Promoter Regions, Genetic / Q000235:genetics
D002412:Cations; D005456:Fluorescent Dyes; D011108:Polymers; D004247:DNA