A membrane-based near-infrared fluorescence assay for detecting DNA methylation and transcription.
DNA methylation is a common epigenetic modification. The epigenetic silencing of a variety of genes by hypermethylation of promoter-associated CpG islands is often associated with diseases. Therefore, a reliable but uncomplicated method for detecting DNA methylation is preferred for research and clinical practice. In this paper, a membrane-based near-infrared fluorescence assay for detecting DNA methylation and its effect on gene transcription was established for this purpose. This assay consisted of two detection systems, i.e., transcription and methylation detection systems. The former detected gene transcription by using biotinylated cDNA and the latter detected DNA methylation by using anti-5-methylcytidine antibody (5MC-Ab). The biotin and 5MC-Ab signals were reported by near-infrared fluorescence-labeled streptavidin and secondary antibody, respectively. The feasibility of the two systems was fully verified with the synthesized biotinylated and methylated oligonucleotides. The reliability of the two systems was demonstrated by successfully detecting the methylation and transcription of a reported hypermethylated gene, p14(ARF), in LOVO cells. This study provides a new method for simultaneously detecting DNA methylation and transcription, which is helpful for exploring the gene expression regulatory role of DNA methylation. The method is free of bisulfite treatment and PCR amplification but has wide dynamic range and high sensitivity.
The State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
Ying
Y
Chen
Jinke
J
Wang
D001483:Base Sequence; D002460:Cell Line; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D006801:Humans; D008567:Membranes, Artificial; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology; D014158:Transcription, Genetic
D008567:Membranes, Artificial; D013447:Sulfites; C042345:hydrogen sulfite
DNA methylation; Gene transcription; Near-infrared fluorescence assay; Nylon membrane