Original Article
A microwell-based near-infrared fluorescence assay of DNA methylation
Abstract
Background: DNA methylation is one of the most common epigenetic modifications, and has profound effects on the mammalian genome. The epigenetic silencing of a variety of genes by hypermethylation of promoter-associated CpG islands is often associated with diseases. This paper describes a method for the detection of DNA methylation.
Methods: This method realized methylation detection in two steps, including capturing the sheared genomic DNA (gDNA) on probe-coupled microplate and detecting methylation by using a simple immunological procedure based on near-infrared fluorescence (NIRF).
Results: This method was validated by detecting the methylation of synthesized oligonucleotides and nine genomic loci in the promoters of three genes, KIR3DL1, p14ARF and TP53BP2 in three cell lines. The detection results of the gDNA were verified by the bisulfite sequencing polymerase chain reaction (BSP) performed in this study. This study also demonstrated that the detected methylation of these promoters reduced the transcriptions of these genes in the detected cells.
Conclusions: This study provides a method for DNA methylation detection that is independent of bisulfite treatment, PCR amplification and immunoprecipitation.
Methods: This method realized methylation detection in two steps, including capturing the sheared genomic DNA (gDNA) on probe-coupled microplate and detecting methylation by using a simple immunological procedure based on near-infrared fluorescence (NIRF).
Results: This method was validated by detecting the methylation of synthesized oligonucleotides and nine genomic loci in the promoters of three genes, KIR3DL1, p14ARF and TP53BP2 in three cell lines. The detection results of the gDNA were verified by the bisulfite sequencing polymerase chain reaction (BSP) performed in this study. This study also demonstrated that the detected methylation of these promoters reduced the transcriptions of these genes in the detected cells.
Conclusions: This study provides a method for DNA methylation detection that is independent of bisulfite treatment, PCR amplification and immunoprecipitation.
