Immobilization of DNA with nitrogen mustard-biotin conjugate for global epigenetic analysis

We report the quantitative analysis of 5-methylcytosine, a representative epigenetic modification in genomic DNA, with an enzyme-linked immunosorbent assay (ELISA). We synthesized a novel hetero-bifunctional linker molecule consisting of nitrogen mustard and biotin to capture DNA on the surface of biosensing devices. The molecule can successfully immobilize genomic DNA on a streptavidin coated 96-well microplate, which was then employed for immunochemical epigenetic assessment. We achieved the sensitive and quantitative detection of 5-mC in genomic DNA samples. The CpG methylation ratios obtained from our system for mouse brain and mouse small intestine genomes were 79% and 82%, respectively. These numbers are in good agreement with the previously reported methylation ratio of 75-85%, which was identified by whole genome bisulfite sequencing. Accordingly, the present technology using our novel bifunctional linker molecule provides a fast, easy, and inexpensive method for epigenetic assessment, without the need for any conventional bisulfite treatment, polymerase chain reaction (PCR), or sequencing.