A simple and rapid agarose gel electrophoresis method to assess CpG methylation of DNA

The presence of 5-methylcytosine at CpG sites in mammalian DNA plays a significant role in various biological processes, both normal and aberrant. Similarly, CpG site methylation status of therapeutic DNAs can affect activity and therapeutic efficacy, as is the case for ADI-100, an immune tolerance-inducing drug candidate for the treatment of type 1 diabetes and other GAD-associated autoimmune diseases. To assess the methylation characteristics of ADI-100 and other therapeutic DNA product candidates, we developed a simple and versatile method, methylation-sensitive restriction enzyme digest and agarose gel electrophoresis (MSRE-AGE), using methylation-sensitive restriction enzyme digest, agarose gel electrophoresis, and custom in silico band pattern analysis for quantitative measurement of CpG methylation levels and patterns. We compared MSRE-AGE with bisulfite pyrosequencing methylation analysis of plasmid DNA and a synthetically-produced closed-linear DNA, Doggybone DNA (dbDNA), demonstrating that MSRE-AGE produces results consistent with bisulfite pyrosequencing but without structure and sequence-dependent artifacts. These findings demonstrate that MSRE-AGE is a robust and practical method for methylation analysis of therapeutic DNA products.