DNA methylation at CpG sites, where a cytosine base is adjacent to a guanine, is a critical mechanism for regulating gene expression, maintaining genome stability, and dictating cell type during development. Aberrant DNA methylation is involved in many human diseases, including immune disorders, developmental abnormalities, and cancer. Currently available methods to investigate DNA methylation patterns suffer from shortcomings, such as sequence or cell-type bias, small genomic target size, and requirement of large volumes of starting DNA material or high quality DNA. This technology describes methods and compounds for determining whether cytosine residues present at predetermined positions within a DNA molecule of known sequence are methylated. This approach is free of sequence or target bias and is compatible with next-generation sequencing methods.
Utilizing S-Adenosyl-L-methionine (AdoMet) analogues and readily available bacterial DNA methyltransferase enzymes (DNMTs) that convert only unmethylated CpG sites to a uracil after exposure to ultraviolet light, this technology can determine the methylation status across a genome. Because only unmethylated CpG sites are modified, the technology facilitates alignment to the genome. In addition, this method results in less DNA damage than bisulfite genome sequencing, in which all unmethylated cytosines are modified. This technology achieves a high efficiency of conversion with high site specificity in targeted approaches. It also requires only a small sample of DNA and has single-base resolution. Furthermore, without the requirement of sodium hydroxide used in bisulfite sequencing, longer DNA fragments are permissible, enabling better read depth with less starting material. The technology is available as a kit and is compatible with next-generation sequencing technology. This process may also be compatible with electrochemistry-based single-molecule sequencing technologies that are currently in development. Analytical software has also been developed and validated.
The technology has been validated using plasmid DNA with known sites of methylation, as well as more complex genetic material from mouse mammary carcinoma cell lines.
Tech Ventures Reference: IR CU13219, IR 2354
