Bisulfite Conversion and DNA Methylation
DNA methylation is widely recognized as a fundamental epigenetic mechanism that is essential for development and gene expression, and most commonly occurs at a cytosine base that is immediately followed by a guanine base (annotated in literature as a “CpG” site). At these CpG sites, cytosines are either methylated (-CH3 group) or unmethylated at the the 5′ carbon. Each genomic copy can be either Methylated or Unmethylated at any given site; therefore the percent methylation is an aggregate of all genomic copies. In addition, “global” or “regional” methylation levels can be determined by analyzing areas that contain multiple “CpG” sites, also known as “CpG Islands”.
In cancer genetic research, hyper- or hypo-methylation of these “CpG Islands – when compared to normal cells – can result in over or under expression of tumor suppressor or oncogenes. To determine the methylation levels at a region of interest, genomic DNA is treated with sodium bisulfite, which converts any unmethylated cytosine to uracil, and any methylated cytosine remains a methylated cytosine. During PCR (which converts any uracil base to thymine) and subsequent pyrosequencing, the ratio of cytosine and thymine present at each CpG site is quantified, and reflects the methylation level of that site in genomic DNA.