Serial Pyrosequencing for Quantitative DNA Methylation Analysis

Reduce, Reuse, Recycle Epigenotyping has become increasingly prevalent as a result of the accumulating evidence linking aberrant DNA methylation with human disease. Specifically, methylation of CpG islands, which are present in the promoters and first exons of many genes, appears to be an early step in tumorigenesis. There are a plethora of techniques for standard genotyping; the same is true for epigenotyping methods. That said, one popular strategy is pyrosequencing of bisulfite-treated DNA. The well-established bisulfite strategy results in the conversion of cytosine, but not methylated cytosine, to uracil via deamination by bisulfite under acidic conditions. Pyrosequencing excels at direct quantitative sequencing, so its application to bisulfite-treated DNA can yield robust measurements of methylation. However, pyrosequencing is limited by sequence read length. As a result, only a few CpGs can be assessed in any one pyrosequencing reaction. This drawback can be expensive; more importantly, if the samp...