Analysis of Promoter Methylation: Bridging the Gap Between Association Studies and Routine Clinical Use

Analysis of gene-specific promoter methylation in human, patient-derived samples is limited by the availability of techniques that are i. feasible within a hierarchy of other tests and ii. routinely provide reliable results. Bisulfite conversion followed by PCR or sequencing provides useful information, but large quantities of patient-derived DNA are required as inputs, and success rates have been sufficient for landmark association studies but less so for individual analyses. Methylation-sensitive restriction enzymes as an alternative to bisulfite conversion introduce the same drawbacks and are not available for all sequences of interest. For these reasons, the conception and design of hybridization-based epigenotyping assays that assess the methylation status of particular gene promoters has been an area of recent effort, with the goal of translating an extensive body of knowledge gleaned from association studies into routine use in the clinical setting. The simplicity of these assays, comparable in complexity to enzyme-linked immunosorbent assays, is appealing, but none of the approaches investigated to date have provided the analytical sensitivity that is required for widespread clinical impact. A single molecule counting approach will be presented, along with the unique challenges of this assay that includes both nucleic acid hybridization and protein-nucleic acid molecular recognition events.