The goal of this project is to develop a sensitive, bisulfite-free assay for detecting methylation of Individual or multiple CpG islands. The assay will use 2 ng or less of genomic DNA, allow detection of 50 or fewer copies of a methylated island, and the analysis of several different islands from a single sample. The assay will first involve isolation? of methylated DNA from total DNA using Ribomed's GST fusion protein containing the methyl CpG binding domain from MBD2b. This protein has high speCificity for methylated DNA. The assay will utilize Ribomed's isothermal signal generation process of Abscription. Signals can be generated for detection using mass spectroscopy, fluorescence detection, thin layer chromatography and UV . or fl uorescence imaging, or fluorescence using real time PCR instruments. The assay wi!! therefore be adaptable for use by basic researchers or clinical labs for the detection of single islands or multiple islands Simultaneously. This type of multi-biomarker analysis with small amounts of patient DNA is important for early disease diagnostics from bodily fluids, molecular profiling of tumors and post-treatment monitoring of patients~ The elimination ofthe harsh bisulfite treatment prevents the destruction of sometimes limited and non-replaceable patient samples.
