Profiling methylation of individual CpG sites

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DESCRIPTION (provided by applicant): The CpG hypermethylation of genes are involved in tumor initiation and progression, and widely accepted as a marker and prognostic indicator of cancer. The density of methylation in a certain CpG island region has been demonstrated to be closely related to gene silencing, but not individual target CpG sites. The heterogeneous methylation patterns are associated with tumorigenesis, histological subtypes and cell origin of cancers. Most of CpG island hypermethylation is assumed based on the study of only 2 to 4 CpG sites within an island region, which may not be conclusive. We will develop a degenerate oligonucleotides ligation assay (DOLA) to map methylation status of a series of CpG sites within a promoter region upon bisulfite treatment. In the assay, we will design and synthesized two pairs of oligos to detect an individual target CpG site for the methylated (C) or unmethylated (T) states in a 40bp region, and subsequently ligate two oligonucleotides. Since the neighbor CpG sites might be very close to the target CpG site and uncertain on methylation status, we will design the degenerated R (G or A), complementary to methylated C or converted T to cover all of possibilities. We will set up the assay for E-cadherin promoter. we will design a series of unique tag sequences to each CpG site and use mircoarray analysis of the tag sequences to simultaneously measure the methylated status of individual CpG sites for five cancer-related genes, E-Cadherin, p15, p16, MGMT, and RASSF1A in five different cancer cell lines including normal and cancer breast cell lines, colon, lymphoma and lung cancer cell lines.