Research Project
6790133
DESCRIPTION (provided by applicant): The goal of this project is to develop a quantitative eDNA genotyping platform for measuring allelic gene expression. This will provide a tool for better understanding transcriptional regulation of the genome. Dysfunction in this regulation leads to a number of disease states including cancer. In particular, cancer genes are often activated or inactivated by genetic and epigenetie mechanisms leading to uncontrolled cell growth. Epigenetie mechanisms leading to aberrant transcriptional control include methylation of CpG islands, loss of imprinting, and chromatin modification. The proposed approach to eDNA genotyping involves direct hybridization of cDNA to a DNA oligonucleotide array and subsequent genotyping by an array-based allele-specific primer extension reaction. In addition to allelic expression, the array's utility in standard gene expression quantitation will also be demonstrated. Whole genome amplification of cDNA will be explored as a means to improve genotyping accuracy for low copy number transcripts. In phase I, a model system employing several hundred eDNA SNPs across a few hundred genes will be used on our Sentrix TM Array of Array TM platform. In phase II, the eDNA array will be scaled to our Sentrix BeadChip platform encompassing whole genome analysis of thousands of genes. To enable easy allele frequency calibration, the direct genotyping of gDNA on the eDNA array will also be explored. The culmination of these efforts will result in the whole genome allelic expression study of matched normal/tumor tissues to identify gene loci involved in tumor development.
