During Phase II of this project we will continue our efforts to develop a rapid and efficient method for detecting mutations on the human p53, msh2 and mlh1 genes. In Phase II we will demonstrate: * the design and synthesis of a single chip each for genotyping each of the genes. Phase I chip designs entail the use of 1, 3 and 2 chips for the p53, msh2 and mlh1 genes, respectively. * the design of specialized probe arrays for each gene which will ensure the detection of the most frequently observed multinucleotide mutations. * the development of multiplex PCR assay conditions for each gene permitting the co-amplification of the 10, 16 and 19 exons and adjoining splice junctions for the p53, msh2 and mlh1 genes, respectively. * the detection of both homozygous and heterozygous mutations using alternative probe designs, hybridization/wash conditions and detection approaches. Previously characterized genomic and cDNA clone mixtures will serve as characterized targets. * the performance of each of the chips to genotype a collection of transformed cell lines and tumor tissues which have also been analyzed by dideoxynucleotide sequencing methodology. Phase II results will provide optimized high density arrays for Phase III studies which will focus on retrospective and prospective genotype analyses of staged colorectal tumors.
Approximately 150,000 new colorectal cancer cases are diagnosed every year. Genotyping of multiple genes will needed to provide information concerning predisposition, prognosis and therapeutic management. Families with hereditary nonpolyposis colorectal cancer (HNPCC) has been characterized to have lesions in the DNA repair genes msh2 and mlh1, as well as the p53 tumor suppressor gene. Genotyping of these three genes can serve as a multigenic surveillance panel for this disease.
