Detection of Mutations in Human P53, MSH2, MLH1 Genes

Our aim is to develop DNA chips which can detect and characterize mutations simultaneously in the psuppressor gene and the MSH2 and MLH1 mismatch repair genes. These chips will be used to correlate ttype of mutations in normal and tumor cells obtained from persons affected with hereditary and sporaIn Phase I, we will 1) design/synthesize DNA chips complementary to intron/exons 5 - 8 of the p53 ge16 of the msh2 gene (2727 bp) and exons 1-19 of the mlh1 gene (2268 bp), 2) develop PCR amplificatioeither genomic (using multiplex PCR) or mRNA (using RT - PCR) copies of each of the genes, 3) developrotocols for the normal alleles for each of the three genes to measure the resequencing accuracy ofPhase II, clones containing mutations for p53, MSH2 and MLH1 will serve as model targets. Detectioncompound heterozygotes will be demonstrated using mixtures of cloned controls. Finally, we will testMLH1 chips against nucleic acids obtained from previously characterized and uncharacterized patienttissues) in a clinical laboratory setting.