IDENTIFYING GENOME CHANGES IN CANCER DEVELOPMENT

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DESCRIPTION: (Applicant's abstract) Human diseases arise from a complex interaction of DNA polymorphisms or mutations and environmental factors. Single nucleotide polymorphisms (SNPs) have recently been identified as potentially powerful means for genetic typing, and are predicted to supersede microsatellite repeat analysis as the standard for genetic association, linkage, and mapping studies. Thus, there is an urgent need to develop robust tools for the rapid analysis of SNPs. Our laboratory has used thermostable ligase to successfully detect and quantify a wide range of polymorphisms and mutations in known oncogenes and tumor suppressor genes. The ligase detection reaction (LDR) combined with multiplex PCR reactions, has also been used to detect multiple polymorphisms for human identification. Thus, the methods developed in our laboratory are ideally suited for rapid scoring of single nucleotide polymorphisms throughout the genome. The first goal of this proposal is to extend quantitative ligase-based detection to score SNPs on a new type of universal DNA array. As a model system, we plan to quantify gene amplification or loss of heterozygosity in the c-myc, K-ras, APC and p53 genes in microdissected tissue from colon tumors using LDR and DNA arrays. This approach will be used to help identify a new tumor suppressor gene at the 7q31 loci which is deleted in 80% of colorectal cancers. A second goal of this proposal is to use a thermostable Endonuclease V mismatch cleavage enzyme / thermostable DNA ligase combination to rapidly identify and precisely characterize unknown coding region SNPs. Such SNPs are present in known cancer genes at low frequency, and carriers are at a significantly higher risk of developing breast, ovarian, prostate or colon cancer. Using enzymatic methods to identify SNPs in single or pooled samples, we expect to greatly accelerate discovery of SNPs in cancer susceptibility genes. Enzymatic discovery/identification combined with universal array-based scoring represents an integrated approach to harness the power of single nucleotide polymorphisms for genetic studies, and may ultimately lead to a reduction of cancer burden through early screening and prevention programs.