DIAGNOSING MUTATIONS W/ THERMOPHILIC DNA REPAIR ENZYMES

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DESCRIPTION: (Adapted from the investigator's abstract) Trevigen has developed a patented technology, the Mismatch Identification DNA Analysis System (MIDAS), for the detection of point mutations without PCR amplification. MIDAS employs DNA mismatch repair enzymes (MREs) to cleave an oligonucleotide probe at sites of mismatches when hybridized to mutated target DNA. The investigators propose to optimize MREs and increase their sensitivity for successful commercialization. Based upon the thermodynamics of enzyme kinetics, their approach is to search for MREs from thermophilic organisms. The investigators have partially purified a MRE from the extreme thermophile, Thermotoga maritima, that recognizes and cuts at all possible base mismatches, operates at high temperatures, survives up to 20s temperature cycles from 65C to 95C, and works well in MIDAS and in heteroduplex analysis for scanning for unknown mutations. Specific Aims 1 and 2 of this Phase 1 SBIR address the need to functionally purify, characterize, and, if necessary, clone this MRE. Specific Aim 3 examines the utility of this thermophilic MRE in the detection of known point mutations in the APC and catenin genes in human colorectal cancer cell lines. The investigators predict that successful optimization of these MREs will quickly translate into commercial products for the detection of known point mutations in a variety of disease-causing genes, without the need for PCR amplification, and for scanning for unknown mutations and single nucleotide polymorphisms by heteroduplex analysis. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE