*** 9560447 Collier This Small Business Innovation Research Phase I project will combine, for the first time, the use of the highly specific mutY cleavage enzyme to the detection of A/G and A/C point mutations in DNA with the highly efficient separation technique of capillary electrophoresis (CE) by UV absorbance detection. In biomedical research and genetic diagnostics, rapid, reliable detection of point mutations in limiting amounts of DNA, as well as, the identification of the location and nature of such point mutations is essential. Automated, direct on-line detection with CE using three orders of magnitude lower quantities of sample, renders CE more appropriate for rapid genetic screening techniques. The majority of previously investigated techniques to detect point mutations in DNA rely on conformational polymorphisms or require additional additives for separation in order to establish the presence of mutations, making them more dependent on DNA sequence and fragment size, as well as, electrophoretic experimental conditions. The use of the highly specific mutY cleavage enzyme will provide more reliable and definitive evidence of point mutations, the presence or absence of cleavage fragments. By conventional slab gel electrophoresis, the mutY enzyme also has higher sensitivity in a direct comparison with sequencing analysis, able to detect as little as 1% mutant in a normal DNA background, confirming the utility of this technique in early detection of disease, particularly, p53 for cancer diagnostics. A newly developed and more advanced polysaccharide polymer solution, TreviSol(TM) CE will be applied as the separation matrix in Phase I, combining enhanced sensitivity of detection and separation selectivity relative to other commercially available polymer solutions. A variety of DNA fragment sizes and sequences will be investigated with the use of mutY enzyme to establish a minimal dependence on these parameters. Results of the new technology will be confirmed with slab gel electrophoresis and sequencing analysis. This new technique has the potential to become a more comprehensive genetic screening technique, since other members of a family of related, highly specific cleavage enzymes with complementary point mutation detection, may be incorporated, as well. This project will provide feasibility testing for later development of a test system for point mutation detection of biological samples, which is faster, more sensitive with limiting amounts of DNA and more accurate for the position of the mutation. This superior product system will have a market advantage in a market of over $800 million, which is a portion of the DNA testing market. ***
