RAPID DETECTION OF GENETIC MUTATIONS BY ELECTROPHORESIS

THIS PROJECT WILL DEVELOP A RELIABLE, TECHNICALLY SIMPLE GEL ELECTROPHORESIS SYSTEM FOR IDENTIFYING MUTATIONS IN CHARACTERIZED GENES. THIS METHOD WILL DETECT SINGLE-BASE CHANGES IN PCR AMPLIFIED GENOMIC DNA AND WILL FACILITATE SCREENING FOR DISEASE RELATED MUTATIONS. AS THE NUMBER OF CLONED GENES RELATED TO HUMAN DISEASE GROWS, THE ABILITY TO IDENTIFY INDIVIDUALS HETEROZYGOTIC AT SPECIFIC ALLELES BECOMES INCREASINGLY MORE IMPORTANT. CURRENT METHODS FOR DETECTING SINGLE-BASE CHANGES EITHER FAIL TO IDENTIFY 50% OF THE MUTATIONS OR REQUIRE HIGHLY SKILLED PERSONNEL, TOXIC ORGANIC SOLVENTS AND EXPENSIVE APPARATUSES. SIMPLER METHODS, BASED ONTHE DETECTION OF SINGLE STRANDED OR HETERODUPLEXED DNA CONFORMATIONAL MOBILITY SHIFTS HAVE BEENDESCRIBED. THE OBSERVED MOBILITY SHIFTS IN NON-DENATURING GELS ARE SLIGHT AND INTERPRETATION OF RESULTS IS POTENTIALLY AMBIGUOUS. IMPROVED RESOLUTION OF HETERODUPLEX DNA HAS BEEN SHOWN WHEN HYDROLINKTM GELS ARE SUBSTITUTED FOR POLYACRYLAMIDE GELS. DURING PHASE I, WE WILL USE ITSLIBRARY OF MONOMERS AND CROSS-LINKERS TO DEVELOP A GEL WITH INCREASED RESOLVING POWER. PHASE II WILL DEVELOP DISEASE SPECIFIC PROTOCOLS AND REAGENTS FOR DIAGNOSTIC APPLICATIONS.