HPLC HYBRIDIZATION ANALYSIS WITH FLUORESCENT DNA PROBES

THE IMMEDIATE GOAL IS TO DEVELOP AND OPTIMIZE AN AUTOMATED, SIMPLE HYBRIDIZATION TECHNIQUE USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) AND LIQUID HYBRIDIZATION TO REPLACE THE TIME-CONSUMING AND LABOR-INTENSIVE PROCEDURES OF GEL ELECTROPHORESIS, BLOTTING,AND SOLID-PHASE HYBRIDIZATION CURRENTLY NECESSARY FOR SUCH A ROUTINE AND BASIC FUNCTION AS HYBRIDIZATION. PRELIMINARY EXPERIMENTS DEMONSTRATED THAT THIS TECHNIQUE, WHICH IS ALSO QUANTITATIVE, WORKS WITH OLIGO-PROBES AND OLIGO-TARGET DNA USING PERMEATION CHROMATOGRAPHY. IT MUST BE DEMONSTRATED THAT IT WILL WORK WITH LARGE, MULTISPECIES NUCLEIC ACID TARGETS. THE NEW TECHNIQUE (AUTOSAMPLER) WOULD ALLOW FOR MULTIPLE EXPERIMENTS, ENABLING MULTIPLE STRINGENCY TESTS PER UNIT TIME THAT WOULD OTHERWISE BE IMPOSSIBLE WITH THE CONVENTIONAL METHOD. THE SYNTHETIC OLIGO-PROBES ARE NONRADIOACTIVE AND HAVE THE ADVANTAGE OF BETTER HYBRIDIZATION DISCRIMINATION OVER CONVENTIONAL ONES. A HEALTH-RELATED, LONG-TERM GOAL IS TO USE THE HPLC HYBRIDIZATION TECHNIQUE TO DEVELOP AN AUTOMATED, SENSITIVE, RAPID, DIRECT DIAGNOSTIC METHOD TO DETECT, QUANTITATE, AND DIFFERENTIATE BETWEEN SIMILAR TYPES (E.G., VIRAL, ONCOGENE, OR GENETIC DEFECT) OF NUCLEIC ACID (DNA OR RNA). CUSTOM-SYNTHESIZED FLUORESCENT OLIGO-PROBES WOULD BE DEVELOPED FOR THE ABOVE PURPOSES TO BE USED IN THIS SYSTEM. THESE WOULD BE COMMERCIALLY AVAILABLE AT THE END OF PHASE II.