NOVEL, RAPID RFLP ANALYSIS WITH FLOURESCENCE DETECTION

GENETIC MARKERS GENERATED BY DNA SEQUENCE POLYMORPHISM WITH RESTRICTION ENZYMES HAVE AIDED IN CONSTRUCTING A DETAILED GENETIC LINKAGE MAP FOR THE HUMAN. MORE THAN 400 POLYMORPHIC DNA MARKERS FOR HUMAN CHROMOSOMES HAVE BEEN DESCRIBED. THESE INCLUDE SOME HUMAN GENETIC DISORDERS SUCH AS DUCHENNE MUSCULAR DYSTROPHY, POLYCYSTIC KIDNEY DISEASE, AND CYSTIC FIBROSIS. AS ADDITIONAL GENETIC MARKERS ARE GENERATED BY RESTRICTION FRAGMENT-LENGTH POLYMORPHISM (RFLP)ANALYSIS, MORE HUMAN GENETIC CHARACTERISTICS WILL BE IDENTIFIED AND DIAGNOSED FOR DISORDERS. A NOVEL METHOD IS PROPOSED FOR EFFICIENT AND NONISOTOPIC RFLP ANALYSES. SYNTHETIC OLIGOMERIC DNA PROBES WILL BE PREPARED AND LABELED WITH FLUOROPHORES THAT ARE UNIQUE TO EACH PROBE. THE LABELED PROBES WILL BE SIMULTANEOUSLY HYBRIDIZED TO GENOMIC DNA FRAGMENTS GENERATED WITH A RESTRICTION ENZYME. THE HYBRIDIZED PROBES WILL SERVE AS PRIMERS FOR THE SEQUENCE TO ELONGATE DNA. THE ELONGATED DNAFRAGMENTS WILL BE SEPARATED BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY, USING A NEWLY INTRODUCED GEN-PAK FAX COLUMN AND WILL BE DETECTED WITH A FLUORESCENCE DETECTOR . THE DATA WILL BE COMPARED TO THE SOUTHERN BLOT TECHNIQUE. THE PROPOSED ANALYTICAL METHOD IS NOVEL, EFFICIENT, AND SAFE FOR LARGE NUMBERS OF ROUTINE RFLP ANALYSES.