IMPROVED PROCEDURE FOR GENETIC ANALYSIS

STRATEGIES ARE PROPOSED FOR DEVELOPING SENSITIVE, RAPID AND EASY TO USE METHODS FOR NONRADIOACTIVE DETECTION OF DNA: DNA HYBRIDS USING BIOTIN LABELED DNA PROBES. THE APPROACHES ARE DIRECTED TOWARD INCORPORATING A GREATLY INCREASED NUMBER OF BIOTIN MOLECULES INTO DNA MOLECULES ASSOCIATED WITH THE HYBRIDIZING PROBE. SINCE THE ENZYMATIC COLOR PRODUCING DETECTION OF HYBRIDIZING SEQUENCES IS DEPENDENT ON DETECTION OF BIOTIN, A MARKED INCREASE IN BIOTIN MOLECULES, I.E., REPORTER MOLECULES, ASSOCIATED WITH HYBRIDIZED PROBE SHOULD RESULT IN A MARKED INCREASE IN SENSITIVITY. ONE APPROACH THAT WE WILL EMPLOY IS A CONCEPT IN WHICH ONE BIOTINYLATED PROBE WILL SUFFICE FOR THE DETECTION OF ANY SEQUENCE CLONED IN THE APPROPRIATE VECTOR ("UNIVERSAL DETECTION"). ANOTHER IS TO DEVELOP METHOD(S) FOR THE USE OF SINGLE STRANDED PHAGE DNA PROBES LABELED WITH BIOTIN BY PRIMER EXTENSION. SINCE SENSITIVITY CAN ALSO BE INCREASED BY IMPROVEMENTS IN THE BIOTIN DETECTING ENZYMATIC COLOR PRODUCING COMPONENT, WE PROPOSE METHODS TO DEVELOP GREATER SENSITIVITY FOR THIS COMPONENT. THESE PROCEDURES ARE BEING DEVELOPED FOR THE USE OF DIRECT EXAMINATION OF DNA FOR GENETIC ANALYSIS USING SOUTHERN BLOT TECHNOLOGY. THE DEVELOPMENT OF SAFE, SENSITIVE AND EASY TO USE SOUTHERN BLOT PROCEDURES WILL GREATLY EXTEND THE CLINICAL USE OF GENETIC ANALYSIS.