CHEMILUMINESCENT MULTIPRIMED DNA SEQUENCING

THE OBJECTIVE OF THIS STUDY IS TO IMPROVE THE EFFICIENCY OF PERFORMANCE OF A NONISOTOPIC DETECTION METHOD FOR DNA SEQUENCING. A CHEMILUMINESCENT DETECTION PROCEDURE USING 1,2-DIOXETANE SUBSTRATES FOR ALKALINE PHOSPHATASE HAS BEEN DEVELOPED. WITH THIS METHOD, DNA SEQUENCING REACTION PRODUCTS LABELED WITH BIOTINYLATED PRIMERS ARE IMAGED FOLLOWING ELECTROPHORESIS BY TRANSFER OF THE SEPARATED DNA FRAGMENTS TO NYLON MEMBRANE, WITH 1,2-DIOXETANE SUBSTRATES. UPON DEPHOSPHORYLATION, THESE ENZYMATIC SUBSTRATES DECOMPOSE AND EMIT LIGHT. IN PHASE I, THE FEASIBILITY OF INCREASING THE AMOUNT OF DNA SEQUENCE INFORMATION THAT CAN BE IMAGED FROM ONE MEMBRANE WILL BE ASSESSED BY SEPARATING MULTIPLE SETS OF DNA SEQUENCING REACTIONS IN EACH LANE OF THE GEL, TRANSFERRING THE DNA TO NYLON MEMBRANE, AND DETECTING EACH SET OF SEQUENCING REACTIONS INDIVIDUALLY. THIS IS ACCOMPLISHED BY UTILIZING MULTIPLE DNA SEQUENCING PRIMERS FOR THE DNA SEQUENCING REACTIONS. THE PRIMER FOR EACH REACTION WILL CONTAIN ITS OWN UNIQUE LABEL. EACH SET OF DNA SEQUENCING REACTIONS IS DETECTED WITH AN ALKALINE PHOSPHATASE CONJUGATE SPECIFIC FOR THE LABEL. THE PERFORMANCE OF VARIOUS LABELS AND ENZYME CONJUGATES IS EVALUATED. THE GOAL OF THE STUDY IS THE DEVELOPMENT OF OPTIMUM PROCEDURES FOR RAPID DETECTION OF EACH SET OF LABELED DNA FRAGMENTS. IF THE RESEARCH IS SUCCESSFUL, NEWLY DEVELOPED PROTOCOLS AND THE INDIVIDUAL REAGENTS WILL BE INCORPORATED INTO A RESEARCH KIT THAT WILL BECOME A USEFUL TOOL FOR MOLECULAR BIOLOGISTS FOR DNA SEQUENCING.