IMPROVEMENTS IN DNA SEQUENCING METHODS BY INCORPORATING CHEMILUMINESCENT DETECTION

PHASE I WILL INVOLVE AN INVESTIGATION OF CHEMILUMINESCENT DETECTION IN DNA SEQUENCING. WITH THE ADVENT OF ENZYMATICALLY INDUCED CHEMILUMINESCENCE FROM STABLE PRECURSORS AND IMPROVED LIGHT METERING INSTRUMENTATION, ULTRASENSITIVE DETECTION OF A VARIETY OF SPECIES UNDER MANY CONDITIONS IS NOW POSSIBLE. THE IMPLEMENTATION OF CHEMILUMINESCENT SUBSTRATE TECHNOLOGY IN DNA SEQUENCING WILLLEAD TO IMPROVEMENTS IN THE EFFICIENCY OF SEQUENCE ANALYSIS,STABILITY OF REAGENTS, SENSITIVITY OF DETECTION, AND COST. TO ASSESS THE VIABILITY OF CHEMILUMINESCENCE TECHNOLOGY IN DNA SEQUENCING, THE INVESTIGATION WILL FOCUS FIRST ON THE PROBLEM OF ALKALINE PHOSPHATASE LABELING. BIOTINYLATED PRIMER STRANDS AND OTHER NECESSARY REAGENTS WILL BE USED IN THE DIDEOXY SEQUENCING REACTIONS. ELECTROPHORETICALLY SEPARATED SEQUENCED DNA WILL BE TRANSFERRED TO MEMBRANES REACTED WITH AVIDIN-ALKALINE PHOSPHATASE AND DETECTED WITH ADAMANTYL- 1,2- DIOXETANE PHOSPHATE. SIMILAR EXPERIMENTS WILL BE PERFORMED USING MAXAM- GILBERT SEQUENCING METHODOLOGY. IN PHASE I, THE LUMINESCENT DETECTION OF NUCLEOTIDES WILL BE ACCOMPLISHED WITH X-RAY AND INSTANT FILMS. THE ANTICIPATED IMPROVEMENTS IN CHEMILUMINESCENCE-BASED DNA SEQUENCING WILL BE FULLY EXPLORED IN PHASE II WITH THE IMPLEMENTATION OF FULL PERFORMANCE PROTOCOLS, COUPLED WITH THE DESIGN AND PROTOTYPEGENERATION OF A SIMPLE, INEXPENSIVE, HIGH THROUGHPUT LUMINESCENT GEL/BLOT SCANNER.