ULTRASENSITIVE NONISOTOPIC DETECTION SYSTEM FOR DNA

PHASE I OF THIS PROJECT RELATES TO THE DEVELOPMENT OF A NONISOTOPIC DETECTION SYSTEM FOR DNA, SPECIFICALLY FOR USE IN SOUTHERN BLOT, GENE-MAPPING EXPERIMENTS. THE SYSTEM WILLUTILIZE A CHEMILUMINESCENT SUBSTRATE FOR ALKALINE PHOSPHATASE, AMPPD, WHICH PROVIDES STATE-OF-THE-ART DETECTION CAPABILITIES. KITS WILL BE DESIGNED TO ENZYMATICALLY INCORPORATE BIOTIN INTO BOTH SHORT OLIGOPROBES AND LARGE DOUBLE-STRANDED PROBES. PROTOCOLS OR REAGENTS DETECTING THESE PROBES USING STREPTAVIDIN ALKALINE PHOSPHATASE AND AMPPD WILL BE PROVIDED. SPECIALIZED BLOCKING REAGENTS AND CONDITIONS DESIGNED TO MINIMIZE BACKGROUND SIGNAL WILL BE DEVELOPED. THE CHEMILUMINESCENT SIGNAL FROM AMPPD WILL BE DETECTED ON BOTH X-RAY FILM AND INSTANT BLACK-AND-WHITE FILM. IT IS EXPECTED TO PROVIDE EQUAL OR LOWER SENSITIVITIES THAN THOSE OBTAINED WITH (32)P- OR (35)S-LABELED PROBES. IN ADDITION, RESULTS WILL BE OBTAINED IN A FEW HOURS, COMPARED TO DAYS WITH (32)P. A MAJOR ADVANTAGE OF A NONISOTOPIC PROBE SYSTEM IS THE STABILITY OF THE REAGENTS. UNLIKE (32)P-LABELED PROBES, BIOTINYLATED PROBES HAVE VERY LONG SHELF LIFES. PHASE II OF THIS PROGRAM WILL INVESTIGATE FURTHER INCREASES IN SENSITIVITY. ALSO, A SYSTEM FOR DIRECT DIGITAL IMAGING OF CHEMILUMINESCENT BLOTS WILL BE DEVELOPED.