ULTRASENSITIVE NON-ISOTOPIC DETECTION SYSTEM FOR DNA

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Phase I of this proposal relates to the development of a non-isotopic detection system for DNA, specifically for use in Southern Blot, gene mapping experiments. The system will utilize 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. We expect to provide equal or lower sensitivities that those obtained with 32P or 35S labeled probes. In addition, we expect to obtain results in a few hours, compared to days with 32P. A major advantage of a non-isotopic probe system is the stability of the reagents. Unlike 32P 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