ULTRASENSITIVE NON-ISOTOPIC DETECTION SYSTEM FOR DNA

该提案的第一阶段涉及开发一种非同位素 DNA检测系统，专用于Southern Blot基因检测 测绘实验。该系统将利用化学发光衬底 对于碱性磷酸酶，AMPPD，提供最先进的检测 能力。试剂盒将被设计成酶促结合生物素 分成短的寡聚探针和大的双链探针。协议或 用链霉亲和素碱性磷酸酶检测这些探针的试剂 将提供AMPPD。专用封闭剂及条件 设计以最大限度地减少背景信号将被开发出来。这个 来自AMPPD的化学发光信号将在X射线胶片和 瞬间黑白胶片。我们希望提供相同或更低的 用32P或35S标记探针获得的灵敏度。在……里面 此外，我们预计在几个小时内就能得到结果，而不是几天 32便士。非同位素探针系统的一个主要优点是 试剂。与32P标记的探针不同，生物素化的探针具有非常 保质期长。 该计划的第二阶段将调查敏感度的进一步增加。 此外，用于化学发光斑点的直接数字成像的系统将是 开发

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