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，特别是用于Southern印迹，基因 绘图实验 该系统将利用一种荧光基质 对于碱性磷酸酶，AMPPD，其提供最先进的检测 能力的 试剂盒将被设计为酶促掺入生物素 分为短寡探针和大双链探针。 协议或 使用链霉亲和素碱性磷酸酶检测这些探针的试剂， 将提供AMPPD。 专用封闭试剂和条件 将开发用于最小化背景信号的设计。 的 来自AMPPD的荧光信号将在X射线胶片和X射线照相机上被检测到。 即成黑白白色胶片。 我们希望提供同等或更低的 灵敏度高于32P或35S标记探针。 在 此外，我们希望在几个小时内获得结果，而不是几天， 32便士。非同位素探针系统的一个主要优点是稳定性， 试剂。 与32P标记的探针不同，生物素化的探针具有非常高的生物活性。 保质期长。 该计划的第二阶段将研究进一步提高灵敏度。 此外，还将介绍一种用于荧光印迹直接数字成像的系统。 发达

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