Research Project
3500503
The objectives of the proposed work is to develop novel chemiluminescent catalysts to enable visualization of oligonucleotides directly in electrophoretic gels. Presently, streptavidin-horseradish peroxidase (MW greater than or equal to 100,000) can be employed to label biotinylated DNA in agarose gels, with a detection limit of 5-10 picograms, but cannot be used in DNA sequencing gels. DNA labeling and detection sensitivity is limited by the size of the affinity-enzyme complex used to catalyze the chemiluminescent reaction. In order to facilitate labeling DNA in sequencing gels, a smaller chemiluminescent catalyzing moiety coupled to a DNA intercalator is proposed. This low molecular weight label will diffuse easily into a gel and bind to the DNA; the chemiluminescence reaction is catalyzed upon addition of chemiluminescent producing reagents. Not only will the DNA not need to be biotinylated prior to electrophoresis, but the chemiluminescent signal can be recorded directly from the gel using film. The anticipated benefits of this technology include the increased expediency of DNA sequencing, the elimination of hazardous labeling methods, and the potential for low cost and rapid automation.
