This proposal describes the application of a novel photon counting device (PCD) to a problem in human genome mapping. The """"""""down-up"""""""" approach of multiplex genome mapping involves hybridization of radiolabeled end- specific DNA probes or oligonucleotides to cloned DNA from YAC, cosmid, lambda, or M13 libraries """"""""dot-blotted"""""""" onto nylon or nitrocellulose membranes. Exposure of the membranes to X-ray film results in hybridization signals signifying the location of overlapping clones. Adjacent negative clone positions are frequently counted as positive because of signal """"""""spill-over"""""""". The PCD will remedy this problem by replacing radioactive detection with chemiluminescent light detection. Rapid hybridization (1 hour) of biotinylated end-specific DNA probes to their targets on nylon filters is followed by binding of avidin-alkaline phosphatase conjugates. A chemiluminescent substrate for the enzyme is supplied and light is produced. The fiber optic component of the PCD is positioned directly over the location of cloned cosmid DNA and light intensity is measured. Since data is received and output as counts per second, an accurate representation of hybridization quality of probes to their targets will be achieved. This data will be useful for positioning cloned DNA segments relative to each other. This system will greatly simplify the current radioactive methods for multiplex genome mapping.
