NSF Award
9220534
We wish to purchase a PhosphorImager instrument and establish a core facility to measure radioactivity with the greater sensitivity and resolution provided by storage phosphor technology. Several investigators at the Beckman Research Institute of the City of Hope have been active in the development of a new technique called ligation-mediated PCR (LMPCR) which, without cloning, gives sequence-level information about DNA nicks and subtleties of DNA structure in specific genes in total mammalian DNA. The procedure combines the use of nested oligonucleotides for specificity and exponential PCR for sensitivity. At our institute LMPCR is already being used for a number of purposes, among which are: (i) Genomic sequencing: (ii) studies on in vivo chromatin structure, including the vivo location or proteins on DNA and the effect of these proteins on DNA structure: (iii) in vivo formation and repair of chemically induced DNA adducts; and (iv) in vivo formation and repair of UV and oxygen radical- induced DNA adducts. The DNA adducts being studied are mutagenic and thus these studies are directly relevant to mutagenesis and cancer; the genomic sequencing studies are relevant to the Gerome Project; and the studies on protein footprints and chromatin structure are relevant to mammalian gene regulation and thus most disease. All of the LMPCR studies are presently limited by difficulties in quantitation. This is caused by the limited sensitivity and rage of responded of film. and by limitations in resolution between adjacent bands if alternatives to film are used. The requested equipment will enable the potential of LMPCR to be more fully reached. In addition to these novel, specific projects, the storage phosphor technology used by the PhosphorImager allows a true multiuser facility to be set up which will enhance the sensitivity, resolution, and accuracy of a great many of the standard experimental techniques such as Southern blots that are now in wide in use in virtually every laboratory at out institute . The rate limiting step, exposure of the storage phosphor screen, is done in each user's laboratory, and scanning the screen, with capture of the digitized image, only takes a few minutes on the core facility instrument. Thus, the bottleneck of current alternatives to film is eliminated and one instrument is able to service many users.
