This proposal describes a set of reagents which will make gene mapping to chromosomes and chromosome bands, as well as gene finding exercises, accessible to any molecular biologist or geneticist familiar with filter hybridization techniques. These reagents, called the Gene.ID system, are a set of """"""""dot-blots"""""""". The """"""""Chromosome.ID"""""""" blot contains DNA corresponding to each chromosome spotted in a dot-blot format and the """"""""Band.ID"""""""" blot contains DNA corresponding to each chromosome band also spotted in a dot blot format. These chromosome-specific and band-specific DNAs will be prepared by dissecting and pooling 10 copies of each chromosome, or each chromosome band, followed by PCR amplification using a universal degenerate primer. The primer is 21 based long and contains 10 nucleotides of degenerate DNA, so that at low annealing and extension temperatures, virtually any DNA is amplifiable. These filters will also users to map any cloned DNA to a chromosome or a chromosome band with standard filter hybridization techniques. The Phase I feasibility demonstration of the Chromosome.ID and Band.ID blots will consist of preparation of several """"""""mini""""""""-Chromosome.ID that contain chromosome- specific DNA prepared from mouse chromosomes 11, along with controls of chromosomes 2 and 9. These blots will be hybridized with a series of single copy gene probes known to map to chromosome 11. Conditions for mapping complex probes such as cosmids, YACs, or lambda clones will also be investigated, thus increasing the spectrum of potential users. Finally, the feasibility of physically removing repetitive sequences from the chromosome-specific DNAs prior to application to the Gene.ID blots will also be examined during Phase I. Removal of the repetitive sequences ultimately will make the blots more """"""""user friendly"""""""" because repeat-free DNA will allow the users to hybridize their probes without prior suppression of repetitive sequences using competitor DNA.
The Gene.ID system will greatly accelerate and simplify gene mapping and gen finding in molecular genetics laboratories worldwide, in part because the blots will be very easy to use, and in part because the system restores independence to laboratories interested in gene mapping.
