Derivation of a single fragment radiation hybrid contig of chromosome 5 from a new mapping panel of 178 radiation hybrids

A radiation hybrid mapping panel for chromosome 5 was created by selecting clones from a fusion of gamma irradiated HHW105, a human hamster hybrid containing an entire chromosome 5 as its only human genetic component with an HPRTCHO cell line. Clones that were positive for human content by RS PCR were harvested in situ on chamber slides for FISH analysis. Metaphase and interphase nuclei were probed with total human DNA to determine the quantity and configuration of human chromosome fragments in each cell line. Image and statistical analyses were done using Oncor`s OIS and V150 software. DNA from hybrids that were positive for human DNA was subjected to IRS-PCR to generate probes for FISH using normal lymphoblast prometaphases as template to locate the chromosomal map positions of the chromosome 5 fragments in each hybrid. A contig panel of approximately 40-50 single fragment radiation hybrids is being derived from this analysis and is expected to span the full length of chromosome 5 in minimally overlapping {approximately}5 Mbp segments. Hybrids with fragments from the centromeric and telomeric regions will be identified using probes specific for the centromeric region of chromosome 5 and the telomeric regions of 5p and 5q. Further tests willmore » determine the degree of overlap and STS content of each hybrid. This contig will provide a new reagent for the Human Genome Project and will be used to quickly generate and delineate regional sets of clones from various sources such as YAC, cosmid and cDNA libraries. The characterization of the complete RH mapping panel by FISH will also enhance its utility by adding a new dimension to standard RH mapping.« less