THE GENERATION AND REGIONAL LOCALIZATION OF 303 NEW CHROMOSOME-5 SEQUENCE-TAGGED SITES

With the ultimate goal of creating a sequence-ready physical map of al l of chromosome 5, 303 new human chromosome 8-specific STS markers hav e been systematically generated and regionally ordered. Chromosome 5 D NA prepared from flow-sorted chromosomes was digested with restriction enzymes BamHI and HindIII and cloned in bacteriophage M13mp18. Random clones were sequenced, and appropriate PCR deoxyoligomers were synthe sized. An acceptable sequence-tagged site (STS)-PCR assay yielded the appropriate size amplification product from both total human DNA and h ybrid cell line DNA containing only human chromosome 5. Each STS has b een regionally localized by breakpoint analysis using a set of hybrid cell panels consisting of natural deletions or translocations of human chromosome 5. This hybrid cell panel was able to localize the STSs to 1 of 51 bins on the short arm and 1 of 15 bins on the long arm. The S TS markers appear to be randomly distributed along the length of this 194-Mb chromosome. The current overall density of these markers (appro ximately 1 STS/640 kb), combined with the numerous PCR-based physical and genetic markers generated by other groups, will provide sufficient ''nucleation points'' for YAC contig assembly and verification in any region of human chromosome 5. (C) 1996 Academic Press, Inc.