title: Constructing chromosome- and region-specific cosmid maps of the human genome.
abstract: A chromosome-specific ordered set of cosmids would be a significant contribution toward understanding human chromosome structure and function. We are developing two parallel approaches for creating an ordered cosmid library of human chromosome 19 and other selected subregions of the human genome. The "bottom up" approach is used to establish sets of overlapping cosmids as islands or "contigs" along the chromosome, while the "top down" approach, using pulsed-field gel electrophoresis and yeast cloning, will establish a large-fragment map and close the inevitable gaps remaining from the "bottom up" approach. Source DNA consists of a single homolog of chromosome 19 from a hamster--human hybrid cell and human fragments cloned in yeast artificial chromosomes. We have constructed cosmid libraries in a vector that facilitates cloning small amounts of DNA, allows transcription of the insert termini, and contains unique sites for partial-digest mapping. Computer simulations of cosmid contig building suggest that near-optimal efficiency can be achieved with high-density restriction fragment digest schemes that can detect 20-30% overlap between cosmids. We developed the chemistry and data analysis tools to compare the ordering efficiencies of several cosmid restriction digest fingerprinting strategies. Restriction fragments from a four-cutter digest are labeled with a fluorochrome, separated by polyacrylamide gel electrophoresis, and detected after laser excitation as they traverse a fixed point in the gel. We have also developed the software to rapidly process the output signal to define and analyze the fragment peaks. Up to three cosmids (or three different digests of the same cosmid) plus a size standard are analyzed simultaneously in a single gel lane.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Biomedical Sciences Division, Lawrence Livermore National Laboratory, CA 94550.
authors.forename: A V
authors.initials: AV
authors.lastname: Carrano
authors.forename: P J
authors.initials: PJ
authors.lastname: de Jong
authors.forename: E
authors.initials: E
authors.lastname: Branscomb
authors.forename: T
authors.initials: T
authors.lastname: Slezak
authors.forename: B W
authors.initials: BW
authors.lastname: Watkins
mesh_terms: D000818:Animals; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D002888:Chromosomes, Human, Pair 19; D003001:Cloning, Molecular / Q000379:methods; D003360:Cosmids; D006224:Cricetinae; D005544:Forecasting; D015723:Gene Library; D015894:Genome, Human; D016045:Human Genome Project; D006801:Humans; D006822:Hybrid Cells; D012441:Saccharomyces cerevisiae / Q000235:genetics