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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
Construction of DNA Libraries From Flow Sorted Human Chromosomes
Larry L. Deaven, Mary K. McCormick, Deborah L. Grady, Donna L. Robinson, Judy M. Buckingham, Nancy C. Brown, Evelyn W. Campbell, Mary L. Campbell, John J. Fawcett, Phil Jewett, Jonathan L. Longmire, Adelmo Martinez, Linda J. Meincke, Pat L. Schor, and Robert K. Moyzis
Center for Human Genome Studies and Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545
We have constructed a series of DNA libraries from flow sorted chromosomes. Small insert, complete digest libraries cloned into the EcoRI insertion site of Charon 21A are available from the American Type Culture Collection, Rockville, MD. Partial digest libraries cloned into cosmid (sCos1) or phage (Charon 40) vectors have been constructed for chromosomes 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, X and Y. Purity estimates by in situ analysis of sorted chromosomes, flow karyotype analysis, and plaque or colony hybridization indicate that most of these libraries are 90-95% pure. Additional cosmid library constructions, 5-10X arrays of libraries into microliter plates, and high density membrane arrays of libraries are in progress.
Recently we have completed YAC libraries for chromosomes 5, 9, 16, and 21. These libraries are made from complete DNA digests using the rare cutters Clal, Sacll, Eagl, or Notl/Nhel. The average insert size is ~200 kb, and chimera frequencies are low (1-10%).
Libraries have also been constructed using M13 or bluescript vectors (chromosomes 5, 7, 17) to generate STS markers for the selection of chromosome-specific inserts from total genomic YAC libraries.
Because of the advantages of insert size and stability associated with BAC and PAC cloning systems, we are currently attempting to adapt pBAC108L and pCYPAC1 vectors for use with flow-sorted chromosomal DNA. This work was supported by the U.S. Department of Energy under contract W-7405-ENG-36.