Progress Towards the Construction of BAC Libraries from Flow Sorted Human Chromosomes*
Jonathan L. Longmire. Nancy C. Brown, Deborah L. Grady, Evelyn W. Campbell, Mary L. Campbell, John J. Fawcett, Phil Jewett, Robert K. Moyzis, and Larry L. Deaven
Life Sciences Division and Center for Human Genome Studies, Los Alamos National Laboratory, Los Alamos, NM 87545
Over the course of the National Laboratory Gene Library Project (NLGLP) we have constructed a series of DNA libraries from flow sorted human chromosomes. Small insert, complete digest libraries cloned into the EcoRI site of Charon 21 A 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 microtiter plates, and high density membrane arrays of libraries are in progress. We have also constructed a limited number of human chromosome-specific YAC libraries. In addition, we have constructed chromosome-specific M13 or pBluescript libraries for generating STS markers and for selection of chromosome-specific inserts from total genomic YAC libraries.
Because of the advantages of large insert size and stability associated with BAC cloning systems, we are currently attempting to adapt the pBelloBAC vector for use with flow sorted human chromosomes. The technical challenges involved in accomplishing this goal include developing methodologies that will allow predictable partial digestion of very small masses of DNA embedded in agarose plugs and improving BAC cloning efficiencies to allow construction of libraries from microgram quantities of chromosomal DNA. Currently, we are making modifications to pBelloBAC that include adding Sacll and Clal restriction sites into the cloning region of the vector. In addition, we have modified and significantly increased the efficiency of methods that are used to recover flow sorted chromosomes into agarose plugs prior to DNA isolation. These improvements together with new methods enabling partial digestion of chromosomal DNA samples (in progress) could allow the construction of BAC libraries from flow sorted human chromosomes.
*This work was supported by the USDOE under contract W-7405-ENG-36.
Abstract scanned from text submitted for January 1996 DOE Human Genome Program Contractor-Grantee Workshop.
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