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Mammalian Genome

, Volume 10, Issue 7, pp 706–709 | Cite as

A 5× genome coverage bovine BAC library: production, characterization, and distribution

  • Baoli  Zhu
  • Judith A.  Smith
  • Simon M.  Tracey
  • Bernard A.  Konfortov
  • Katrin  Welzel
  • Leonard C.  Schalkwyk
  • Hans  Lehrach
  • Sonja  Kollers
  • Julio  Masabanda
  • Johannes  Buitkamp
  • Ruedi  Fries
  • John L.  Williams
  • J. Ross  Miller

Abstract.

A bovine large-insert DNA library has been constructed in a Bacterial Artificial Chromosome (BAC) vector. The source DNA was derived from lymphocytes of a Jersey male. High-molecular-weight DNA fragments were produced by treatment with EcoRI/EcoRI methylase and cloned into the EcoRI site of pBACe3.6. In total, 157,240 individual BACs have been picked into 384-well plates. Approximately 190 randomly chosen clones have been characterized by Pulsed Field Gel Electrophoresis (PFGE) and have an average insert size of 105 kb, suggesting library coverage representing 5–6 genome equivalents. The frequency of clones without inserts is 4%. The chromosomal location of 51 BACs was studied by FISH; 3 showed more than one signal, indicating a chimerism frequency of roughly 6%. Approximately 50% of the clones in the library contain Simple Repeat Sequences (microsatellites), and 4% of the clones contain centromeric repeats. Insert stability was assessed by restriction digestion of DNA prepared from 20 clones after serial culture for one and three nights. Only one clone showed any evidence of an altered restriction pattern. Clones from 360 × 384-well plates (138,240 colonies) were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Both membranes and superpools are available from the RZPD, Berlin (http://www.rzpd.de). PCR 4-D superpools have been prepared from an additional 23,000 clones. The library has been screened for a total of 24 single-copy sequences; positive clones have been obtained in all cases.

Keywords

Bacterial Artificial Chromosome Genome Coverage Bacterial Artificial Chromosome Library Genome Equivalent Simple Repeat 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag New York Inc. 1999

Authors and Affiliations

  • Baoli  Zhu
    • 1
  • Judith A.  Smith
    • 2
  • Simon M.  Tracey
    • 1
  • Bernard A.  Konfortov
    • 1
  • Katrin  Welzel
    • 3
  • Leonard C.  Schalkwyk
    • 4
  • Hans  Lehrach
    • 4
  • Sonja  Kollers
    • 5
  • Julio  Masabanda
    • 5
  • Johannes  Buitkamp
    • 5
  • Ruedi  Fries
    • 5
  • John L.  Williams
    • 2
  • J. Ross  Miller
    • 1
  1. 1.Department of Immunology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UKGB
  2. 2.Roslin Institute, Roslin, Midlothian EH25 9PS, UKGB
  3. 3.RZPD, Heubnerweg 6, 14059 Berlin-Charlottenburg, GermanyDE
  4. 4.Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D14195 Berlin-Dahlem, GermanyDE
  5. 5.Lehrstuhl für Tierzucht, Technische Universität München, Alte Akademie 12, 85350 Freising-Weihenstephan, GermanyDE

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