Fosmid Libraries for Genomic Structural Variation Detection

William F. Donahue1, Heather M. Ebling1

1 Agencourt Bioscience, Beverly, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.20
DOI:  10.1002/0471142905.hg0520s54
Online Posting Date:  July, 2007
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Fosmid libraries have demonstrated their utility for a number of applications. These include filling gaps between BACs and small insert libraries in sequence assemblies, performing hybridization/screening studies to isolate functional elements within the genome (Vergin et al., 1998), and detecting insertions, deletions, and rearrangements in structural variation studies (Tuzun et al., 2005). This unit covers the basic methodologies for the construction of fosmid libraries with tight insert sizes suitable for these applications. Basic Protocol 1 covers the shearing, size selection, and recovery of DNA from a pulsed‐field gel. Basic Protocol 2 covers the cloning of insert DNA into the fosmid vector, packaging of DNA into infective phage particles, and the infection/transformation of bacteria. A commentary section is provided, which outlines many of the critical parameters involved in fosmid library construction, along with some additional background information and a section discussing anticipated results. Curr. Protoc. Hum. Genet. 54:5.20.1‐5.20.18. © 2007 by John Wiley & Sons, Inc.

Keywords: fosmid cloning; fosmid library; fosmid; fosmid protocol; structural variation detection

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Table of Contents

  • Basic Protocol 1: Preparing DNA for Ligation into pCC2FOS Vector
  • Basic Protocol 2: Vector Ligation, DNA Packaging, and Cell Transformation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparing DNA for Ligation into pCC2FOS Vector

  • Genomic DNA
  • 3 M sodium acetate, pH 5.5 (Sigma)
  • 70% and 100% ethanol (Sigma)
  • GlycoBlue (Ambion)
  • End‐It DNA End‐Repair kit (Epicentre) containing:
    • End‐Repair enzyme mix: T4 DNA polymerase and T4 polynucleotide kinase
    • 10× End‐Repair buffer
    • dNTP solution
    • ATP
  • LoTE buffer, pH 7.5 and 8 (see recipe)
  • Phenol/chloroform (Sigma; also see appendix 3C)
  • Chloroform/isoamyl alcohol (Sigma; also see appendix 3C)
  • 10× TBE buffer ( appendix 2D)
  • Agarose (Seakem Gold, Cambrex or equivalent)
  • 10× BlueJuice gel loading buffer (Invitrogen)
  • Monocut λ DNA marker (New England Biolabs)
  • 1‐kb extension ladder (Invitrogen)
  • SYBR Gold gel dye (Invitrogen)
  • 1× TAE buffer ( appendix 2D)
  • HydroShear and large shearing assembly (Genomic Solutions)
  • 1.5‐ and 1.7‐ml microcentrifuge tubes
  • Refrigerated centrifuge (Eppendorf or equivalent)
  • PCR strip tubes
  • Thermal cycler
  • CHEF Mapper XA pulsed‐field electrophoresis system (BioRad)
  • 10‐well comb (4‐cm wide, 1.5‐mm thick, adjustable height)
  • Razor blades (VWR)
  • Platform shaker, 37°C
  • Gel reader (e.g., DarkReader)
  • Dialysis membrane tubing (Spectra‐Por 15,000 MWCO, VWR)
  • 10‐ml serological pipet
  • Weighted tubing closures (VWR)
  • Gel box (e.g., Owl model B3)
  • Microcon spin columns (YM‐100, Millipore)
  • UV spectrophotometer
  • Additional reagents and equipment for extraction and precipitation of DNA ( appendix 3C)

Basic Protocol 2: Vector Ligation, DNA Packaging, and Cell Transformation

  • CopyControl HTP Fosmid library production kit (CCFOS059, Epicentre) containing:
    • CopyControl pCC2FOS vector
    • End‐Repair enzyme mix
    • End‐Repair 10× buffer
    • dNTP mix
    • Fast‐Link DNA ligase
    • Fast‐Link 10× ligation buffer
    • ATP solution
    • GELase gel‐digesting preparation
    • GELase 50× reaction buffer
    • MaxPlax lambda packaging extracts
    • Control DNA
    • Ligated lambda control DNA
    • EPI300 plating strain
    • Control lambda plating strain
    • CopyControl induction solution
  • T4 DNA ligase (Invitrogen)
  • 10× T4 DNA ligation buffer (see recipe)
  • Phage dilution buffer (see recipe)
  • Chloroform (Sigma)
  • LB medium ( appendix 2D) with 10 mM MgSO 4 ( appendix 2D)
  • 10 mM MgSO 4
  • 16°, 30°, and 70°C water bath
  • Microcon YM‐100 column (100 k MWCO; Millipore)
  • 15‐ and 50‐ml centrifuge tubes (VWR)
  • 37°C shaker
  • 2‐ml cryotubes (Nunc)
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Literature Cited

Literature Cited
   Collins, J. and Hohn, B. 1978. Cosmids: A type of plasmid gene‐cloning vector that is packageable in vitro in bacteriophage λ heads. Proc. Natl. Acad. Sci. U.S.A. 75:4242‐4246.
   EPICENTER Product Literature (2006). EPICENTER Product Literature #171: CopyControl TM Fosmid Library Production Kit, 2006, EPICENTER Technologies Corporation. All rights reserved.
   Feiss, M., Kobayashi, I., and Widner, W. 1983. Separate sites for binding and nicking of bacteriophage λ DNA by terminase. Proc. Natl. Acad. Sci. U.S.A. 80:955‐959.
   Hohn, B. and Murray, K. 1977. Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proc. Natl. Acad. Sci. U.S.A. 74:3259‐3263.
   Jones, P.G., VanBogelen, R.A., and Neidhardt, F.C. 1987. Induction of proteins in response to low temperature in Escherichia coli. J. Bacteriol. 169:2092‐2095.
   Kandror, O. and Goldberg, A.L. 1997. Trigger factor is induced upon cold shock and enhances viability of Escherichia coli at low temperatures. Proc. Natl. Acad. Sci. U.S.A. 94:4978‐4981.
   Kandror, O., Deleon, A., and Goldberg, A.L. 2002. Trehelose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures. Proc. Natl. Acad. Sci. U.S.A. 99:9727‐9732.
   Kim, U., Shizuya, H., deJong, P.J., Birren, B., and Simon, M.I. 1992. Stable propagation of cosmid sized human DNA inserts in an F–factor‐based vector. Nucl. Acids Res. 20:1083‐1085.
   Phadtare, S. and Inouye, M. 2004. Genome‐wide transcriptional analysis of the cold shock response in wild‐type and cold‐sensitive, quadruple‐csp‐deletion strains of Escherichia coli. J. Bacteriol. 186:7007‐7014.
   Shizuya, H., Birren, B., Kim, U., Mancino, V., Slepak, T., Tachiiri, Y., and Simon, M. 1992. Cloning and maintenance of 300‐kilobase‐pair fragments of human DNA in Escherichia coli using an F‐factor‐based vector. Proc. Natl. Acad. Sci. U.S.A. 89:8794‐8797.
   Stenberg, N., Tiemeir, D., Enquist, L. 1977. In vitro packaging of a lambda Dam vector containing EcoRI fragments of Escherichia coli and phage P1. Gene. 1:255‐280.
   Thomas, M., Cameron, J.R., and Davis, R.W. 1974. Viable molecular hybrid of bacteriophage lambda and eukaryotic DNA. Proc. Natl. Acad. Sci. U.S.A. 71:4579‐4583.
   Tuzun, E., Sharp, A.J., Bailey, J.A., Kaul, R., Morrison, V.A., Pertz, L.M., Haugen, E., Hayden, H., Albertson, D., Pinkel, D., Olson, M.V., and Eichler, E.E. 2005. Fine scale structural variation of the human genome. Nat. Genet. 37:727‐732.
   Weiczorek, D.J. and Feiss, M. 2001. Defining cosQ, the site requirement for termination of bacteriophage λ DNA packaging. Genetics 158:495‐506.
   Wild, J., Hradenca, Z., and Szybalski, W. 2002. Switching from single‐copy to high‐copy vectors and genomic clones. Genome Res. 12:1434‐1444.
   Vergin, K.L., Urbach, E., Stein, J.L., DeLong, E.F., Lanoil, B.D., and Giovannoni, S.J. 1998. Screening of a fosmid library of marine environmental genomic DNA fragments reveals four clones related to members of the order Planctomycetales. Appl. Environ. Microbiol. 64:3075‐3078.
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