Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries

Kazutoyo Osoegawa1, Pieter J. de Jong1, Eirik Frengen2, Panayiotis A. Ioannou3

1 Children's Hospital Oakland Research Institute, Oakland, California, 2 The Biotechnology Centre of Oslo University of Oslo, Oslo, Norway, 3 The Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Melbourne, Australia
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 5.9
DOI:  10.1002/0471142727.mb0509s55
Online Posting Date:  August, 2001
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Abstract

Large‐insert genomic libraries are necessary for physical mapping of large chromosomal regions, for isolation of complete genes, and for use as intermediates in DNA sequencing of entire genomes. Construction of BAC and PAC libraries is detailed in the unit, including preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, dephosphorylation with alkaline phosphatase, and purification through pulsed‐field gel electrophoresis (PFGE). For the preparation of high‐molecular weight DNA for cloning, procedures for embedding total genomic DNA from lymphocytes or animal tissue cells are also provided. Other protocols detail partial digestion of genomic DNA with MboI or with a combination of EcoRI endonuclease and EcoRI methylase (including methods for optimizing the extent of digestion), and subsequent size fractionation by preparative PFGE. Finally, the isolation of BAC and PAC plasmid DNA for analyzing clones is also presented.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of BAC/PAC Clones Using pCYPAC2, pPAC4, or pBACe3.6 Vector
  • Support Protocol 1: Preparation of BAC/PAC Vector for Cloning
  • Support Protocol 2: Preparation of High‐Molecular‐Weight DNA from Lymphocytes in Agarose Blocks
  • Support Protocol 3: Preparation of High‐Molecular‐Weight DNA from Animal Tissue Cells in Agarose Blocks
  • Support Protocol 4: Partial Digestion and Size Fractionation of Genomic DNA
  • Support Protocol 5: Modified Alkaline Lysis Miniprep for Recovery of DNA from BAC/PAC Clones
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of BAC/PAC Clones Using pCYPAC2, pPAC4, or pBACe3.6 Vector

  Materials
  • ≥2 to 10 ng/µl size‐fractionated MboI‐ or EcoRI‐digested genomic DNA (see protocol 5)
  • 10 to 50 ng/µl pBACe3.6, pCYPAC2, or pPAC4 vector DNA prepared for cloning (see protocol 2)
  • 1 Weiss U/µl T4 DNA ligase (Life Technologies) and recipe5× buffer (see recipe)
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 10 mg/ml proteinase K
  • recipe100 mM phenylmethylsulfonyl fluoride solution (PMSF; see recipe)
  • TE/PEG solution: 0.5× TE buffer, pH 8.0 ( appendix 22) containing 30% (w/v) polyethylene glycol 8000 (PEG 8000)
  • Electrocompetent bacterial cells (ElectroMAX DH10B; Life Technologies)
  • SOC medium (Life Technologies; also see unit 1.8, but reduce yeast extract to 0.5%)
  • LB plates (unit 1.1) containing 5% (w/v) sucrose and either 25 µg/ml kanamycin (for PAC clones) or 20 µg/ml chloramphenicol (for BAC clones):
  •  100 × 15–mm petri dishes for test transformation
  •  22 × 22–cm trays for picking colonies
  • LB medium (unit 1.1) containing 20 µg/ml chloramphenicol (BAC clones) or 25 µg/ml kanamycin (PAC clones)
  • TE buffer, pH 8.0 ( appendix 22)
  • NotI restriction endonuclease and buffer (New England Biolabs)
  • 1% (w/v) ultrapure agarose solution (Life Technologies)
  • 0.5× TBE buffer ( appendix 22)
  • Low‐range PFG markers in agarose containing a mixture of lambda HindIII fragments and lambda concatemers (New England Biolabs)
  • 0.5 µg/ml ethidium bromide in 0.5× TBE buffer ( appendix 22)
  • 80% (v/v) glycerol, sterile
  • Dry ice/ethanol bath
  • 16° and 37°C water baths
  • 0.025‐µm‐pore‐size microdialysis filters (Millipore): 25‐mm diameter for small‐scale test ligation and 47‐mm diameter for large‐scale ligation
  • Wide‐bore pipet tips, sterile
  • Disposable microelectroporation cuvettes with a 0.15‐cm gap (Life Technologies or equivalent)
  • Electroporator (Cell Porator equipped with a voltage booster; Life Technologies or equivalent)
  • 15‐ml snap‐cap polypropylene tubes, sterile
  • Orbital shaker, 37°C
  • Automated plasmid isolation system (AutoGen 740, Integrated Separation Systems, optional)
  • Flexible plastic 96‐well plate (Falcon or equivalent)
  • Contour‐clamped homogeneous electrical field (CHEF; unit 2.5) apparatus (Bio‐Rad) or field‐inversion gel electrophoresis (FIGE; unit 2.5) apparatus (Bio‐Rad or equivalent)
  • Digital imager (Alpha Innotech IS1000 or equivalent)
  • 50‐ml disposable centrifuge tube (Corning or equivalent)
  • Additional reagents and equipment for modified alkaline lysis preparation of BAC or PAC clone DNA (see protocol 6; optional)
CAUTION: To prevent shearing, use sterile wide‐bore pipet tips for all steps involving the handling of genomic DNA.

Support Protocol 1: Preparation of BAC/PAC Vector for Cloning

  • pBACe3.6, pCYPAC2, or pPAC4 stock in E. coli DH10B cells (P. de Jong; )
  • LB plates (unit 1.1) containing:
  •  25 µg/ml kanamycin (for PAC) or 20 µg/ml chloramphenicol (for BAC)
  •  5% (w/v) sucrose and either kanamycin or chloramphenicol
  •  5% (w/v) sucrose, 100 µg/ml ampicillin, and either kanamycin or chloramphenicol
  • BamHI and EcoRI restriction endonucleases and 10× buffers (New England Biolabs or equivalent)
  • 0.7% (w/v) agarose gels (for standard electrophoresis)
  • Calf intestine alkaline phosphatase (AP; Boehringer Mannheim)
  • 10 mg/ml proteinase K (Boehringer Mannheim) stock solution
  • 95% (v/v) ethanol
  • 1.0% (w/v) ultrapure agarose solution (Life Technologies; for CHEF system)
  • 6× loading buffer (unit 2.5), not containing xylene cyanol FF or SDS
  • 1‐kb ladder or lambda HindIII markers
  • T4 polynucleotide kinase (New England Biolabs)
  • 30% (w/v) polyethylene glycol (PEG) 8000
  • 1.5‐mm‐thick electrophoresis comb for CHEF apparatus
  • Dialysis tubing of 3/4 in. diameter, mol. wt. exclusion limit of 12,000 to 14,000 daltons (Life Technologies or equivalent)
  • Dialysis clip
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5), plasmid extraction (e.g., unit 1.7), CsCl/ethidium bromide equilibrium centrifugation (e.g., unit 1.8), electroelution (see protocol 5), and ethanol precipitation (unit 2.1)

Support Protocol 2: Preparation of High‐Molecular‐Weight DNA from Lymphocytes in Agarose Blocks

  Materials
  • Healthy human volunteer
  • PBS ( appendix 22), ice cold
  • recipe1× RBC lysis solution (see recipe)
  • InCert agarose (FMC Bioproducts)
  • recipeProteinase K lysis solution (see recipe)
  • TE 50 buffer: 10 mM Tris⋅Cl, pH 8.0 ( appendix 22)/50 mM EDTA
  • 0.1 mM PMSF solution: recipe100 mM PMSF solution (see recipe) diluted 1/1000 in TE 50 buffer immediately before use
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • Blood‐drawing equipment
  • Blood collection tubes containing EDTA
  • Automated hematology counter
  • 50‐ml conical screw‐cap polypropylene tubes, sterile
  • Refrigerated centrifuge with rotor/adapters for 50‐ml tubes (e.g., Sorvall RI6000D centrifuge with H‐1000B swinging‐bucket rotor or equivalent)
  • Roller mixer (Robbins Scientific or equivalent)
  • 50°C water bath
  • 10 × 5 × 1.5–mm disposable DNA plug mold (Bio‐Rad)

Support Protocol 3: Preparation of High‐Molecular‐Weight DNA from Animal Tissue Cells in Agarose Blocks

  • Healthy animal (e.g., ∼5‐week‐old mice, rats)
  • Sterile dissecting tools
  • Sterile Dounce homogenizer
  • 15‐ml conical screw‐cap polypropylene tubes, sterile (Corning or equivalent)
  • Counting chambers (VWR)
  • Additional reagents and equipment for euthanasia with CO 2 (Donavan and Brown, )

Support Protocol 4: Partial Digestion and Size Fractionation of Genomic DNA

  Materials
  • Agarose plugs with embedded high‐molecular‐weight genomic DNA stored in 0.5 M EDTA (see Support Protocols protocol 32 and protocol 43)
  • 0.5× TBE buffer ( appendix 22), sterile
  • 95% (v/v) ethanol
  • 1% (w/v) ultrapure agarose gel (Life Technologies) in 0.5× TBE buffer
  • Low‐range PFG markers in agarose containing a mixture of lambda HindIII fragments and lambda concatemers (New England Biolabs)
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 0.5 µg/ml ethidium bromide solution in 0.5× TBE buffer ( appendix 22)
  • recipe1× MboI buffer (see recipe)
  • 10 U/µl MboI restriction endonuclease (Life Technologies)
  • 1 M MgCl 2
  • 10 mg/ml proteinase K (Boehringer Mannheim) stock solution
  • 10% (w/v) N‐lauroylsarcosine
  • TE 50 buffer: 10 mM Tris⋅Cl, pH 8.0 ( appendix 22)/50 mM EDTA
  • recipe100 mM PMSF solution (see recipe)
  • 10 mg/ml BSA (New England Biolabs)
  • recipe10× EcoRI endonuclease/methylase buffer (see recipe)
  • 0.1 M spermidine
  • 20 U/µl EcoRI endonuclease (New England Biolabs)
  • 40 U/µl EcoRI methylase (New England Biolabs)
  • TE buffer, pH 8.0 ( appendix 22)
  • 1× TAE buffer ( appendix 22; optional)
  • Lambda DNA
  • 50‐ml conical screw‐cap polypropylene tubes, sterile
  • Contour‐clamped homogeneous electrical field (CHEF) apparatus (Bio‐Rad or equivalent; unit 2.5) with 1.5‐mm‐thick, 20‐well comb
  • Digital imager (Alpha Innotech IS1000 or equivalent)
  • Disposabler γ‐ray‐sterilized inoculating loops
  • 15‐ml conical screw‐cap polypropylene tubes, sterile
  • Dialysis tubing, 3/4‐in. diameter, mol. wt. exclusion limit 12,000 to 14,000 daltons (Life Technologies or equivalent)
  • Dialysis clips
  • Submarine gel electrophoresis apparatus (Bio‐Rad Sub‐Cell GT DNA Electrophoresis Cell, 31‐cm length × 16‐cm width, or equivalent)
  • Wide‐bore pipet tips
  • Additional reagents and equipment for analytical pulsed‐field gel electrophoresis (unit 2.5)
CAUTION: To prevent shearing, use sterile wide‐bore pipet tips in all steps involving the handling of genomic DNA.

Support Protocol 5: Modified Alkaline Lysis Miniprep for Recovery of DNA from BAC/PAC Clones

  Materials
  • BAC or PAC clones (see protocol 1)
  • LB medium or terrific broth (unit 1.1) containing 25 µg/ml kanamycin (for PAC clones) or 20 µg/ml chloramphenicol (for BAC clones)
  • recipeResuspension solution (see recipe)
  • recipeAlkaline lysis solution (see recipe)
  • recipePrecipitation solution (see recipe)
  • Isopropanol
  • 70% (v/v) ethanol
  • TE buffer, pH 8.0 ( appendix 22)
  • NotI restriction endonuclease and buffer (New England Biolabs)
  • Toothpicks, sterile
  • 12‐ to 15‐ml snap‐cap polypropylene tubes
  • Forceps, sterile
  • Orbital shaker, 37°C
  • 1.5‐ml microcentrifuge tubes or 2‐ml screw‐cap tubes
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)
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Figures

Videos

Literature Cited

Literature Cited
   Chalker, A.F., Leach, D.R., and Lloyd, R.G. 1988. Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome. Gene 71:201‐205.
   Chu, G., Vollrath, D., and Davis, R.W. 1986. Separation of large DNA molecules by contour‐clamped homogeneous electric field. Science 234:1582‐1585.
   Donovan, J. and Brown, P. 1995. Euthanasia. In Current Protocols in Immunology. (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevock, and W. Strober, eds.) pp. 1.8.1‐1.8.4. John Wiley & Sons, New York.
   Frengen, E., Weichenhan, D., Zhao, B., Osoegawa, K., van Geel, M., and de Jong, P.J. 1999. A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites. Genomics In press.
   Fukushige, S. and Sauer, B. 1992. Genomic targeting with a positive‐selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 89:7905‐7909.
   Gibson, T.J., Coulson, A.R., Sulston, J.E., and Little, P.F. 1987. Lorist2, a cosmid with transcriptional terminators insulating vector genes from interference by promoters within the insert: Effect on DNA yield and cloned insert frequency. Gene 53:275‐281.
   Gibson, F.P., Leach, D.R., and Lloyd, R.G. 1992. Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K‐12. J. Bacteriol. 174:1221‐1228.
   Grant, S.G., Jessee, J., Bloom, F.R., and Hanahan, D. 1990. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation‐restriction mutants. Proc. Natl. Acad. Sci. U.S.A. 87:4645‐4649.
   Hanahan, D., Jessee, J., and Bloom, F.R. 1991. Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 204:63‐113.
   Ioannou, P.A., Amemiya, C.T., Garnes, J., Kroisel, P.M., Shizuya, H., Chen, C., Batzer, M.A., and de Jong, P.J. 1994. A new bacteriophage P1‐derived vector for the propagation of large human DNA fragments. Nature Genet. 6:84‐89.
   Osoegawa, K., Woon, P.‐Y., Zhao, B., Frengen, E., Tateno, M., Catanese, J.J., and de Jong, P.J. 1998. An improved approach for construction of bacterial artificial chromosome libraries. Genomics 52:1‐8.
   Pierce, J.C., Sauer, B., and Sternberg, N. 1992. A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: Improved cloning efficacy. Proc. Natl. Acad. Sci. U.S.A. 89:2056‐2060.
   Raleigh, E.A. 1987. Restriction and modification in vivo by Escherichia coli K12. Methods Enzymol. 152:130‐141.
   Schwartz, D.C., Li, X., Hernandez, L.I., Ramnarain, S.P., Huff, E.J., and Wang, Y.K. 1993. Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping. Science 262:110‐114.
   Sheng, Y.L., Mancino, V., and Birren, B. 1995. Transformation of Escherichia coli with large DNA molecules by electroporation. Nucl. Acids Res. 23:1990‐1996.
   Shizuya, H., Birren, B., Kim, U.‐J., Mancino, V., Stepak, T., Tachiiri, Y., and Simon, M. 1992. Cloning and stable 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.
   Sternberg, N. 1990. Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs. Proc. Natl. Acad. Sci. U.S.A. 87:103‐107.
   Strong, S.J., Ohta, Y., Litman, G.W., and Amemiya, C.T. 1997. Marked improvement of PAC and BAC cloning is achieved using electroelution of pulsed‐field gel‐separated partial digests of genomic DNA. Nucl. Acids Res. 25:3959‐3961.
   Wang, M., Chen, X.N., Shouse, S., Manson, J., Wu, Q., Li, R., Wrestler, J., Noya, D., Sun, Z.G., Korenberg, J., and Lai, E. 1994. Construction and characterization of a human chromosome 2‐specific BAC library. Genomics 24:527‐534.
   Wang, G.L., Holsten, T.E., Song, W.Y., Wang, H.P., and Ronald, P.C. 1995. Construction of a rice bacterial artificial chromosome library and identification of clones linked to the Xa‐21 disease resistance locus. Plant J. 7:525‐533.
   Woo, S.S., Jiang, J., Gill, B.S., Paterson, A.H., and Wing, R.A. 1994. Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor. Nucl. Acids Res. 22:4922‐4931.
   Woon, P.‐Y., Osoegawa, K., Kaisaki, P.J., Zhao, B., Catanese, J.J., Gauguier, D., Cox, R., Levy, E. R., Lathrop, G.M., Monaco, A.P., and de Jong, P.J. 1998. Construction and characterization of a 10‐fold genome equivalent rat P1‐derived artificial chromosomal library. Genomics 50:306‐316.
   Wyman, A.R. and Wertman, K.F. 1987. Host strains that alleviate underrepresentation of specific sequences: Overview. Methods Enzymol. 152:173‐180.
Key References
   Albertsen, H.M., Abderrahim, H., Cann, H.M., Dausset, J., Le Paslier, D., and Cohen, D. 1990. Construction and characterization of a yeast artificial chromosome library containing seven haploid equivalents. Proc. Natl. Acad. Sci. U.S.A. 87:4256‐4260.
  The first description of use of PFGE to size select DNA for ligation.
   Ioannou et al., 1994. See above.
  The first paper describing PAC cloning.
   O'Connor, M., Peifer, M., and Bender, W. 1989. Construction of large DNA segments in Escherichia coli. Science 244:1307‐1312.
  Describes first large BAC clone constructed by a combination of in vivo recombination and recombinant DNA technology.
   Osoegawa et al., 1998. See above.
  An improved BAC/PAC cloning approach.
   Pierce et al., 1992. See above.
  Development of an elegant and efficient P1 cloning vector, a direct predecessor to the pCYPAC vectors.
   Shizuya et al., 1992. See above.
  The development of efficient procedures for constructing large‐insert plasmids by bacterial transformation.
   Sternberg, 1990. See above.
  The first large‐insert P1 plasmids constructed using in vitro packaging and viral infection to transform E. coli.
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