Molecular Biology > Cloning and Recombination Methods

User Ratings

Your rating: None
Your rating: None (1 vote)
Your rating: None
 Add your comments

Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries

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

1Children's Hospital Oakland Research Institute, Oakland, California
2The Biotechnology Centre of Oslo University of Oslo, Oslo, Norway
3The Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Melbourne, Australia, Australia



Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
Unit 5.9
DOI: 
10.1002/0471142727.mb0509s55
Online Posting Date: 
August, 2001
GO TO THE FULL TEXT:
PDF or HTML at Wiley Online Library
Are you the author of this protocol? Login or register and return to this page.

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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Unit Introduction
  • Basic Protocol: 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
  • Bibliography
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

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

 Materials
  • ³2 to 10 ng/µl size-fractionated MboI- or EcoRI-digested genomic DNA (see Support Protocol 4)
  • 10 to 50 ng/µl pBACe3.6, pCYPAC2, or pPAC4 vector DNA prepared for cloning (see Support Protocol 1)
  • 1 Weiss U/µl T4 DNA ligase (Life Technologies) and 5× buffer (see recipe)
  • 0.5 M EDTA, pH 8.0 (APPENDIX 2)
  • 10 mg/ml proteinase K
  • 100 mM phenylmethylsulfonyl fluoride solution (PMSF; see recipe)
  • TE/PEG solution: 0.5× TE buffer, pH 8.0 (APPENDIX 2) 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 2)
  • NotI restriction endonuclease and buffer (New England Biolabs)
  • 1% (w/v) ultrapure agarose solution (Life Technologies)
  • 0.5× TBE buffer (APPENDIX 2)
  • 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 2)
  • 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 2.5B) apparatus (Bio-Rad) or field-inversion gel electrophoresis (FIGE; UNIT 2.5B) 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 Support Protocol 5; 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

 Additional Materials (also see Basic Protocol)
  • pBACe3.6, pCYPAC2, or pPAC4 stock in E. coli DH10B cells (P. de Jong; pieter@dejong.med.buffalo.edu)
  • 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 2.5A), 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 2.5A), plasmid extraction (e.g., UNIT 1.7), CsCl/ethidium bromide equilibrium centrifugation (e.g., UNIT 1.8), electroelution (see Support Protocol 4), and ethanol precipitation (UNIT 2.1A)

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

 Materials
  • Healthy human volunteer
  • PBS (APPENDIX 2), ice cold
  • 1× RBC lysis solution (see recipe)
  • InCert agarose (FMC Bioproducts)
  • Proteinase K lysis solution (see recipe)
  • TE50 buffer: 10 mM Tris×Cl, pH 8.0 (APPENDIX 2)/50 mM EDTA
  • 0.1 mM PMSF solution: 100 mM PMSF solution (see recipe) diluted 1/1000 in TE50 buffer immediately before use
  • 0.5 M EDTA, pH 8.0 (APPENDIX 2)
  • 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

 Additional Materials (also see Support Protocol 2)
  • 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 CO2 (Donavan and Brown, 1995)

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 2 and 3)
  • 0.5× TBE buffer (APPENDIX 2), 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 2)
  • 0.5 µg/ml ethidium bromide solution in 0.5× TBE buffer (APPENDIX 2)
  • 1× MboI buffer (see recipe)
  • 10 U/µl MboI restriction endonuclease (Life Technologies)
  • 1 M MgCl2
  • 10 mg/ml proteinase K (Boehringer Mannheim) stock solution
  • 10% (w/v) N-lauroylsarcosine
  • TE50 buffer: 10 mM Tris×Cl, pH 8.0 (APPENDIX 2)/50 mM EDTA
  • 100 mM PMSF solution (see recipe)
  • 10 mg/ml BSA (New England Biolabs)
  • 10× 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 2)
  • 1× TAE buffer (APPENDIX 2; optional)
  • Lambda DNA
  • 50-ml conical screw-cap polypropylene tubes, sterile
  • Contour-clamped homogeneous electrical field (CHEF) apparatus (Bio-Rad or equivalent; UNIT 2.5B) with 1.5-mm-thick, 20-well comb
  • Digital imager (Alpha Innotech IS1000 or equivalent)
  • Disposabler -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 2.5B)

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 Basic Protocol)
  • LB medium or terrific broth (UNIT 1.1) containing 25 µg/ml kanamycin (for PAC clones) or 20 µg/ml chloramphenicol (for BAC clones)
  • Resuspension solution (see recipe)
  • Alkaline lysis solution (see recipe)
  • Precipitation solution (see recipe)
  • Isopropanol
  • 70% (v/v) ethanol
  • TE buffer, pH 8.0 (APPENDIX 2)
  • 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 2.5A)
Looking for materials?  Suppliers Guide
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  • Figure 5.9.1
    (at left) The (A) pCYPAC2, (B) pBACe3.6, and (C) pPAC4 vectors contain common features for positive selection of cloned inserts. The insert-containing E. coli cells can be grown in preference over cells containing plasmids without an insert by including sucrose in the medium. Sucrose is converted into toxic metabolites by levansucrase encoded by the sacBII gene present in all three vectors. The pUC-link segment contains a functional high-copy-number plasmid that includes the ampicillin resistance gene (Apr; not shown). The pUC-link sequence is removed in the cloning procedure and is not present in the PAC and BAC clones. The digested genomic DNA fragment replaces the fragment containing the pUC-link in the PAC or BAC vectors. The loxP recombination sites are not used in cloning. A detailed description of vector elements is given elsewhere (see, Background Information, Vector Elements). (A) The pCYPAC2 vector contains the P1 plasmid replicon, which maintains recombinant clones at around one copy per cell. Alternatively, the P1 lytic replicon can be induced to provide higher copy numbers by adding the lac inducer IPTG into the medium. The kanamycin resistance gene (Kmr) is also present in this vector. (B) The pBACe3.6 vector contains the chloramphenicol resistance gene (Cmr) and the F plasmid replicon, maintaining recombinant clones at one copy per cell. The multiple cloning sites flanking the pUC-link segment allow positive selection for cloned inserts using any of the restriction enzymes BamHI, SacII, EcoRI, SacI, MluI, or NsiI. The Tn7att sequence permits specific Tn7-based retrofitting of BAC clones. (C) The pPAC4 vector contains four elements from pCYPAC2: the P1 plasmid replicon, the kanamycin resistance gene, the sacBII gene, and the pUC-link. The P1 plasmid replicon ensures low-copy-number maintenance of recombinant clones; the P1 lytic replicon is removed. In addition, theoriP and bsrr cassette have been included to facilitate the use of PAC clones for functional analysis of genes carried on the cloned inserts. The Tn7att sequence is also present, enabling specific retrofitting of PACs.

    View Image
  • Figure 5.9.2
    Isolation of insert DNA from agarose blocks by PFGE. (A) Agarose blocks containing partially digested DNA are applied to the center wells of a 1% agarose gel and low-range PFG markers are applied to flanking wells. (B-D) Size fractionation is performed in a CHEF apparatus. The DNA separation occurs in three stages, all with pulse directions at a 120° angle. (B) The initial direction of the field allows the DNA to migrate from the wells toward the nearest gel edge (1 cm away from the well), using 0.5× TBE at 14°C and 5.0 V/cm for 6 hr with 15-sec pulse time. (C) The same conditions are then used after turning the direction of the gel to bring all fragments remaining in the gel back to the original starting wells. (D) After the second run, new marker DNA is applied to additional flanking wells that were not previously used. The old markers are useful for checking the electrophoresis condition for the first run by comparing the new markers. The new markers are used to identify the size range after the final electrophorsis. High-molecular-weight fragments are then resolved in the third run, which is performed at 6 V/cm for 16 hr with 0.1- to 40-sec pulse time. After this procedure (not shown), the flanking marker lanes are removed from the gel and stained with ethidium bromide to indicate the location of the size ranges. Gel slices are then cut from the genomic DNA lanes by horizontal cuts at 0.5-cm intervals to obtain gel slices in the range of 150 to 500 kb.

    View Image

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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.