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Construction and screening of BAC libraries made from Brachypodium genomic DNA

Nature Protocols volume 2, pages 1661–1674 (2007)Cite this article

Abstract

Bacterial artificial chromosome (BAC) libraries are the large DNA insert libraries of choice and valuable tools for the map-based cloning of target quantitative trait loci, physical mapping, molecular cytogenetics and comparative genomics. The protocol reported here is a simplified method used to produce and screen BAC libraries from Brachypodium species and other related grasses. Intact nuclei, containing high molecular weight (HMW) DNA, are isolated and embedded in agarose plugs. The HMW DNA is digested using an appropriate restriction enzyme and size-fractionated using pulsed-field gel electrophoresis. The DNA is isolated by dialysis, ligated into pre-prepared vector and electroporated into competent Escherichia coli cells. A PCR-based method for screening the library is also described. The entire protocol takes at least 6 weeks to complete.

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Figure 1: Example of a trial digestion of high molecular weight (HMW) DNA in agarose plugs with 0, 0.1, 0.2, 0.5, 1, 2, 5 or 10 U of HindIII restriction enzyme per half-plug.
Figure 2: Reconstructed gel after removal of gel slices containing target bacterial artificial chromosome (BAC) insert DNA after high molecular weight (HMW) DNA was subjected to a single pulsed-field gel electrophoresis (PFGE).
Figure 3: Reconstructed gel after removal of gel slices containing target bacterial artificial chromosome (BAC) insert DNA (100–150 kb on left, 150–200 kb on right) after high molecular weight (HMW) DNA was subjected to pulsed-field gel electrophoresis (PFGE) for a second time.
Figure 4: DNA from randomly selected bacterial artificial chromosome (BAC) clones was restricted using NotI and fragments separated by pulsed-field gel electrophoresis (PFGE).
Figure 5: Representation of the 3D pooling strategy for PCR screening of the bacterial artificial chromosome (BAC) library.
Figure 6: PCR-based screen of BAC DNA pools using gene specific primers.
Figure 7: PCR-based screen of individual BACs using gene-specific primers.

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References

  1. Shizuya, H. et al. 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. USA 89, 8794–8797 (1992).

    Article  CAS  Google Scholar 

  2. Woo, S.S., Jiang, J., Gill, B.S., Paterson, A.H. & Wing, R.A. Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor . Nucleic Acids Res. 22, 4922–4931 (1994).

    Article  CAS  Google Scholar 

  3. Zhang, H.B. & Wu, C. BAC as tools for genome sequencing. Plant Physiol. Biochem. 39, 195–209 (2001).

    Article  CAS  Google Scholar 

  4. Draper, J. et al. Brachypodium distachyon. A new model system for functional genomics in grasses. Plant Physiol. 127, 1539–1555 (2001).

    Article  CAS  Google Scholar 

  5. Christiansen, P., Andersen, C.H., Didion, T., Folling, M. & Nielsen, K.K. A rapid and efficient transformation protocol for the grass Brachypodium distachyon . Plant Cell Rep. 23, 751–758 (2005).

    Article  CAS  Google Scholar 

  6. Vogel, J.P., Garvin, D.F., Leong, O.M. & Hayden, D.M. Agrobacterium-mediated transformation and inbred line development in the model grass Brachypodium distachyon . Plant Cell Tissue Organ Cult. 85, 199–211 (2006).

    Article  Google Scholar 

  7. Vogel, J.P. et al. EST sequencing and phylogenetic analysis of the model grass Brachypodium distachyon . Theor. Appl. Genet. 113, 186–195 (2006).

    Article  CAS  Google Scholar 

  8. Hasterok, R., Dulawa, J., Jenkins, G., Leggett, M. & Langdon, T. Multi-substrate chromosome preparations for high throughput comparative FISH. BMC Biotechnol. 6, 20–24 (2006).

    Article  Google Scholar 

  9. Jenkins, G. & Hasterok, R. BAC 'landing' on chromosomes of Brachypodium distachyon for comparative genome alignment. Nat. Protoc. 2, 88–98 (2007).

    Article  CAS  Google Scholar 

  10. Engvild, K.C. Mutagenesis of the model grass Brachypodium distachyon with sodium azide. Risø Report R-1510 http://www.risoe.dk/rispubl/BIO/ris-r-1510.htm (2005).

  11. Hasterok, R. et al. Alignment of the genomes of Brachypodium distachyon and temperate cereals and grasses using BAC landing with fluorescent in situ hybridization. Genetics 173, 349–362 (2006).

    Article  CAS  Google Scholar 

  12. Farrar, K. et al. Construction of two Lolium perenne BAC libraries and identification of BACs containing candidate genes for disease resistance and forage quality. Mol. Breed. 19, 15–23 (2006).

    Article  Google Scholar 

  13. Donnison, I.S. et al. Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines. Theor. Appl. Genet. 110, 846–851 (2005).

    Article  CAS  Google Scholar 

  14. Calderini, O. et al. Molecular cytogenetics and DNA sequence analysis of an apomixis-linked BAC in Paspalum simplex reveal a non pericentromere location and partial microcolinearity with rice. Theor. Appl. Genet. 112, 1179–1191 (2006).

    Article  CAS  Google Scholar 

  15. Zhang, H.B., Zhao, X.P., Ding, X.L., Paterson, A.H. & Wing, R.A. Preparation of megabase-size DNA from plant nuclei. Plant J. 7, 175–184 (1995).

    Article  CAS  Google Scholar 

  16. Huo, N. et al. Construction and characterization of two BAC libraries from Brachypodium distachyon, a new model for grass genomics. Genome 49, 1099–1108 (2006).

    Article  CAS  Google Scholar 

  17. Foote, T., Griffiths, S., Allouis, S. & Moore, G. Construction and analysis of a BAC library in the grass Brachypodium sylviticum: its use as a tool to bridge the gap between rice and wheat in elucidating gene content. Funct. Integr. Genomics 4, 26–33 (2004).

    Article  CAS  Google Scholar 

  18. Chalhoub, B., Belcram, H. & Caboche, M. Efficient cloning of plant genomes into bacterial artificial chromosome (BAC) libraries with larger and more uniform insert size. Plant Biotech. J. 2, 181–188 (2004).

    Article  CAS  Google Scholar 

  19. Peterson, D.G., Tomkins, J.P., Frisch, D.A., Wing, R.A. & Paterson, A.H. Construction of plant bacterial artificial chromosome (BAC) libraries: an illustrated guide 2nd Edition. (2002) http://www.mgel.msstate.edu/pubs/bacman2.pdf.

  20. Kim, U.J. et al. Construction and characterization of a human bacterial artificial chromosome library. Genomics 34, 213–218 (1996).

    Article  CAS  Google Scholar 

  21. Frengen, E. et al. A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites. Genomics 58, 250–253 (1999).

    Article  CAS  Google Scholar 

  22. Wild, J., Hradecna, Z. & Szybalski, W. Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones. Genome 12, 1434–1444 (2007).

    Article  Google Scholar 

  23. Hamilton, C.M., Frary, A., Lewis, C. & Tanksley, S.D. Stable transfer of intact high molecular weight DNA into plant chromosomes. Proc. Natl. Acad. Sci. USA 93, 9975–9979 (1996).

    Article  CAS  Google Scholar 

  24. Takken, F.L. et al. A one-step method to convert vectors into binary vectors suited for Agrobacterium-mediated transformation. Curr. Genet. 45, 242–248 (2004).

    Article  CAS  Google Scholar 

  25. Chang, Y.-L., Henriquez, X., Preuss, D., Copenhaver, G.P. & Zhang, H.-B. A plant-transformation-competent BIBAC library from the Arabidopsis thaliana Landsberg ecotype for functional and comparative genomics. Theor. Appl. Genet. 106, 269–276 (2003).

    Article  CAS  Google Scholar 

  26. Song, J., Bradeen, J.M., Naess, S.K., Helgeson, J.P. & Jiang, J. BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium . Theor. Appl. Genet. 107, 958–964 (2003).

    Article  CAS  Google Scholar 

  27. She, K. So you want to work with giants: the BAC vector. BioTech. J. 1, 69–74 (2003).

    Google Scholar 

  28. Sheng, Y., Mancino, V. & Birren, B. Transformation of Escherichia coli with large DNA molecules by electroporation. Nucleic Acids Res. 23, 1990–1996 (1995).

    Article  CAS  Google Scholar 

  29. Bennett, M.D. & Leitch, I.J. Nuclear DNA amounts in angiosperms: progress, problems and prospects. Ann. Bot. (Lond.) 95, 45–90 (2005).

    Article  CAS  Google Scholar 

  30. Southern, E. Southern blotting. Nat. Protoc. 1, 518–525 (2006).

    Article  CAS  Google Scholar 

  31. Sambrook, J. & Russel, D.W. Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

    Google Scholar 

  32. O'Sullivan, D.M., Ripoll, P.J., Rodgers, M. & Edwards, K.J. A maize bacterial artificial chromosome (BAC) library from the European flint inbred line F2 . Theor. Appl. Genet. 103, 425–432 (2001).

    Article  CAS  Google Scholar 

  33. Whatling, C.A. & Thomas, C.M. Pre electrophoresis of agarose plugs containing bacterial chromosomal DNA prepared for analysis by pulsed field gel electrophoresis can improve the clarity of restriction patterns. Anal. Biochem. 210, 98–101 (1993).

    Article  CAS  Google Scholar 

  34. Ma, Z., Song, W., Sharp, P.J. & Liu, C. Non-gridded library: a new approach for BAC (bacterial artificial chromosome) exploitation in hexaploid wheat (Triticum aestivum). Nucleic Acids Res. 28, e106 (2000).

    Article  CAS  Google Scholar 

  35. Strong, S.J., Ohta, O., Litman, G.W. & Amemiya, C.T. Marked improvement of PAC and BAC cloning is achieved using electroelution of pulsed-field gel-separated partial digests of genomic DNA. Nucleic Acids Res. 25, 3959–3961 (1997).

    Article  CAS  Google Scholar 

  36. Zhu, H. & Dean, R.A. A novel method for increasing the transformation efficiency of Escherichia coli-application for bacterial artificial chromosome library construction. Nucleic Acids Res. 27, 910–911 (1999).

    Article  CAS  Google Scholar 

  37. Clarke, L. & Carbon, J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome . Cell 9, 91–99 (1976).

    Article  CAS  Google Scholar 

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Acknowledgements

The Institute of Grassland and Environmental Research (IGER) is sponsored by the UK Biotechnology and Biological Sciences Research Council of the United Kingdom.

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  1. Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, SY23 4AR, UK

    Kerrie Farrar & Iain S Donnison

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  1. Kerrie Farrar
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Correspondence to Kerrie Farrar.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Organisation of super pools (PDF 16 kb)

Supplementary Table 2

List of plate pools making up each super pool (PDF 15 kb)

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Farrar, K., Donnison, I. Construction and screening of BAC libraries made from Brachypodium genomic DNA. Nat Protoc 2, 1661–1674 (2007). https://doi.org/10.1038/nprot.2007.204

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