Use of Rolling-Circle Amplification for Large-Scale Yeast Two-Hybrid Analyses

  • Xiaodong Ding
  • Yan Zhang
  • Wen-Yuan Song
Part of the Methods in Molecular Biology book series (MIMB, volume 354)


Detection of protein–protein interactions on a large-scale has become a major focus of functional genomics after the completion of genome sequencing. The information generated from these studies not only assembles proteins into signaling networks, but also reveals potential functions of uncharacterized proteins when their interacting partners have known functions. We have developed a rolling circle amplification-based yeast two-hybrid scheme that allows one to test reproducibility and specificity of the interactions on a large scale. Using this scheme, technical false-positives from yeast two-hybrid analyses can be efficiently minimized.

Key Words

Protein—protein interactions yeast two-hybrid rolling-circle amplification high throughput plasmids 



We thank Dr. M. Vidal for providing the yeast strain MaV203, Dr. P. C. Ronald for the precursors of the pXDGATcy86 and pXDGATU86 vectors, and Lisa Nodzon for critical reading of the manuscript. This research was supported by the Florida Agricultural Experiment Station and a grant from the National Science Foundation Plant Genome Research to W-Y S. This work was approved for publication as Journal Series No. R-10865.


  1. 1.
    Fields, S. and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–246.PubMedCrossRefGoogle Scholar
  2. 2.
    Brent, R., Jr. and Finley, R. L. (1997) Understanding gene and allele function with two-hybrid methods. Annu. Rev. Genet. 31, 663–704.PubMedCrossRefGoogle Scholar
  3. 3.
    Nodzon, L. and Song, W.-Y. (2004). Yeast two-hybrid technology, in Encyclopedia of Plant and Crop Science (Goodman, R. M., ed.), Marcel Dekker, Inc.: New York, pp. 1302–1304.Google Scholar
  4. 4.
    Xenarios, I., Salwinski, L., Duan, X. J., Higney, P., Kim, S. M., and Eisenberg, D. (2002) DIP, the Database of Interacting Proteins: a research tool for studying cellular networks of protein interactions. Nucleic Acids Res. 30, 303–305.PubMedCrossRefGoogle Scholar
  5. 5.
    Vidalain, P. O., Boxem, M., Ge, H., Li, S., and Vidal, M. (2004) Increasing specificity in high-throughput yeast two-hybrid experiments. Methods 32, 363–370.PubMedCrossRefGoogle Scholar
  6. 6.
    Kornberg, A. and Baker, T. A. (1992) DNA Replication. W. H. Freeman and Company, San Francisco.Google Scholar
  7. 7.
    Fire, A. and Xu, S.-Q. (1995) Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. USA 92, 4641–4645.PubMedCrossRefGoogle Scholar
  8. 8.
    Dean, F. B., Nelson, J. R., Giesler, T. L., and Lasken, R. S. (2001) Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multipleprimed rolling circle amplification. Genome Res. 11, 1095–1099.PubMedCrossRefGoogle Scholar
  9. 9.
    Blanco, L., Bernad, A., Lazaro, J. M., Martin, G., Garmendia, C., and Salas, M. (1989) Highly efficient DNA synthesis by the phage phi29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264, 8935–8940.PubMedGoogle Scholar
  10. 10.
    Garmendia, C., Bernad, A., Esteban, J. A., Blanco, L., and Salas, M. (1992) The bacteriophage phi29 DNA polymerase, a proofreading enzyme. J. Biol. Chem. 267, 2594–2599.PubMedGoogle Scholar
  11. 11.
    Esteban, J. A., Salas, M., and Blanco, L. (1993) Fidelity of phi 29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization. J. Biol. Chem. 268, 2719–2726.PubMedGoogle Scholar
  12. 12.
    Ding, X., Cory, G., and Song, W.-Y. (2004) A high-throughput system to verify candidate interactors from yeast two-hybrid screening using rolling circle amplification. Anal. Biochem. 331, 195–197.PubMedGoogle Scholar
  13. 13.
    Chen, X., Ding, X., and Song, W.-Y. (2003) Isolation of plasmid DNA rescued from single colonies of Agrobacterium tumefaciens by means of rolling circle amplification. Plant Mol. Biol. Rep. 21, 411–415.CrossRefGoogle Scholar
  14. 14.
    Ding, X., Snyder, A. K., Shaw, R., Farmerie, W.G., and Song, W.-Y. (2003) Direct retransformation of yeast with plasmid DNA isolated from single yeast colonies using rolling circle amplification. Biotechniques 35, 774–779.PubMedGoogle Scholar
  15. 15.
    Toyn, J. H., Gunyuzlu, P. L., White, W. H., Thompson, L. A., and Hollis, G. F. (2000) A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance. Yeast 16, 553–560.PubMedCrossRefGoogle Scholar
  16. 16.
    Walhout, A. J. and Vidal, M. (2001) High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. Methods 24, 297–306.PubMedCrossRefGoogle Scholar
  17. 17.
    Soellick, T. R. and Uhrig, J. F. (2001) Development of an optimized interactionmating protocol for large-scale yeast two-hybrid analyses. Genome Biol. 2, research0052.1–0052.7.Google Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Xiaodong Ding
    • 1
  • Yan Zhang
    • 2
  • Wen-Yuan Song
    • 1
  1. 1.Department of Plant PathologyUniversity of FloridaGainesville
  2. 2.University of FloridaGainesville

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