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The Polymerase Incomplete Primer Extension (PIPE) Method Applied to High-Throughput Cloning and Site-Directed Mutagenesis

  • Heath E. Klock
  • Scott A. Lesley
Part of the Methods in Molecular Biology book series (MIMB, volume 498)

Summary

Significant innovations in molecular biology methods have vastly improved the speed and efficiency of traditional restriction site and ligase-based cloning strategies. “Enzyme-free” methods eliminate the need to incorporate constrained sequences or modify Polymerase Chain Reaction (PCR)-generated DNA fragment ends. The Polymerase Incomplete Primer Extension (PIPE) method further condenses cloning and mutagenesis to a very simple two-step protocol with complete design flexibility not possible using related strategies. With this protocol, all major cloning operations are achieved by transforming competent cells with PCR products immediately following amplification. Normal PCRs generate mixtures of incomplete extension products. Using simple primer design rules and PCR, short, overlapping sequences are introduced at the ends of these incomplete extension mixtures which allow complementary strands to anneal and produce hybrid vector/insert combinations. These hybrids are directly transformed into recipient cells without any post-PCR enzymatic manipulations. We have found this method to be very easy and fast as compared to other available methods while retaining high efficiencies. Using this approach, we have cloned thousands of genes in parallel using a minimum of effort. The method is robust and amenable to automation as only a few, simple processing steps are needed.

Key words:

Cloning Ligase independent Enzyme free Site-directed mutagenesis PIPE Incomplete primer extension 

References

  1. 1.
    Scharf, S.J., Horn, G.T., Erlich, H.A. (1986) Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 233, 1076–78.CrossRefPubMedGoogle Scholar
  2. 2.
    Costa, G.L., Grafsky, A., Weiner, M.P. (1994) Cloning and analysis of PCR-gener-ated DNA fragments. PCR Methods Appl. 6,338–45.Google Scholar
  3. 3.
    Aslanidis, C., de Jong, P.J. (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069–74.CrossRefPubMedGoogle Scholar
  4. 4.
    Hsiao, K. (1993) Exonuclease III induced ligase-free directional subcloning of PCR products. Nucleic Acids Res. 21, 5528–9.CrossRefPubMedGoogle Scholar
  5. 5.
    Boyd, A.C. (1993) Turbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids. Nucleic Acids Res. 21, 817–821.CrossRefPubMedGoogle Scholar
  6. 6.
    Bubeck, P., Winkler, M., Bautsch, W. (1993) Rapid cloning by homologous recombination in vivo. Nucleic Acids Res. 21, 3601–2.CrossRefPubMedGoogle Scholar
  7. 7.
    Liu, Q., Li, M.Z., Leibham, D., Cortez, D., Elledge, S.J. (1998) The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr Biol. 8, 1300–9.CrossRefPubMedGoogle Scholar
  8. 8.
    Oliner, J.D., Kinzler, K.W., Vogelstein, B.(1993) In vivo cloning of PCR products in E. coli. Nucleic Acids Res. 21, 5192–7.CrossRefGoogle Scholar
  9. 9.
    Hartley, J.L., Temple, G.F., Brasch, M.A. (2000) DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–95.CrossRefPubMedGoogle Scholar
  10. 10.
    Tillett, D., Neilan, B.A. (1999) Enzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sites. Nucleic Acids Res. 27, e26.CrossRefPubMedGoogle Scholar
  11. 11.
    Chiu, J., March, P.E., Lee, R., Tillett, D. (2004) Site-directed, ligase-independent mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h. Nucleic Acids Res. 32, e174.CrossRefPubMedGoogle Scholar
  12. 12.
    Kirsch, R.D., Joly, E. (1998) An improved PCR-mutagenesis strategy for two-site muta-genesis or sequence swapping between related genes. Nucleic Acids Res. 26, 1848–50.CrossRefPubMedGoogle Scholar
  13. 13.
    Sawano, A., Miyawaki, A. (2000) Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res. 28, e78.CrossRefPubMedGoogle Scholar
  14. 14.
    Hanke, M., Wink, M. (1995) Direct DNA sequencing of PCR-amplified vector inserts following enzymatic degradation of primer and dNTPs. Biotechniques.18, 636.Google Scholar
  15. 15.
    Olsen, D.B., Eckstein, F. (1989) Incomplete primer extension during in vitro DNA amplification catalyzed by Ta q polymerase; exploitation for DNA sequencing. Nucleic Acids Res. 23, 9613–20.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Heath E. Klock
    • 1
  • Scott A. Lesley
    • 1
  1. 1.The Genomics Institute of the Novartis Research FoundationSan DiegoUSA

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