In-Fusion® Cloning with Vaccinia Virus DNA Polymerase

  • Chad R. Irwin
  • Andrew Farmer
  • David O. Willer
  • David H. EvansEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 890)


Vaccinia virus DNA polymerase (VVpol) encodes a 3′-to-5′ proofreading exonuclease that can degrade the ends of duplex DNA and expose single-stranded DNA tails. The reaction plays a critical role in promoting virus recombination in vivo because single-strand annealing reactions can then fuse molecules sharing complementary tails into recombinant precursors called joint molecules. We have shown that this reaction can also occur in vitro, providing a simple method for the directional cloning of PCR products into any vector of interest. A commercial form of this recombineering technology called In-Fusion® that facilitates high-throughput directional cloning of PCR products has been commercialized by Clontech. To effect the in vitro cloning reaction, PCR products are prepared using primers that add 16–18 bp of sequence to each end of the PCR amplicon that are homologous to the two ends of a linearized vector. The linearized vector and PCR products are coincubated with VVpol, which exposes the complementary ends and promotes joint molecule formation. Vaccinia virus single-stranded DNA binding protein can be added to enhance this reaction, although it is not an essential component. The resulting joint molecules are used to transform E. coli, which convert these noncovalently joined molecules into stable recombinants. We illustrate how this technology works by using, as an example, the cloning of the vaccinia N2L gene into the vector pETBlue-2.

Key words

Vaccinia virus Recombineering In-Fusion® cloning DNA polymerase PCR cloning 


  1. 1.
    Shuman S (1992) Two classes of DNA end-joining reactions catalyzed by vaccinia topoisomerase I. J Biol Chem 267:16755–16758PubMedGoogle Scholar
  2. 2.
    Shuman S (1992) DNA strand transfer reactions catalyzed by vaccinia topoisomerase I. J Biol Chem 267:8620–8627PubMedGoogle Scholar
  3. 3.
    Marsischky G, LaBaer J (2004) Many paths to many clones: a comparative look at high-throughput cloning methods. Genome Res 14:2020–2028PubMedCrossRefGoogle Scholar
  4. 4.
    Gammon DB, Evans DH (2009) The 3′-to-5′ exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination. J Virol 83:4236–4250PubMedCrossRefGoogle Scholar
  5. 5.
    Willer DO et al (1999) Vaccinia virus DNA polymerase promotes DNA pairing and strand-transfer reactions. Virology 257:511–523PubMedCrossRefGoogle Scholar
  6. 6.
    Willer DO et al (2000) In vitro concatemer formation catalyzed by vaccinia virus DNA polymerase. Virology 278:562–569PubMedCrossRefGoogle Scholar
  7. 7.
    Yao XD, Evans DH (2003) Characterization of the recombinant joints formed by single-strand annealing reactions in vaccinia virus-infected cells. Virology 308:147–156PubMedCrossRefGoogle Scholar
  8. 8.
    Yao XD, Evans DH (2001) Effects of DNA structure and homology length on vaccinia virus recombination. J Virol 75:6923–6932PubMedCrossRefGoogle Scholar
  9. 9.
    Hamilton MD et al (2007) Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase. Nucleic Acids Res 35:143–151PubMedCrossRefGoogle Scholar
  10. 10.
    Hamilton MD, Evans DH (2005) Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase. Nucleic Acids Res 33:2259–2268PubMedCrossRefGoogle Scholar
  11. 11.
    Sleight SC et al (2010) In-Fusion BioBrick assembly and re-engineering. Nucleic Acids Res 38:2624–2636PubMedCrossRefGoogle Scholar
  12. 12.
    Zhu B et al (2007) In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. BioTechniques 43:354–359PubMedCrossRefGoogle Scholar
  13. 13.
    Streisinger G et al (1966) Frameshift mutations and the genetic code. This paper is dedicated to Professor Theodosius Dobzhansky on the occasion of his 66th birthday. Cold Spring Harb Symp Quant Biol 31:77–84PubMedCrossRefGoogle Scholar
  14. 14.
    Benoit RM et al (2006) An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids. Protein Exp Purif 45:66–71CrossRefGoogle Scholar
  15. 15.
    Park J et al (2005) Building a human kinase gene repository: bioinformatics, molecular cloning, and functional validation. Proc Natl Acad Sci USA 102:8114–8119PubMedCrossRefGoogle Scholar
  16. 16.
    Berrow NS et al (2007) A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res 35:e45PubMedCrossRefGoogle Scholar
  17. 17.
    McDonald WF, Traktman P (1994) Overexpression and purification of the vaccinia virus DNA polymerase. Protein Exp Purif 5:409–421CrossRefGoogle Scholar
  18. 18.
    Tseng M et al (1999) DNA binding and aggregation properties of the vaccinia virus I3L gene product. J Biol Chem 274:21637–21644PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Chad R. Irwin
    • 1
  • Andrew Farmer
    • 2
  • David O. Willer
    • 3
  • David H. Evans
    • 1
    Email author
  1. 1.Department of Medical Microbiology and Immunology, Li Ka Shing Institute of VirologyUniversity of AlbertaEdmontonCanada
  2. 2.Clontech Laboratories, Inc.Mountain ViewUSA
  3. 3.Department of MicrobiologyMt. Sinai HospitalTorontoCanada

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