Assembling Linear DNA Templates for In Vitro Transcription and Translation

  • Viktor Stein
  • Miriam Kaltenbach
  • Florian HollfelderEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 815)


Cell-free expression systems provide straightforward access from genes to the corresponding proteins, involving fewer handling steps than in vivo procedures. A quick procedure to assemble a gene of interest into a linear DNA template together with 3′- and 5′-untranslated regions using a coupled uracil-excision–ligation strategy based on USER Enzyme and T4 DNA ligase. This methodology will be useful for repeated cycles of expression and in vitro selection, in which gene libraries are repeatedly assembled and their products and templates regenerated.

Key words

Linear DNA template assembly USER friendly cloning In vitro screening and selection In vitro transcription/translation 


  1. 1.
    Leemhuis, H., Stein, V., Griffiths, A.D. and Hollfelder, F. (2005) New genotype-phenotype linkages for directed evolution of functional proteins. Curr Opin Struct Biol, 15, 472–478.PubMedCrossRefGoogle Scholar
  2. 2.
    He, M. (2008) Cell-free protein synthesis: applications in proteomics and biotechnology. New Biotechnol, 25, 126–132.CrossRefGoogle Scholar
  3. 3.
    Rashtchian, A. (1995) Novel methods for cloning and engineering genes using the polymerase chain reaction. Curr Opin Biotechnol, 6, 30–36.PubMedCrossRefGoogle Scholar
  4. 4.
    Nisson, P.E., Rashtchian, A. and Watkins, P.C. (1991) Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase. PCR Methods Appl, 1, 120–123.PubMedGoogle Scholar
  5. 5.
    Smith, C., Day, P.J. and Walker, M.R. (1993) Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors. PCR Methods Appl, 2, 328–332.PubMedGoogle Scholar
  6. 6.
    Nour-Eldin, H.H., Hansen, B.G., Norholm, M.H., Jensen, J.K. and Halkier, B.A. (2006) Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res, 34, e122.PubMedCrossRefGoogle Scholar
  7. 7.
    Nour-Eldin, H.H., Geu-Flores, F. and Halkier, B.A. USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. Methods Mol Biol, 643, 185–200.Google Scholar
  8. 8.
    Geu-Flores, F., Nour-Eldin, H.H., Nielsen, M.T. and Halkier, B.A. (2007) USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res, 35, e55.PubMedCrossRefGoogle Scholar
  9. 9.
    Gronemeyer, T., Chidley, C., Juillerat, A., Heinis, C. and Johnsson, K. (2006) Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling. Protein Eng Des Sel, 19, 309–316.PubMedCrossRefGoogle Scholar
  10. 10.
    Connolly, B.A., Fogg, M.J., Shuttleworth, G. and Wilson, B.T. (2003) Uracil recognition by archaeal family B DNA polymerases. Biochem Soc Trans, 31, 699–702.PubMedCrossRefGoogle Scholar
  11. 11.
    Norholm, M.H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol, 10, 21.Google Scholar
  12. 12.
    Hofacker, I.L. (2003) Vienna RNA secondary structure server. Nucleic Acids Res, 31, 3429–3431.PubMedCrossRefGoogle Scholar
  13. 13.
    SantaLucia, J., Jr. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA, 95, 1460–1465.PubMedCrossRefGoogle Scholar
  14. 14.
    Stein, V. and Hollfelder, F. (2009) An efficient method to assemble linear DNA templates for in vitro screening and selection systems. Nucleic Acids Res, 37, e122.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Viktor Stein
    • 1
  • Miriam Kaltenbach
    • 1
  • Florian Hollfelder
    • 1
    Email author
  1. 1.Department of BiochemistryUniversity of CambridgeCambridgeUK

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