DNA Cloning and Engineering by Uracil Excision

Jurate Bitinaite1, Nicole M. Nichols1

1 New England Biolabs, Ipswich, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.21
DOI:  10.1002/0471142727.mb0321s86
Online Posting Date:  April, 2009
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Abstract

This unit describes a simple and efficient DNA engineering method that combines nucleotide sequence alteration, multiple PCR fragment assembly, and directional cloning. PCR primers contain a single deoxyuracil residue (dU), and can be designed to accommodate nucleotide substitutions, insertions, and/or deletions. The primers are then used to amplify DNA in discrete fragments that incorporate a dU at each end. Excision of deoxyuracils results in PCR fragments flanked by unique, overlapping, single‐stranded extensions that allow the seamless and directional assembly of customized DNA molecules into a linearized vector. In this way, multi‐fragment assemblies, as well as various mutagenic changes, can all be accomplished in a single‐format experiment. Two basic protocols on the methods of uracil excision‐based engineering are presented, and special attention is given to primer design. The use of a commercially available cloning vector and the preparation of custom vectors are also presented. Curr. Protoc. Mol. Biol. 86:3.21.1‐3.21.16. © 2009 by John Wiley & Sons, Inc.

Keywords: uracil excision; UDG; USER enzyme; DNA mutagenesis; directional cloning; DNA assembly; nicking endonuclease

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: General Methods for DNA Cloning and Engineering by Uracil Excision
  • Basic Protocol 2: Construction and Linearization of Custom‐Made Vectors Compatible with DNA Cloning by Uracil Excision
  • Alternate Protocol 1: Site‐Specific Mutagenesis
  • Alternate Protocol 2: Sequence Insertions
  • Alternate Protocol 3: Sequence Deletions
  • Alternate Protocol 4: Nucleotide Sequence Assembly, Domain Substitution, and Multiple Sequence Manipulations
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: General Methods for DNA Cloning and Engineering by Uracil Excision

  Materials
  • Template DNA
  • Oligonucleotide primers, each containing a single dU residue (see discussion on primer design, above; see units 2.11& 2.12 for synthesis and purification of oligonucleotides)
  • Pfu Turbo C x Hotstart DNA Polymerase (Stratagene) and 10× Pfu C x Reaction Buffer (Stratagene)
  • 20 U/µl restriction endonuclease DpnI
  • 20 ng/µl linearized vector pNEB206A (Fig. ; New England Biolabs) or linearized custom‐made vector compatible with DNA cloning by uracil excision ( protocol 2)
  • 1 U/µl USER Enzyme (New England Biolabs)
  • Chemically competent E. coli cells (available from various molecular biology suppliers; also see unit 1.8)
  • 2 U/µl restriction endonuclease BbvCI and 10× buffer for BbvCI
  • Additional reagents and equipment for DNA amplification by PCR (unit 15.1), agarose gel electrophoresis (unit 2.5), restriction endonuclease digestion (unit 3.1), chemical transformation of DNA (unit 1.8), plasmid DNA minipreps (unit 1.6), DNA sequencing (Chapter 7), and subcloning of DNA (unit 3.16)

Basic Protocol 2: Construction and Linearization of Custom‐Made Vectors Compatible with DNA Cloning by Uracil Excision

  Materials
  • 0.1 pmol appropriate phosphorylated, blunt‐ended DNA vector
  • 2.5 pmol/µl Universal USER Cassette (New England Biolabs)
  • T4 DNA Ligase (2000 U/µl; New England Biolabs) and 10× T4 DNA ligase buffer
  • Chemically competent E. coli cells (available from various molecular biology suppliers; also see unit 1.8)
  • 10× XbaI buffer
  • Restriction endonuclease XbaI
  • Nt.BbvCI nicking endonuclease (New England Biolabs)
  • TE buffer, pH 8.0 ( appendix 22)
  • Additional reagents and equipment for transformation of DNA (unit 1.8), plasmid DNA purification (Chapter 1), phenol/chloroform extraction and ethanol precipitation of DNA (unit 2.1), restriction enzyme digestion (unit 3.1), and determination of DNA concentration ( appendix 3D)
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Figures

Videos

Literature Cited

   Bitinaite, J., Rubino, M., Hingorani‐Vaarma, K., Schildkraut, I., Vaisvila, R., and Vaiskunaite, R. 2007. USER friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res. 35:1992‐2002.
   Braman, J., Papworth, C., and Greener, A. 1996. Site‐directed mutagenesis using double‐stranded plasmid DNA templates. Methods Mol. Biol. 57:31‐44.
   Booth, P.M., Buchman, G.W., and Rashtchian, A. 1994. Assembly and cloning of coding sequences for neurotrophic factors directly from genomic DNA using polymerase chain reaction and uracil DNA glycosylase. Gene 146:303‐308.
   Fogg, M.J., Pearl, L.H., and Connolly, BA. 2002. Structural basis for uracil recognition by archaeal family B DNA polymerases. Nat. Struct. Biol. 9:922‐927.
   Greagg, M.A., Fogg, M.J., Panayotou, G., Evans, S.J., Connolly, B.A., and Pearl, L.H. 1999. A read‐ahead function in archaeal DNA polymerases detects promutagenic template‐strand uracil. Proc. Natl Acad. Sci. U.S.A. 96:9045‐9050.
   Hemsley, A., Arnheim, N., Toney, M.D., Cortopassi, G., and Galas, D.J. 1989. A simple method for site‐specific mutagenesis using the polymerase chain reaction. Nucleic Acids Res. 17:6545‐6551.
   Ho, S.F., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R. 1989. Site‐directed mutagenesis by overlap extension using polymerase chain reaction. Gene 77:51‐59.
   Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., and Pease, L.R. 1989. Engineering hybrid genes without the use of restriction enzymes: Gene splicing by overlap extension. Gene 77:61‐68.
   Lasken, R.S., Schuster, D.M., and Rashtchian, A. 1996. Archaebacterial DNA polymerases tightly bind uracil‐containing DNA. J. Biol. Chem. 271:17692‐17696.
   Lundberg, K.S., Shoemaker, D.D., Adams, M.W.W., Short, J.M., Sorge, J.A., and Mathur, E.J. 1991. High‐fidelity amplification using thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108:1‐6.
   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.
   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.
   Rashtchian, A., Thornton, C.G., and Heidecker, G. 1992. A novel method for site‐directed mutagenesis using PCR and uracil DNA glycosylase. PCR Methods Appl. 2:124‐130.
   Weiner, M.P., Costa, G.L., Schoetlin, W., Cline, J., Mathur, E., and Bauer, J.C. 1994. Site‐directed mutagenesis of double‐stranded DNA by the polymerase chain reaction. Gene 151:119‐123.
Key Reference
   Bitinaite et al., 2007. See above.
  Provides specific examples of DNA engineering by uracil excision.
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