UNIT 3.21 DNA Cloning and Engineering by Uracil Excision

  1. Jurate Bitinaite,
  2. Nicole M. Nichols

Published Online: 1 APR 2009

DOI: 10.1002/0471142727.mb0321s86

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Bitinaite, J. and Nichols, N. M. 2009. DNA Cloning and Engineering by Uracil Excision. Current Protocols in Molecular Biology. 86:V:3.21:3.21.1–3.21.16.

Author Information

  1. New England Biolabs, Ipswich, Massachusetts

Publication History

  1. Published Online: 1 APR 2009
  2. Published Print: APR 2009


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.


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