Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

Nucleic Acids Res. 2006;34(18):e122. doi: 10.1093/nar/gkl635. Epub 2006 Sep 25.

Abstract

The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular / methods*
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyribonucleases, Type II Site-Specific
  • Genetic Vectors
  • Polymerase Chain Reaction*
  • Uracil / metabolism*

Substances

  • Uracil
  • DNA-Directed DNA Polymerase
  • Deoxyribonucleases, Type II Site-Specific
  • endodeoxyribonuclease PacI