A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering

BMC Biotechnol. 2010 Mar 16;10:21. doi: 10.1186/1472-6750-10-21.

Abstract

Background: The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR.

Results: Here, the addition of a highly efficient DNA polymerase and a low-background-, large-insertion- compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision DNA engineering.

Conclusions: The different uracil-excision based molecular tools that have been developed in an open-source fashion, constitute a comprehensive, yet simple and inexpensive toolkit for any need in molecular cloning.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaeal Proteins / genetics
  • Cloning, Molecular / methods*
  • DNA / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Directed DNA Polymerase / chemistry*
  • Mutagenesis, Site-Directed / methods*
  • Polymerase Chain Reaction
  • Uracil / chemistry*

Substances

  • Archaeal Proteins
  • DNA-Binding Proteins
  • Sso7d protein, Sulfolobus
  • Uracil
  • DNA
  • Pfu DNA polymerase
  • DNA-Directed DNA Polymerase