A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering.
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.
Nørholm
Morten H H
MH
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden. norholm@gmail.com
D019843:Archaeal Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D016133:Polymerase Chain Reaction; D014498:Uracil / Q000737:chemistry*
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; C093228:Sso7d protein, Sulfolobus; D014498:Uracil; D004247:DNA; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase