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Updated: Jun 15, 2026

Homemade Site Directed Mutagenesis of Whole Plasmids
Published on: May 11, 2009
A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering
1Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden. norholm@gmail.com
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.
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