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Open Access Highly Accessed Methodology article

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

Morten HH Nørholm

Author Affiliations

Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden

BMC Biotechnology 2010, 10:21  doi:10.1186/1472-6750-10-21

Published: 16 March 2010

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.