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

• Main Author: Nørholm Morten HH
• Format: Article
• Language: English
• Published: BMC 2010-03-01
• Series: BMC Biotechnology
• Online Access: http://www.biomedcentral.com/1472-6750/10/21

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p>