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A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2010 (English)In: BMC Biotechnology, ISSN 1472-6750, Vol. 10, 21-27 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2010. Vol. 10, 21-27 p.
National Category
Natural Sciences
URN: urn:nbn:se:su:diva-49108DOI: 10.1186/1472-6750-10-21ISI: 000276299200001OAI: diva2:376840
authorCount :1Available from: 2010-12-13 Created: 2010-12-10 Last updated: 2010-12-29Bibliographically approved

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Nørholm, Morten H. H.
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