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Nucleic Acids Research Advance Access originally published online on September 25, 2006
Nucleic Acids Research 2006 34(18):e122; doi:10.1093/nar/gkl635
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Nucleic Acids Research, 2006, Vol. 34, No. 18 e122
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

Hussam H. Nour-Eldin, Bjarne G. Hansen, Morten H. H. Nørholm, Jacob K. Jensen and Barbara A. Halkier*

Plant Biochemistry Laboratory, Center for Molecular Plant Physiology, The Royal Veterinary and Agricultural University Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

*To whom correspondence should be addressed. Tel: +45 35283342; Fax: +45 35283333; Email: bah{at}kvl.dk

Received May 22, 2006. Revised August 11, 2006. Accepted August 15, 2006.

The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed.

The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors


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