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Nucleic Acids Research Advance Access originally published online on March 27, 2007
Nucleic Acids Research 2007 35(7):e55; doi:10.1093/nar/gkm106
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Nucleic Acids Research, 2007, Vol. 35, No. 7 e55
© 2007 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

USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products

Fernando Geu-Flores, Hussam H. Nour-Eldin, Morten T. Nielsen and Barbara A. Halkier*

Plant Biochemistry Laboratory, Institute of Plant Biology, Faculty of Life Sciences, University of Copenhagen, 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 December 14, 2006. Revised January 30, 2007. Accepted February 6, 2007.

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5' end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3' overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications.


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