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Nucleic Acids Research Advance Access published online on March 6, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm041
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© 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.

Nucleic Acid Enzymes

USERTM friendly DNA engineering and cloning method by uracil excision

Jurate Bitinaite1,*, Michelle Rubino2, Kamini Hingorani Varma3, Ira Schildkraut4, Romualdas Vaisvila1 and Rita Vaiskunaite1

1New England Biolabs, Inc., 240 County Road, Ipswich MA 01938, USA, 2Department of Radiology, Maine Medical Center, 22 Bramhall Street, Portland, ME 04102, USA, 3Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404, USA and 4CerroSci LLC, PO Box 177, Cerrillos, NM 87010, USA

*To whom correspondence should be addressed. Tel: +1 978 927 5054; Fax: +1 978 921 1350; Email: bitinait{at}neb.com

Received October 27, 2006. Revised December 22, 2006. Accepted January 9, 2007.

Here we report a PCR-based DNA engineering technique for seamless assembly of recombinant molecules from multiple components. We create cloning vector and target molecules flanked with compatible single-stranded (ss) extensions. The vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The spacer sequences between the nicking and restriction sites are tailored to create ss extensions of custom sequence. The vector is then linearized by digestion with nicking and restriction endonucleases. To generate target molecules, a single deoxyuridine (dU) residue is placed 6–10 nt away from the 5'-end of each PCR primer. 5' of dU the primer sequence is compatible either with an ss extension on the vector or with the ss extension of the next-in-line PCR product. After amplification, the dU is excised from the PCR products with the USER enzyme leaving PCR products flanked by 3' ss extensions. When mixed together, the linearized vector and PCR products directionally assemble into a recombinant molecule through complementary ss extensions. By varying the design of the PCR primers, the protocol is easily adapted to perform one or more simultaneous DNA manipulations such as directional cloning, site-specific mutagenesis, sequence insertion or deletion and sequence assembly.


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