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Nucleic Acids Research Advance Access originally published online on July 17, 2009
Nucleic Acids Research 2009 37(18):e122; doi:10.1093/nar/gkp589
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Nucleic Acids Research, 2009, Vol. 37, No. 18 e122
© 2009 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

An efficient method to assemble linear DNA templates for in vitro screening and selection systems

Viktor Stein and Florian Hollfelder*

Department of Biochemistry, University of Cambridge, CB2 1GA, Cambridge, UK

*To whom correspondence should be addressed. Tel: +44 1223 766048; Fax: +44 1223 766002; Email: fh111{at}mole.bio.cam.ac.uk

Received January 19, 2009. Revised June 24, 2009. Accepted June 26, 2009.

A method is presented to assemble a gene of interest into a linear DNA template with all the components necessary for in vitro transcription and translation in ~90 min. Assembly is achieved using a coupled uracil excision–ligation strategy based on USER Enzyme and T4 DNA ligase, which allows the simultaneous and seamless assembly of three different PCR products. The method is suitable for screening and selection systems of very high throughput as up to 1011 molecules can be efficiently assembled and purified in reaction volumes of 100 µl. The method is exemplified with the gene coding for a mutant version of O6-alkylguanine alkyltransferase, which is efficiently assembled with an N-terminal peptide tag and its 5'- and 3'-untranslated regions that include a T7 promoter, ribosome binding site and T7 terminator. The utility of the method is further corroborated by assembling error-prone PCR libraries and regenerating templates following model affinity selections. This fast and robust method should find widespread application in directed evolution for the assembly of gene libraries and the regeneration of linear DNA templates between successive screening and selection cycles.


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