Published online 17 July 2009 Nucleic Acids Research, 2009, Vol. 37, No. 18 e122 doi:10.1093/nar/gkp589 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 Received January 19, 2009; Revised June 24, 2009; Accepted June 26, 2009 ABSTRACT A method is presented to assemble a gene of interest into a linear DNA template with all the com- ponents 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 effi- ciently assembled and purified in reaction volumes of 100 ml. 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 tem- plates following model affinity selections. This fast and robust method should find widespread applica- tion in directed evolution for the assembly of gene libraries and the regeneration of linear DNA tem- plates between successive screening and selection cycles. INTRODUCTION Directed evolution mimics evolution through natural selection in the test tube with the aim of creating genes that code for proteins with new or improved functional traits (1). For this purpose, a number of in vitro screening and selection systems have been developed over the past 10 years (2–4). Nucleic acid display systems are now routinely used for the directed evolution of protein binders (5–9) and water-in-oil emulsions provide a high- throughput platform for the screening and selection of catalytic activities (10–13) and binding interactions (14–16). Their main advantages over screening and selec- tion systems that depend on steps in vivo, such as genetic screening and selection systems and surface display meth- ods based on phage, yeast or Escherichia coli, are as follows: (i) greater sensitivity, (ii) controllable assay con- ditions with less interference from cellular functions, (iii) rapid successive selection cycles, (iv) subcloning- independent processing of DNA, (v) ﬂexible in vitro expression systems that facilitate selections with non- natural amino acids and (vi) libraries that are neither lim- ited by toxicity nor transformation eﬃciencies (2–4). Between successive in vitro screening and selection cycles, the gene of interest (GOI) is propagated by PCR. Only the diversiﬁed region is usually ampliﬁed. This is to minimise the formation of non-speciﬁc by-products, regenerate sites for binding or conjugation and prevent the accumulation of mutations in constant parts, e.g. in regulatory regions, specialized linkers or fusion genes required for covalent or non-covalent conjugation (6–16). As a result, the GOI needs to be re-assembled into a functional template for in vitro transcription and translation (TS–TL). The preferred assembly methods are either based on overlap extension PCR (9,10) or sequential restriction–digestion and ligation reactions (7,8,12–15). Both strategies are, however, laborious, time- consuming and typically take an entire day to complete— in many cases longer than the selection itself. For instance, restriction–digestion–ligation protocols alone require sev- eral hours of sequential incubations. Furthermore, both strategies entail relatively long PCRs featuring 25–30 ampliﬁcation cycles to amplify the desired template from an overlap extension or ligation reaction (7–10,12–15). While the purity of templates is critical for the success of a screening and selection process, obtaining pure tem- plates after PCR ampliﬁcation is not necessarily a trivial task in practice (8). Ampliﬁcation from overlap extension and ligation reactions generates additional non-speciﬁc by-products. Therefore templates frequently need to be further puriﬁed by agarose gel electrophoresis (7–10,12). These additional steps signiﬁcantly add to the work-up *To whom correspondence should be addressed. Tel: +44 1223 766048; Fax: +44 1223 766002; Email: fh111@mole.bio.cam.ac.uk 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.