e122 Nucleic Acids Research, 2009, Vol. 37, No. 18 separately. Depending on the type of modiﬁcation, this may signiﬁcantly add to the costs. Furthermore, PCR ampliﬁcation of the whole template is expected to accu- mulate mutations in constant parts, particularly in the coding regions of long and essential fusion genes, which can signiﬁcantly compromise the performance of a screening and selection process (9). An exception relates to streptavidin that forms the basis of a non-covalent DNA display system (5) and has also been the subject of evolutionary optimization to improve binding towards a biotin analogue (16). In both cases, the entire streptavidin gene was ampliﬁed for at least ﬁve successive selection cycles with no detrimental eﬀect on the selection process suggesting that streptavidin can tolerate this level of muta- tion. This may, however, be a protein-speciﬁc eﬀect and does not necessarily apply to systems that rely on less robust proteins. In summary, the assembly protocol presented here should be applicable to many diﬀerent in vitro screening and selection systems, especially those that feature rela- tively long fusion genes that are susceptible to mutations and thus rely on nested PCRs. The protocol saves time, labour and enables many rapid successive selection cycles. This is highly practical when mimicking evolutionary processes such as genetic drifts. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank Yolanda Scha¨ rli for helpful advice with experi- mental methods and comments on the manuscript. We would also like to thank the reviewers for their helpful comments and suggestions. FUNDING EU NEST project MiFem; an EPSRC Platform grant; ﬁnancial support to Trinity College, Cambridge and the BBSRC (to V.S.). F.H. is an European Research Council (ERC) Starting Investigator. Funding for open access charge: EU NEST MiFem. REFERENCES 1. Jackel,C., Kast,P. and Hilvert,D. 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