Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly

Hans Jasper Genee, Mads Tvillinggaard Bonde, Frederik Otzen Bagger, Jakob Berg Jespersen, Morten Sommer, Rasmus Wernersson, Lars Rønn Olsen

37 Citationer (Scopus)

Abstract

USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/ .

OriginalsprogEngelsk
TidsskriftA C S Synthetic Biology
Vol/bind4
Udgave nummer3
Sider (fra-til)342-9
Antal sider8
ISSN2161-5063
DOI
StatusUdgivet - 20 mar. 2015

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