Journal article
Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly
The Novo Nordisk Foundation Center for Biosustainability1
Bioinformatics Centre, Department of Biology2
Faculty of Health Sciences, Copenhagen3
Copenhagen4
Center for Biological Sequence Analysis, Department of Systems Biology5
Department of Chemistry6
Department of Systems Biology7
Intomics A/S, Lyngby8
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/.
Language: | English |
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Publisher: | American Chemical Society |
Year: | 2014 |
ISSN: | 21615063 |
Types: | Journal article |
DOI: | 10.1021/sb500194z |