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 O. A. Sommer; Rasmus Wernersson; Lars Rønn Olsen

doi:10.1021/sb500194z

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 O. A. Sommer, Rasmus Wernersson, Lars Rønn Olsen

University of Copenhagen

Research output: Contribution to journal › Journal article › Research › peer-review

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 .

Original language	English
Journal	A C S Synthetic Biology
Volume	4
Issue number	3
Pages (from-to)	342-349
Number of pages	8
ISSN	2161-5063
DOIs	https://doi.org/10.1021/sb500194z
Publication status	Published - 2015

Keywords

DNA assembly
USER cloning
Primer design
Site-directed mutagenesis
Point mutation
Web server

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10.1021/sb500194z

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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 .",

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AU - Genee, Hans Jasper

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AU - Jespersen, Jakob Berg

AU - Sommer, Morten O. A.

AU - Wernersson, Rasmus

AU - Olsen, Lars Rønn

PY - 2015

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N2 - 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 .

AB - 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 .

KW - DNA assembly

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KW - Primer design

KW - Site-directed mutagenesis

KW - Point mutation

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

Abstract

Keywords

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