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

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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 .
Original languageEnglish
JournalA C S Synthetic Biology
Volume4
Issue number3
Pages (from-to)342-349
Number of pages8
ISSN2161-5063
DOIs
Publication statusPublished - 2015

Keywords

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

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