Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.
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/.
†The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Hans Jasper
HJ
Genee
†The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Mads Tvillinggaard
MT
Bonde
‡Bioinformatics Centre, Department of Biology, University of Copenhagen, Copenhagen, Denmark.|§The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, Copenhagen, Denmark.|∥Biotech Research and Innovation Center (BRIC), Copenhagen, Denmark.
Frederik Otzen
FO
Bagger
⊥Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark.|#Department of Chemistry, Technical University of Denmark, Lyngby, Denmark.
Jakob Berg
JB
Jespersen
†The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.|∇Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark.
Morten O A
MO
Sommer
⊥Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark.|○Intomics A/S, Lyngby, Denmark.
Rasmus
R
Wernersson
‡Bioinformatics Centre, Department of Biology, University of Copenhagen, Copenhagen, Denmark.|∥Biotech Research and Innovation Center (BRIC), Copenhagen, Denmark.
Lars Rønn
LR
Olsen
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020407:Internet; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D017354:Point Mutation; D012984:Software; D058615:Synthetic Biology / Q000379:methods
D017931:DNA Primers; D004247:DNA
DNA assembly; USER cloning; Web server; point mutation; primer design; site-directed mutagenesis