title: One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.
abstract: Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis.
authors.affiliation: Biomarkers in Cancer Unit, Biocruces Health Research Institute, Barakaldo, Spain.
authors.forename: Janire
authors.initials: J
authors.lastname: Mingo
authors.affiliation: Quantitative Biomedicine Unit, Biocruces Health Research Institute, Barakaldo, Spain.
authors.forename: Asier
authors.initials: A
authors.lastname: Erramuzpe
authors.affiliation: Biomarkers in Cancer Unit, Biocruces Health Research Institute, Barakaldo, Spain.
authors.forename: Sandra
authors.initials: S
authors.lastname: Luna
authors.affiliation: Biomarkers in Cancer Unit, Biocruces Health Research Institute, Barakaldo, Spain.
authors.forename: Olaia
authors.initials: O
authors.lastname: Aurtenetxe
authors.affiliation: Biomarkers in Cancer Unit, Biocruces Health Research Institute, Barakaldo, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Amo
authors.affiliation: Quantitative Biomedicine Unit, Biocruces Health Research Institute, Barakaldo, Spain.
authors.forename: Ibai
authors.initials: I
authors.lastname: Diez
authors.affiliation: Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Jan T G
authors.initials: JT
authors.lastname: Schepens
authors.affiliation: Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Wiljan J A J
authors.initials: WJ
authors.lastname: Hendriks
authors.affiliation: Quantitative Biomedicine Unit, Biocruces Health Research Institute, Barakaldo, Spain.|IKERBASQUE, Basque Foundation for Science, Bilbao, Spain.
authors.forename: Jesús M
authors.initials: JM
authors.lastname: Cortés
authors.affiliation: Biomarkers in Cancer Unit, Biocruces Health Research Institute, Barakaldo, Spain.|IKERBASQUE, Basque Foundation for Science, Bilbao, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Pulido
mesh_terms: D019943:Amino Acid Substitution; D017931:DNA Primers / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5 / Q000235:genetics / Q000378:metabolism; D012984:Software
chemical_list: D017931:DNA Primers; C516895:PTPRZ1 protein, human; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human