One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections
| Autor: | Sandra Luna, Ibai Diez, Asier Erramuzpe, Wiljan Hendriks, Jesus M. Cortes, Olaia Aurtenetxe, Laura Amo, Janire Mingo, Jan Schepens, Rafael Pulido |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Mutant Gene Expression lcsh:Medicine Artificial Gene Amplification and Extension medicine.disease_cause Polymerase Chain Reaction Biochemistry 0302 clinical medicine Plasmid Amino Acids Site-directed mutagenesis lcsh:Science Genetics Mutation Multidisciplinary Receptor-Like Protein Tyrosine Phosphatases Class 5 Organic Compounds Complementary DNA Nucleic acids Chemistry Template 030220 oncology & carcinogenesis Physical Sciences Plasmids Research Article Substitution Mutation Forms of DNA Nucleotide Sequencing Mutagenesis (molecular biology technique) DNA construction Biology Research and Analysis Methods 03 medical and health sciences Escherichia coli medicine Humans Molecular Biology Techniques Sequencing Techniques Molecular Biology DNA Primers Point mutation Organic Chemistry lcsh:R PTEN Phosphohydrolase Chemical Compounds Biology and Life Sciences Proteins DNA 030104 developmental biology Amino Acid Substitution Mutagenesis Plasmid Construction Mutagenesis Site-Directed lcsh:Q Primer (molecular biology) Nanomedicine Radboud Institute for Molecular Life Sciences [Radboudumc 19] Software |
| Zdroj: | PLoS One, 11 PLoS ONE, Vol 11, Iss 8, p e0160972 (2016) PLoS One, 11, 8 PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | Contains fulltext : 171086.PDF (Publisher’s version ) (Open Access) 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. |
| Databáze: | OpenAIRE |
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