ArticlePloS one2012
PFunkel: efficient, expansive, user-defined mutagenesis.
Article in PloS one, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 72 papers.
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Who cites it
72 citing papers in PubMed.
- Rapid directed evolution guided by protein language models and epistatic interactions.Science (New York, N.Y.) · 2026Article
- Massively parallel functional genomic assays in endocrinology: from promise to delivery.Journal of molecular endocrinology · 2026Review
- Environmental and mutational modulation of collateral fitness effects informs their mechanisms.Molecular biology and evolution · 2026Article
- Origins and breadth of pairwise epistasis in an α-helix of β-lactamase TEM-1.Nature communications · 2026Article
- Deep-mutational scanning libraries using Tiled-Region Exchange mutagenesis.G3 (Bethesda, Md.) · 2026Article
- Deep Mutational Scanning in Immunology: Techniques and Applications.Pathogens (Basel, Switzerland) · 2025Review
- Variant scoring tools for deep mutational scanning.Molecular systems biology · 2025Review
- CRISPR/Cas system-guided plasmid mutagenesis without sequence restriction.Fundamental research · 2025Article
- Epitope mapping via in vitro deep mutational scanning methods and its applications.The Journal of biological chemistry · 2025Review
- Article
- Deep Mutational Scanning in Disease-related Genes with Saturation Mutagenesis-Reinforced Functional Assays (SMuRF).bioRxiv : the preprint server for biology · 2024Article
- Genes Vary Greatly in Their Propensity for Collateral Fitness Effects of Mutations.Molecular biology and evolution · 2023Article
- Mutational fitness landscape and drug resistance.Current opinion in structural biology · 2023Review
- Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis and Rolling Circle Amplification.Methods in molecular biology (Clifton, N.J.) · 2023Article
- SUNi mutagenesis: Scalable and uniform nicking for efficient generation of variant libraries.PloS one · 2023Article
- Deep mutational scanning: A versatile tool in systematically mapping genotypes to phenotypes.Frontiers in genetics · 2023Review
- Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.Nature catalysis · 2022Article
- Local and Global Protein Interactions Contribute to Residue Entrenchment in Beta-Lactamase TEM-1.Antibiotics (Basel, Switzerland) · 2022Article
- Oligo Pools as an Affordable Source of Synthetic DNA for Cost-Effective Library Construction in Protein- and Metabolic Pathway Engineering.Chembiochem : a European journal of chemical biology · 2022Review
- OverFlap PCR: A reliable approach for generating plasmid DNA libraries containing random sequences without a template bias.PloS one · 2022Article
12 more citing papers are in PubMed but not listed here.
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Authors and funding
2 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We introduce PFunkel, a versatile method for extensive, researcher-defined DNA mutagenesis using a ssDNA or dsDNA template. Once the template DNA is prepared, the method can be completed in a single day in a single tube, and requires no intermediate DNA purification or sub-cloning. PFunkel can be used for site-directed mutagenesis at an efficiency approaching 100%. More importantly, PFunkel allows researchers the unparalleled ability to efficiently construct user-defined libraries. We demonstrate the creation of a library with site-saturation at four distal sites simultaneously at 70% efficiency. We also employ PFunkel to create a comprehensive codon mutagenesis library of the TEM-1 ß-lactamase gene. We designed this library to contain 18,081 members, one for each possible codon substitution in the gene (287 positions in TEM-1 x 63 possible codon substitutions). Deep sequencing revealed that ∼97% of the designed single codon substitutions are present in the library. From such a library we identified 18 previously unreported adaptive mutations that each confer resistance to the ß-lactamase inhibitor tazobactam. Three of these mutations confer resistance equal to or higher than that of the most resistant reported TEM-1 allele and have the potential to emerge clinically.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.