ArticleApplied microbiology and biotechnology2008
Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates.
Article in Applied microbiology and biotechnology, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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29 citing papers in PubMed, 127 citations in OpenAlex.
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- A Haloalkane Dehalogenase from Saccharomonospora viridis Strain DSM 43017, a Compost Bacterium with Unusual Catalytic Residues, Unique (Applied and environmental microbiology · 2020Article
- Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties.Biomolecules · 2020Article
- Twin enzymes, divergent control: The cholesterogenic enzymes DHCR14 and LBR are differentially regulated transcriptionally and post-translationally.The Journal of biological chemistry · 2020Article
- Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic BacteriumMicroorganisms · 2019Article
- Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution.Nature communications · 2019Article
- Asp305Gly mutation improved the activity and stability of the styrene monooxygenase for efficient epoxide production in Pseudomonas putida KT2440.Microbial cell factories · 2019Article
- Converting Galactose into the Rare Sugar Talose with Cellobiose 2-Epimerase as Biocatalyst.Molecules (Basel, Switzerland) · 2018Article
- Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy.Applied microbiology and biotechnology · 2018Article
- A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.The Journal of biological chemistry · 2017Article
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- Glucosylglycerate Phosphorylase, an Enzyme with Novel Specificity Involved in Compatible Solute Metabolism.Applied and environmental microbiology · 2017Article
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- Bioinformatics for the synthetic biology of natural products: integrating across the Design-Build-Test cycle.Natural product reports · 2016Review
- Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing.Molecular biotechnology · 2015Article
- The Regulatory Domain of Squalene Monooxygenase Contains a Re-entrant Loop and Senses Cholesterol via a Conformational Change.The Journal of biological chemistry · 2015Article
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Authors and funding
17 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.
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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.