ArticleApplied microbiology and biotechnology2018
Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy.
Article in Applied microbiology and biotechnology, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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11 citing papers in PubMed, 38 citations in OpenAlex.
- High-Throughput Methods for Variant Functional Assessment in Cardiac Disease.Circulation. Genomic and precision medicine · 2026Review
- Engineering 2-Deoxy-D-ribose-5-phosphate Aldolase for anti- and syn-Selective Epoxidations of α,β-Unsaturated Aldehydes.Angewandte Chemie (International ed. in English) · 2025Article
- Article
- Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.PLoS genetics · 2024Article
- Optimal trade-off control in machine learning-based library design, with application to adeno-associated virus (AAV) for gene therapy.Science advances · 2024Article
- Asymmetrical dose responses shape the evolutionary trade-off between antifungal resistance and nutrient use.Nature ecology & evolution · 2022Article
- Article
- Deep Mutational Scanning of Protein-Protein Interactions Between Partners Expressed from Their Endogenous Loci In Vivo.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Protein context shapes the specificity of SH3 domain-mediated interactions in vivo.Nature communications · 2021Article
- Targeted mutagenesis of multiple chromosomal regions in microbes.Current opinion in microbiology · 2020Review
- Evolving a Peptide: Library Platforms and Diversification Strategies.International journal of molecular sciences · 2019Review
Corrections and comments
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Authors and funding
3 authors at 2 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Site-saturation mutagenesis (SSM) has been used in directed evolution of proteins for a long time. As a special form of saturation mutagenesis, it involves individual randomization at a given residue with formation of all 19 amino acids. To date, the most efficient embodiment of SSM is a one-step PCR-based approach using NNK codon degeneracy. However, in the case of difficult-to-randomize genes, SSM may not deliver all of the expected 19 mutants, which compels the user to invest further efforts by applying site-directed mutagenesis for the construction of the missing mutants. To solve this problem, we developed a two-step PCR-based technique in which a mutagenic primer and a non-mutagenic (silent) primer are used to generate a short DNA fragment, which is recovered and then employed as a megaprimer to amplify the whole plasmid. The present two-step and older one-step (partially overlapped primer approach) procedures were compared by utilizing cytochrome P450-BM3, which is a "difficult-to-randomize" gene. The results document the distinct superiority of the new method by checking the library quality on DNA level based on massive sequence data, but also at amino acid level. Various future applications in biotechnology can be expected, including the utilization when constructing mutability landscapes, which provide semi-rational information for identifying hot spots for protein engineering and directed evolution.
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