ArticleNucleic acids research2018
Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis.
Article in Nucleic acids research, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 37 citations in OpenAlex.
- High-throughput synthesis of DNA fragments by molecular self-assembly of overlapping oligonucleotides.Nature biotechnology · 2026Article
- Reversal of protein chemical aging by enzymatic deglycation.Nature communications · 2026Article
- Rapid directed evolution guided by protein language models and epistatic interactions.Science (New York, N.Y.) · 2026Article
- Assembling unregulated DNA segments bypasses synthesis screening: regulate fragments as select agents.Nature communications · 2026Article
- Rational evolution for altering the ligand preference of estrogen receptor alpha.Protein science : a publication of the Protein Society · 2024Article
- Fast and efficient template-mediated synthesis of genetic variants.Nature methods · 2023Article
- Antha-Guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries.Methods in molecular biology (Clifton, N.J.) · 2022Article
- OverFlap PCR: A reliable approach for generating plasmid DNA libraries containing random sequences without a template bias.PloS one · 2022Article
- Regio- and Stereoselective Steroid Hydroxylation at C7 by Cytochrome P450 Monooxygenase Mutants.Angewandte Chemie (International ed. in English) · 2020Article
- Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins.Nucleic acids research · 2020Article
- iFLinkC: an iterative functional linker cloning strategy for the combinatorial assembly and recombination of linker peptides with functional domains.Nucleic acids research · 2020Article
- User-defined single pot mutagenesis using unamplified oligo pools.Protein engineering, design & selection : PEDS · 2019Article
- Golden Mutagenesis: An efficient multi-site-saturation mutagenesis approach by Golden Gate cloning with automated primer design.Scientific reports · 2019Article
- Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides.Interface focus · 2019Review
- Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot.PloS one · 2019Article
- The evolving art of creating genetic diversity: From directed evolution to synthetic biology.Biotechnology advancesReview
Corrections and comments
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Authors and funding
2 authors at 2 institutions in 1 country.
Funding
Abstract
Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations - particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation.
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Registered trials
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.