ArticleBiotechnology and bioengineering2023
A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation.
Article in Biotechnology and bioengineering, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed.
- The Single-Stranded DNA Platform: A Potential Broad-Spectrum Vector for Editing Genes in Prokaryotes: ssDNA May Break Through the Difficulties of Genetic Manipulation on Non-Model Bacteria.BioEssays : news and reviews in molecular, cellular and developmental biology · 2026Review
- Generation of high-quality single-stranded DNA for full-length and truncated genome standards of recombinant adeno-associated viruses.Molecular therapy. Methods & clinical development · 2025Article
- An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries.Nature communications · 2024Article
- An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries.bioRxiv : the preprint server for biology · 2024Article
- Standardizing cassette-based deep mutagenesis by Golden Gate assembly.Biotechnology and bioengineering · 2024Article
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Authors and funding
5 authors.
Funding
Abstract
Construction of user-defined long circular single stranded DNA (cssDNA) and linear single stranded DNA (lssDNA) is important for various biotechnological applications. Many current methods for synthesis of these ssDNA molecules do not scale to multikilobase constructs. Here we present a robust methodology for generating user-defined cssDNA employing Golden Gate assembly, a nickase, and exonuclease degradation. Our technique is demonstrated for three plasmids with insert sizes ranging from 2.1 to 3.4 kb, requires no specialized equipment, and can be accomplished in 5 h with a yield of 33%-43% of the theoretical. To produce lssDNA, we evaluated different CRISPR-Cas9 cleavage conditions and reported a 52 ± 8% cleavage efficiency of cssDNA. Thus, our current method does not compete with existing protocols for lssDNA generation. Nevertheless, our protocol can make long, user-defined cssDNA readily available to biotechnology researchers.
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Registered trials
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