ArticleNucleic acids research2015
Solid-phase cloning for high-throughput assembly of single and multiple DNA parts.
Article in Nucleic acids research, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed.
- Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping.Communications biology · 2024Article
- Systematic use of synthetic 5'-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories.Nucleic acids research · 2020Article
- Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins.Nucleic acids research · 2020Article
- SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping.Nucleic acids research · 2019Article
- Machine learning in computational biology to accelerate high-throughput protein expression.Bioinformatics (Oxford, England) · 2017Article
- Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering.Nucleic acids research · 2016Article
- Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells.Scientific reports · 2015Article
Corrections and comments
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Authors and funding
9 authors.
Funding
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Abstract
We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.
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