ArticleNucleic acids research2009
An efficient method to assemble linear DNA templates for in vitro screening and selection systems.
Article in Nucleic acids research, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed.
- Established and Emerging Methods for Protecting Linear DNA in Cell-Free Expression Systems.Methods and protocols · 2023Review
- Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins.Nucleic acids research · 2020Article
- BioBrick-based 'Quick Gene Assembly'Synthetic biology (Oxford, England) · 2017Article
- Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.Protein engineering, design & selection : PEDS · 2015Article
- Towards the systematic mapping and engineering of the protein prenylation machinery in Saccharomyces cerevisiae.PloS one · 2015Article
- Non-enzymatic DNA cleavage reaction induced by 5-ethynyluracil in methylamine aqueous solution and application to DNA concatenation.PloS one · 2014Article
- Simultaneous splicing of multiple DNA fragments in one PCR reaction.Biological procedures online · 2013Article
- Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology.Nucleic acids research · 2013Article
- In vitro evolution of enzymes.Methods in molecular biology (Clifton, N.J.) · 2013Article
- Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand.Nucleic acids research · 2012Article
- PHUSER (Primer Help for USER): a novel tool for USER fusion primer design.Nucleic acids research · 2011Article
- A calibrated diversity assay for nucleic acid libraries using DiStRO--a Diversity Standard of Random Oligonucleotides.Nucleic acids research · 2010Article
- A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments.Avicenna journal of medical biotechnologyArticle
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2 authors.
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Abstract
A method is presented to assemble a gene of interest into a linear DNA template with all the components necessary for in vitro transcription and translation in approximately 90 min. Assembly is achieved using a coupled uracil excision-ligation strategy based on USER Enzyme and T4 DNA ligase, which allows the simultaneous and seamless assembly of three different PCR products. The method is suitable for screening and selection systems of very high throughput as up to 10(11) molecules can be efficiently assembled and purified in reaction volumes of 100 microl. The method is exemplified with the gene coding for a mutant version of O(6)-alkylguanine alkyltransferase, which is efficiently assembled with an N-terminal peptide tag and its 5'- and 3'-untranslated regions that include a T7 promoter, ribosome binding site and T7 terminator. The utility of the method is further corroborated by assembling error-prone PCR libraries and regenerating templates following model affinity selections. This fast and robust method should find widespread application in directed evolution for the assembly of gene libraries and the regeneration of linear DNA templates between successive screening and selection cycles.
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