ArticleNucleic acids research2005
Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution.
Article in Nucleic acids research, 2005. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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17 citing papers in PubMed.
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- In vitro generation of genetic diversity for directed evolution by error-prone artificial DNA synthesis.Communications biology · 2024Article
- Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds.Nature biotechnology · 2022Article
- From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma.RSC chemical biology · 2022Review
- Continuous evolution of SpCas9 variants compatible with non-G PAMs.Nature biotechnology · 2020Article
- Tailoring Proteins to Re-Evolve Nature: A Short Review.Molecular biotechnology · 2018Review
- Molecular barcoding of viral vectors enables mapping and optimization of mRNARNA (New York, N.Y.) · 2018Article
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- A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing.Scientific reports · 2016Article
- Methods for the directed evolution of proteins.Nature reviews. Genetics · 2015Review
- Article
- A simple and reproducible method for directed evolution: combination of random mutation with dITP and DNA fragmentation with endonuclease V.Molecular biotechnology · 2013Article
- A general strategy for the evolution of bond-forming enzymes using yeast display.Proceedings of the National Academy of Sciences of the United States of America · 2011Article
- Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting.Nucleic acids research · 2009Article
- Effects of target length on the hybridization efficiency and specificity of rRNA-based oligonucleotide microarrays.Applied and environmental microbiology · 2007Article
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6 authors.
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Abstract
DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs.
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