Abstract
Here we report the development of a gene-synthesis technology, circular assembly amplification. In this approach, we first constructed exonuclease-resistant circular DNA via simultaneous ligation of oligonucleotides. Exonuclease- and subsequent mismatch cleaving endonuclease–mediated degradation of the resulting ligation mixture eliminated error-rich products, thereby substantially improving gene-synthesis quality. We used this method to construct genes encoding a small thermostable DNA polymerase, a highly repetitive DNA sequence and large (>4 kb) constructs.
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Acknowledgements
We thank M. Umbarger, M. Price and J. Aach for critical comments on the manuscript; F. Issacs for comments on experimental design; an anonymous reviewer's suggestion to test repetitive DNA sequence synthesis. We acknowledge funding from US Department of Energy for GTL Center support. D.B is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG#1911-06).
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G.M.C. is a scientific advisory board member of Codon Devices.
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Bang, D., Church, G. Gene synthesis by circular assembly amplification. Nat Methods 5, 37–39 (2008). https://doi.org/10.1038/nmeth1136
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DOI: https://doi.org/10.1038/nmeth1136
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