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.
Bibliographical noteFunding Information:
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).
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology