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Gene synthesis by circular assembly amplification

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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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Figure 1: Schematic representation of circular assembly amplification.
Figure 2: Dpo4 gene constructed by circular assembly amplification of 48 oligonucleotides.
Figure 3: Circular assembly amplification for the synthesis of Pfu DNA polymerase.

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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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Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA

    Duhee Bang & George M Church

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  1. Duhee Bang
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  2. George M Church
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Correspondence to Duhee Bang.

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Competing interests

G.M.C. is a scientific advisory board member of Codon Devices.

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Supplementary Figures 1–5, Supplementary Table 1, Supplementary Methods (PDF 1555 kb)

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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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