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Enzymatic assembly of DNA molecules up to several hundred kilobases

Nature Methods volume 6, pages 343–345 (2009)Cite this article

Abstract

We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.

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Figure 1: One-step isothermal in vitro recombination.
Figure 2: Examples of the one-step isothermal assembly method.

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Acknowledgements

This work was supported by the Office of Science (Biological and Environmental Research) United States Department of Energy grant number DE-FG02-02ER63453, and Synthetic Genomics, Inc.

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Authors and Affiliations

  1. The J. Craig Venter Institute, Synthetic Biology Group, Rockville, Maryland, USA

    Daniel G Gibson, Lei Young, Ray-Yuan Chuang & J Craig Venter

  2. The J. Craig Venter Institute, Synthetic Biology Group, San Diego, California, USA

    J Craig Venter, Clyde A Hutchison III & Hamilton O Smith

Authors
  1. Daniel G Gibson
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  2. Lei Young
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  3. Ray-Yuan Chuang
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  4. J Craig Venter
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  5. Clyde A Hutchison III
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  6. Hamilton O Smith
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Contributions

D.G.G., L.Y., R.-Y.C., J.C.V., C.A.H. and H.O.S. designed research; D.G.G., L.Y., R.-Y.C., C.A.H. and H.O.S. performed research; D.G.G., L.Y., R.-Y.C., J.C.V., C.A.H. and H.O.S. analyzed data; and D.G.G., C.A.H. and H.O.S. wrote the paper.

Corresponding author

Correspondence to Daniel G Gibson.

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Competing interests

J.C.V is chief executive officer and co-chief scientific officer of Synthetic Genomics, Inc (SGI), a privately held entity that develops genomic-driven strategies to address global energy and environmental challenges. H.O.S. is co-chief scientific officer and a member of the board of directors of SGI. C.A.H. is chairman of the SGI scientific advisory board. All three of these authors hold SGI stock.

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Supplementary Figures 1–6, Supplementary Tables 1–2, Supplementary Results (PDF 2997 kb)

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Gibson, D., Young, L., Chuang, RY. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6, 343–345 (2009). https://doi.org/10.1038/nmeth.1318

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