Brief Communication | Published:

Enzymatic assembly of DNA molecules up to several hundred kilobases

Nature Methods volume 6, pages 343345 (2009) | Download Citation

Subjects

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from $8.99

All prices are NET prices.

References

  1. 1.

    Proc. Natl. Acad. Sci. USA 57, 148–155 (1967).

  2. 2.

    & J. Mol. Biol. 51, 379–391 (1970).

  3. 3.

    , & J. Biol. Eng. 2, 5 (2008).

  4. 4.

    , , & J. Virol. 76, 11065–11078 (2002).

  5. 5.

    , , & Biotechniques 8, 528–535 (1990).

  6. 6.

    Mol. Biotechnol. 3, 93–99 (1995).

  7. 7.

    & Nat. Methods 5, 37–39 (2008).

  8. 8.

    , , & Nucleic Acids Res. 35, e55 (2007).

  9. 9.

    & Nucleic Acids Res. 18, 6069–6074 (1990).

  10. 10.

    & Nat. Methods 4, 251–256 (2007).

  11. 11.

    et al. Science 319, 1215–1220 (2008).

  12. 12.

    & J. Biol. Chem. 265, 18311–18317 (1990).

  13. 13.

    , & Nucleic Acids Res. 23, 1990–1996 (1995).

  14. 14.

    Nature 438, 449–453 (2005).

  15. 15.

    , & Genes Dev. 21, 242–254 (2007).

  16. 16.

    , , & Plasmid 48, 149–159 (2002).

  17. 17.

    Gene 28, 351–359 (1984).

  18. 18.

    , , & Proc. Natl. Acad. Sci. USA 102, 17332–17336 (2005).

Download references

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.

Author information

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. Search for Daniel G Gibson in:

  2. Search for Lei Young in:

  3. Search for Ray-Yuan Chuang in:

  4. Search for J Craig Venter in:

  5. Search for Clyde A Hutchison in:

  6. Search for Hamilton O Smith in:

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.

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.

Corresponding author

Correspondence to Daniel G Gibson.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6, Supplementary Tables 1–2, Supplementary Results

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.1318