Brief Communication abstract

Nature Methods 6, 343 - 345 (2009)
Published online: 12 April 2009 | doi:10.1038/nmeth.1318

Enzymatic assembly of DNA molecules up to several hundred kilobases

Daniel G Gibson1, Lei Young1, Ray-Yuan Chuang1, J Craig Venter1,2, Clyde A Hutchison III2 & Hamilton O Smith2


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.

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

Correspondence to: Daniel G Gibson1 e-mail:


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