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Brief Communication

Nature Methods 6, 343–345 (1 May 2009) | doi:10.1038/nmeth.1318

Enzymatic assembly of DNA molecules up to several hundred kilobases

Daniel G Gibson , Lei Young , Ray-Yuan Chuang , J Craig Venter , Clyde A Hutchison & Hamilton O Smith

We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5|[prime]| 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.