Gibson Assembly®

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  • Daniel G. Gibson, of the J. Craig Venter Institute, described a robust exonuclease-based method to assemble DNA seamlessly and in the correct order, eponymously known as Gibson Assembly. The reaction is carried out under isothermal conditions using three enzymatic activities: a 5’ exonuclease generates long overhangs, a polymerase fills in the gaps of the annealed single strand regions, and a DNA ligase seals the nicks of the annealed and filled-in gaps. This method has been widely adopted and is a major workhorse of synthetic biology projects worldwide. Applying this methodology, the 16.3 kb mouse mitochondrial genome was assembled from 600 overlapping 60-mers(1). In combination with in vivo assembly in yeast, Gibson Assembly was used to synthesize the 1.1 Mbp Mycoplasma mycoides genome. The synthesized genome was transplanted to a M. capricolum recipient cell, creating new self-replicating M. mycoides cells(2).

    • Introduction to Gibson Assembly®

      Watch an interactive tutorial that details the process by which Gibson Assembly® joins DNA fragments in a single tube, isothermal reaction.

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    • Primer Design and Fragment Assembly Using Gibson Assembly®

      Watch an interactive tutorial on primer design to see how simple it really is to clone with the Gibson Assembly® Cloning Kit.

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    Gibson Assembly Workflow

    Gibson Assembly employs three enzymatic activities in a single-tube reaction:
    5´ exonuclease, the 3´ extension activity of a DNA polymerase and DNA ligase activity. The 5´ exonuclease activity chews back the 5´ end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed regions. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. The NEB Gibson Assembly Master Mix (NEB #E2611) and Gibson Assembly Cloning Kit (NEB #E5510S) enable rapid assembly at 50˚C.


    1. Gibson, D.G., et al. (2009) Nat. Methods 6, 343-345.
    2. Gibson, D.G., et al. (2010) Science 329, 52-56.

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