Article

Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases

The J. Craig Venter Institute, Synthetic Biology Group, Rockville, Maryland, USA.
Nature Methods (Impact Factor: 32.07). 05/2009; 6(5):343-5. DOI: 10.1038/nmeth.1318
Source: PubMed

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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Available from: Daniel G Gibson, May 12, 2014
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    • "The same PCR conditions were used for a further 30 rounds and the product was purified using a QiaQuick PCR cleanup kit (Qiagen, Hilden, Germany ) and eluted in EB. Gibson assembly [47] was used to insert the assembled gene into gelpurified pETFLAG vector digested with NdeI/XhoI (NEB, Waltham, MA). A 1.5μL aliquot of cut vector at 20ng/μL was added to 1μL of assembled gene and 7.5μL of Gibson enzyme mix (all enzymes from NEB, Waltham, MA). "
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