JCVI: Enzymatic Assembly of DNA Molecules Up to Several Hundred Kilobases
 
 
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Citation

Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., 3rd, Smith, H. O.

Enzymatic Assembly of DNA Molecules Up to Several Hundred Kilobases

Nat Methods. 2016 Feb 12; 6(5): 343-345.

PubMed Citation

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.