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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Gellert, M. Proc. Natl. Acad. Sci. USA 57, 148–155 (1967).
Smith, H.O. & Wilcox, K.W. J. Mol. Biol. 51, 379–391 (1970).
Shetty, R.P., Endy, D. & Knight, T.F. Jr. J. Biol. Eng. 2, 5 (2008).
Yount, B., Denison, M.R., Weiss, S.R. & Baric, R.S. J. Virol. 76, 11065–11078 (2002).
Horton, R.M., Cai, Z.L., Ho, S.N. & Pease, L.R. Biotechniques 8, 528–535 (1990).
Horton, R.M. Mol. Biotechnol. 3, 93–99 (1995).
Bang, D. & Church, G.M. Nat. Methods 5, 37–39 (2008).
Geu-Flores, F., Nour-Eldin, H.H., Nielsen, M.T. & Halkier, B.A. Nucleic Acids Res. 35, e55 (2007).
Aslanidis, C. & de Jong, P.J. Nucleic Acids Res. 18, 6069–6074 (1990).
Li, M.Z. & Elledge, S.J. Nat. Methods 4, 251–256 (2007).
Gibson, D.G. et al. Science 319, 1215–1220 (2008).
Sayers, J.R. & Eckstein, F. J. Biol. Chem. 265, 18311–18317 (1990).
Sheng, Y., Mancino, V. & Birren, B. Nucleic Acids Res. 23, 1990–1996 (1995).
Endy, D. Nature 438, 449–453 (2005).
Drubin, D.A., Way, J.C. & Silver, P.A. Genes Dev. 21, 242–254 (2007).
Lartigue, C., Duret, S., Garnier, M. & Renaudin, J. Plasmid 48, 149–159 (2002).
Henikoff, S. Gene 28, 351–359 (1984).
Hutchison, C.A., III, Smith, H.O., Pfannkoch, C. & Venter, J.C. Proc. Natl. Acad. Sci. USA 102, 17332–17336 (2005).
This work was supported by the Office of Science (Biological and Environmental Research) United States Department of Energy grant number DE-FG02-02ER63453, and Synthetic Genomics, Inc.
J.C.V is chief executive officer and co-chief scientific officer of Synthetic Genomics, Inc (SGI), a privately held entity that develops genomic-driven strategies to address global energy and environmental challenges. H.O.S. is co-chief scientific officer and a member of the board of directors of SGI. C.A.H. is chairman of the SGI scientific advisory board. All three of these authors hold SGI stock.
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Gibson, D., Young, L., Chuang, RY. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6, 343–345 (2009). https://doi.org/10.1038/nmeth.1318
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