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

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

Research output: Contribution to journal › Article › peer-review

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.

Original language	English (US)
Pages (from-to)	343-345
Number of pages	3
Journal	Nature Methods
Volume	6
Issue number	5
DOIs	https://doi.org/10.1038/nmeth.1318
State	Published - 2009
Externally published	Yes

ASJC Scopus subject areas

Biotechnology
Biochemistry
Molecular Biology
Cell Biology

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10.1038/nmeth.1318

Cite this

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title = "Enzymatic assembly of DNA molecules up to several hundred kilobases",

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.",

author = "Gibson, \{Daniel G.\} and Lei Young and Chuang, \{Ray Yuan\} and Venter, \{J. Craig\} and Hutchison, \{Clyde A.\} and Smith, \{Hamilton O.\}",

note = "Funding Information: 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.",

year = "2009",

doi = "10.1038/nmeth.1318",

language = "English (US)",

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pages = "343--345",

journal = "Nature Methods",

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AU - Gibson, Daniel G.

AU - Young, Lei

AU - Chuang, Ray Yuan

AU - Venter, J. Craig

AU - Hutchison, Clyde A.

AU - Smith, Hamilton O.

N1 - Funding Information: 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.

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

UR - https://www.scopus.com/pages/publications/67349270900

UR - https://www.scopus.com/pages/publications/67349270900#tab=citedBy

U2 - 10.1038/nmeth.1318

DO - 10.1038/nmeth.1318

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VL - 6

SP - 343

EP - 345

JO - Nature Methods

JF - Nature Methods

IS - 5

ER -

Enzymatic assembly of DNA molecules up to several hundred kilobases

Abstract

ASJC Scopus subject areas

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