Enzymatic assembly of DNA molecules up to several hundred kilobases

@article{Gibson2009EnzymaticAO,
  title={Enzymatic assembly of DNA molecules up to several hundred kilobases},
  author={Daniel G. Gibson and Lei Young and R Y Chuang and J. Craig Venter and Clyde A. Hutchison and Hamilton O. Smith},
  journal={Nature Methods},
  year={2009},
  volume={6},
  pages={343-345}
}
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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References

SHOWING 1-10 OF 34 REFERENCES
Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC
TLDR
A new cloning method, sequence and ligation–independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing, which allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology. Expand
PCR-mediated recombination and mutagenesis
  • R. Horton
  • Biology, Medicine
  • Molecular biotechnology
  • 1995
Gene Splicing by Overlap Extension (gene SOEing) is a sequence-independent method for site-directed mutagenesis and/or recombination of DNA molecules. It is based on the idea that a PCR product canExpand
Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction.
Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro.Expand
Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.
A method is described for the rapid generation and cloning of deletion derivatives well-suited for the sequencing of long stretches of DNA. This method is based on two useful features of exonucleaseExpand
Cell-free cloning using φ29 DNA polymerase
TLDR
Conditions for rolling-circle amplification (RCA) of individual DNA molecules 5–7 kb in size by >109-fold, using φ29 DNA polymerase is described, which allows cell-free cloning of individual synthetic DNA molecules that cannot be cloned in Escherichia coli, and may also speed genome sequencing by eliminating the need for biological cloning. Expand
Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome
TLDR
The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments. Expand
Properties of overexpressed phage T5 D15 exonuclease. Similarities with Escherichia coli DNA polymerase I 5'-3' exonuclease.
TLDR
The D15 gene of the bacteriophage T5, thought to encode an exonuclease, was cloned into an M13 phage on a 1344-base pair fragment, allowing the production of large amounts of enzyme for physical characterization and crystallization trials and was purified to homogeneity. Expand
Transformation of Escherichia coli with large DNA molecules by electroporation.
TLDR
It is demonstrated that conditions may be selected which increase the average size of BAC clones generated by electroporation and compared the overall efficiency of each of the conditions tested, to increase the yield of transformants which have taken up large DNA relative to the number incorporating smaller molecules. Expand
Ligation-independent cloning of PCR products (LIC-PCR).
TLDR
A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones, and the procedure is applied for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones. Expand
A restriction enzyme from Hemophilus influenzae. I. Purification and general properties.
Abstract Extracts of Hemophilus influenzae strain Rd contain an endonuclease activity which produces a rapid decrease in the specific viscosity of a variety of foreign native DNA's; the specificExpand
...
1
2
3
4
...