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Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC

Nature Methods volume 4, pages 251256 (2007) | Download Citation

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Abstract

We describe 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. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro. SLIC inserts can also be prepared by incomplete PCR (iPCR) or mixed PCR. SLIC allows efficient and reproducible assembly of recombinant DNA with as many as 5 and 10 fragments simultaneously. SLIC circumvents the sequence requirements of traditional methods and functions much more efficiently at very low DNA concentrations when combined with RecA to catalyze homologous recombination. This flexibility allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.

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Acknowledgements

We thank B. Wanner, G. Hannon and T. Moore for providing plasmids, bacterial strains and advice concerning their use. We thank M. Schlabach for comments on the manuscript. This work was supported by a grant from US National Institutes of Health. S.J.E. is an investigator with the Howard Hughes Medical Institute.

Author information

Affiliations

  1. Howard Hughes Medical Institute, Department of Genetics, Harvard Partners Center for Genetics and Genomics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.

    • Mamie Z Li
    •  & Stephen J Elledge

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Contributions

M.Z.L. performed all experiments. S.J.E. helped in experimental design. M.Z.L. and S.J.E. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Stephen J Elledge.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Effect of insert to vector ratio on SLIC.

  2. 2.

    Supplementary Fig. 2

    Effect of insert size on SLIC.

  3. 3.

    Supplementary Methods

  4. 4.

    Supplementary Protocol 1

    SLIC Sub-cloning using T4 DNA polymerase treated inserts without RecA.

  5. 5.

    Supplementary Protocol 2

    SLIC Sub-cloning using T4 DNA polymerase treated inserts with RecA.

  6. 6.

    Supplementary Protocol 3

    SLIC Sub-cloning using iPCR or mixed PCR products.

Word documents

  1. 1.

    Supplementary Table 1

    T4 DNA polymerase is the most efficient and reproducible exonuclease for SLIC cloning.

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DOI

https://doi.org/10.1038/nmeth1010

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