Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC

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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Figure 1: In vitro recombination of MAGIC vectors mediated by RecA.
Figure 2: The dependency on RecA can be overcome by increasing DNA concentration.
Figure 3: iPCR and mixed PCR can be used to prepare inserts for SLIC cloning without nuclease treatment.
Figure 4: Multi-fragment assembly using SLIC.

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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.

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Authors

Contributions

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

Corresponding author

Correspondence to Stephen J Elledge.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Effect of insert to vector ratio on SLIC. (PDF 34 kb)

Supplementary Fig. 2

Effect of insert size on SLIC. (PDF 35 kb)

Supplementary Table 1

T4 DNA polymerase is the most efficient and reproducible exonuclease for SLIC cloning. (DOC 20 kb)

Supplementary Methods (PDF 70 kb)

Supplementary Protocol 1

SLIC Sub-cloning using T4 DNA polymerase treated inserts without RecA. (PDF 56 kb)

Supplementary Protocol 2

SLIC Sub-cloning using T4 DNA polymerase treated inserts with RecA. (PDF 55 kb)

Supplementary Protocol 3

SLIC Sub-cloning using iPCR or mixed PCR products. (PDF 51 kb)

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Li, M., Elledge, S. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4, 251–256 (2007). https://doi.org/10.1038/nmeth1010

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