Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Smith, H.O. & Wilcox, K.W. A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J. Mol. Biol. 51, 379–391 (1970).

    CAS  Article  Google Scholar 

  2. 2

    Danna, K. & Nathans, D. Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc. Natl. Acad. Sci. USA 68, 2913–2917 (1971).

    CAS  Article  Google Scholar 

  3. 3

    Cohen, S.N., Chang, A.C., Boyer, H.W. & Helling, R.B. Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. USA 70, 3240–3244 (1973).

    CAS  Article  Google Scholar 

  4. 4

    Backman, K. & Ptashne, M. Maximizing gene expression on a plasmid using recombination in vitro. Cell 13, 65–71 (1978).

    CAS  Article  Google Scholar 

  5. 5

    Hutchison, C.A. et al. Mutagenesis at a specific position in a DNA sequence. J. Biol. Chem. 253, 6551–6560 (1978).

    CAS  PubMed  Google Scholar 

  6. 6

    Rumsby, G. An introduction to PCR techniques. PCR Methods Mol. Biol. 324, 75–89 (2006).

    CAS  PubMed  Google Scholar 

  7. 7

    Saiki, R.K. et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354 (1985).

    CAS  Article  Google Scholar 

  8. 8

    Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    CAS  Article  Google Scholar 

  9. 9

    Liu, Q., Li, M.Z., Liebham, D., Cortez, D. & Elledge, S.J. The univector plasmid fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr. Biol. 8, 1300–1309 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Hartley, J.L., Temple, G.F. & Brasch, M.A. DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–1795 (2000).

    CAS  Article  Google Scholar 

  11. 11

    Walhout, A.J. et al. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 328, 575–592 (2000).

    CAS  Article  Google Scholar 

  12. 12

    Bethke, B. & Sauer, B. Segmental genomic replacement by Cre-mediated recombination: Genotoxic stress activation of the p53 promoter in single-copy transformants. Nucleic Acids Res. 25, 2828–2834 (1997).

    CAS  Article  Google Scholar 

  13. 13

    Nebert, D.W., Dalton, T.P., Stuart, G.W. & Carvan, M.J. Gene-swap knock-in cassette in mice to study allelic differences in human genes. Ann. NY Acad. Sci. 919, 148–170 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Siegel, R.W. et al. Recombinatorial cloning using heterologous lox sites. Genome Res. 14, 1119–1129 (2004).

    CAS  Article  Google Scholar 

  15. 15

    Li, M.Z. & Elledge, S.J. MAGIC: An in vivo genetic method for the rapid construction of recombinant DNA molecules. Nat. Genet. 37, 311–319 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Amundsen, S.K. & Smith, G.R. Interchangeable parts of the Escherichia coli recombination machinery. Cell 112, 741–744 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Kuzminov, A. Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. Microbiol. Mol. Biol. Rev. 63, 751–813 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Konforti, B.B. & Davis, R.W. 3′ homologous free ends are required for stable joint molecule formation by the RecA and single-stranded binding proteins of Escherichia coli. Proc. Natl. Acad. Sci. USA 84, 690–694 (1987).

    CAS  Article  Google Scholar 

  19. 19

    Tillett, D. & Neilan, B.A. Enzyme-free cloning: A rapid method to clone PCR products independent of vector restriction enzyme sites. Nucleic Acids Res. 27, e26 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Aslanidis, C. & de Jong, P.J. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069–6074 (1990).

    CAS  Article  Google Scholar 

  21. 21

    Haun, R.S., Servanti, I.M. & Moss, J. Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors. Biotechniques 13, 515–518 (1992).

    CAS  PubMed  Google Scholar 

  22. 22

    Aslanidis, C., de Jong, P.J. & Schmitz, G. Minimal length requirement of the single-stranded tails for ligation-independent cloning (LIC) of PCR products. PCR Methods Appl. 4, 172–177 (1994).

    CAS  Article  Google Scholar 

  23. 23

    Cheo, D.L. et al. Concerted assembly and cloning of multiple DNA fragments using in vitro site-specific recombination: Functional analysis of multi-site expression clones. Gen. Res. 14, 2111–2120 (2004).

    CAS  Article  Google Scholar 

Download references


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




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.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing