Nature Methods
- 4, 251 - 256 (2007)
Published online: 11 February 2007; | doi:10.1038/nmeth1010
Harnessing homologous recombination in vitro to generate recombinant DNA via SLICMamie Z Li & Stephen J Elledge
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.
Correspondence should be addressed to Stephen J Elledge selledge@genetics.med.harvard.edu 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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