Investigation of T7 exonuclease dependent one-step cloning

Abstract

Objective To test whether T7 exonuclease could mediate DNA fragment assembly by homologous recombination. Methods DNA fragments generated by PCR were digested with T7 exonuclease and the cocktail was transfected into E.coli for colony counting. Results Without purification, T7 exonuclease treated DNA fragments with homologous ends could be transfected into E.coli directly for colony formation. Similar to other homologous recombination dependent cloning, efficacy of EDOC increased with the length of homologous ends, minimal requirement of which was 10 bp. Recombination was significantly prevented by mutations in homologous ends. EDOC was proved to be seamless and mutant-free. Conclusion Our data proved that T7 exonuclease was capable to induce the assembly of recombinant, and a new method, T7 exonuclease dependent one-step cloning, was successfully established. Key words: Molecular cloning; T7 exonuclease; Homologous recombination