Fig. 1

Comparison of classical Gibson and Gibson Deletion assembly. During Gibson reaction two pieces of linear DNA with homologous sequences at their end can recombine into an assembled DNA (left panel). A 5′- > 3′ exonuclease (T5) chews back the DNA ends exposing the homologous sequences (orange and yellow lines) then able to anneal (a-b). A Phusion DNA polymerase extends the DNA from the 3′ ends (c) and a Taq ligase will ligate the nicks (d). Gibson Deletion (right panel) is an alternative application of the classical Gibson assembly method that takes advantage of the homologous sequences being present several bases away from the DNA ends (e; red and yellow = homologous DNA; blue and brown = non-homologous DNA). This allows for deletion of the non-homologous sequences and assembly of the two linear pieces (f-h). The created flap-DNA (g, brown line) is removed most likely by the 3′- > 5′ exonuclease activity of the Phusion polymerase in the Gibson mix and the nick filled-in and ligated