Isothermal cloning, more commonly known as Gibson assembly, efficiently joins multiple overlapping DNA fragments in a single-tube. It takes advantage of the interplay of 3 enzymes:
- The 5’-exonuclease creates single-stranded 3´ overhangs into DNA fragments that facilitate the annealing of fragments that share complementarity at one end (overlap region).
- The polymerase fills in gaps within each overlap region.
- The DNA ligase seals nicks in the assembled DNA.
The final is a double-stranded fully sealed DNA molecule that can serve as template for PCR, RCA or a variety of other molecular biology applications, including direct transformation. The downside of Gibson Assembly is that it does not allow to easily excise and replace DNA pieces once the full-length construct is assembled making post-synthesis manipulation, such as combinatorial library construction and fragment replacement, cumbersome.