During the past few years a novel approach based on the use of type IIS restriction enzymes in combination with T4 DNA ligase has become popular (known as Golden Gate cloning, PLoS ONE 3, e3647, 2008). Briefly, the type IIS restriction enzymes, a subset of type II restriction endonucleases, have the particularity of cleaving the DNA a few base pairs away from the recognition site. Recognition sites are strategically placed at the ends of the fragments that one intends to clone in such a way that upon DNA cleavage: i) exogenous sequences including the restriction site are removed, and ii) compatible overhangs are generated (Figure 1A).
Once ligated, the fragments become immune to further digestion by the restriction enzyme as the recognition sites have been eliminated. Thus, the restriction and ligation reactions can be consolidated into a single reaction. In the final construct the junctions between any pair of adjacent fragments carry no added or deleted sequences, thereby representing a true scarless assembly.
The system can be utilized i) for the seamless assembly of multiple fragments into a vector in a predetermined order, using PCR fragments or plasmids as donors (Figure 1B), or ii) for transferring fragments from a central repository donor clone into different customized vectors geared toward different applications (Figure 1C).