- Requires 25 bp of homology between vector and insert
- Low-fidelity DNA polymerase fills in cloning junctions
- Ligation-based cloning mechanism
The Gibson method (Gibson et al. 2009) uses a three-enzyme mix to go from linear DNA fragments to finished plasmid. As with other seamless cloning methods, inserts are amplified by PCR such that they include homologous overlaps with a linear vector or other adjacent fragments. The required overlap is longer than for In-Fusion Cloning (see below), with recommendations set at 25 bp. Once linear fragments are prepared, a 5’ exonuclease chews back the DNA to create cohesive ends, and a low-fidelity polymerase fills in any nucleotide gaps. The more recent NEBuilder HiFi DNA Assembly uses this same technology but relies on a high-fidelity polymerase to fill in gaps. Regardless, for both versions of the Gibson method, DNA ligase then seals the nicks in the annealed fragments, before transformation. While this technology does yield directional, scarless clones, it can be error-prone due to mismatches in base joining, and background can be high due to the use of ligase. If scaling up for a high-throughput project, be mindful of the potential error rate and background levels, as these problems will be magnified when cloning number increases.