• 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.

• Similar to traditional digest/ligation cloning
• Not completely sequence-independent
• Ligation-based cloning mechanism

Type IIS restriction enzymes (RE) cleave DNA outside of their recognition sites, facilitating a unique strategy for seamless cloning (Toth et al. 2014). Inserts and vectors can be designed so that the recognition site is removed by the enzyme itself. In this way, the overhang resulting from the digest is not independent of the enzyme used, and no scar sequence need be included between fragments. This also means that resulting ligations will not be cleavable by the original RE. Overhangs can be specifically designed to ensure directional cloning of multiple fragments in the same reaction, but it is important to check that none of the final cloning fragments include additional instances of the recognition sites. If restriction sites are unavailable, they may need to be built into the insert(s) and/or vector beforehand. The commercially available Golden Gate Assembly is based on a Type IIS RE and T4 DNA Ligase cloning mix. If working on a higher-throughput project, this particular cloning method may be problematic, as it can suffer from low efficiency and is difficult to scale up.

• One-step, 15-minute reaction, even with large vectors/inserts
• No sequence errors at cloning junctions
• Ligase-free cloning enzyme mix
• Amenable to high-throughput cloning projects

In-Fusion Cloning technology (Irwin et al. 2012) provides ligation-free cloning of PCR products into any linear vector, at any locus. Because this method does not use ligase, there is no need for enzymatic treatment of the PCR fragment (e.g., restriction digest, phosphorylation), and background (i.e., the presence of self-ligated empty vectors) is extremely low. Typical issues arising from ligation inefficiency are therefore also eliminated, allowing researchers to skip subcloning steps, even with large vectors and/or inserts. Linear vectors can be prepared either with restriction digest or by inverse PCR, offering a truly sequence-independent option. The single-step, 15-minute cloning reaction relies on 15–20 bp of homology between DNA fragment ends, ensuring that each reaction is directional and accurate. Exonuclease activity chews back one strand of each fragment, creating compatible ends for joining and annealing in vitro. Cohesive bonds are formed in vivo, following transformation, protecting against the introduction of any sequence errors or nucleotide mismatches. High accuracy and a streamlined workflow make this technology easily adaptable to high-throughput workflows.

Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343–345 (2009).

Irwin, C .R., Farmer, A., Willer, D. O., Evans D. H. In-Fusion Cloning with Vaccinia Virus DNA Polymerase. Vaccinia Virus and Poxvirology (Methods and Protocols) 890, 23–35 (2012).

Toth, E. et al. Restriction Enzyme Body Doubles and PCR Cloning: On the General Use of Type IIS Restriction Enzymes for Cloning. PLOS One 9, e90896 (2014).