Traditional restriction enzyme cloning has been supplemented — and in many labs replaced — by recombinatorial and modular assembly methods that are faster, scarless, and compatible with high-throughput workflows.
Gateway Cloning
Gateway cloning uses the site-specific recombination system of bacteriophage lambda. Two sequential recombination reactions move a DNA sequence of interest between vectors without restriction enzymes or ligase.
In the BP reaction, a PCR product flanked by attB sites recombines with a donor vector containing attP sites, creating an entry clone. The LR reaction then transfers the insert from the entry clone into any destination vector containing attR sites. The destination vector provides the appropriate expression elements for the target organism.
Gateway cloning is directional, does not change the reading frame, and allows the same entry clone to be shuttled into multiple destination vectors (e.g., for expression in bacteria, mammalian cells, or plants). Its main limitation is the size of the recombination sites (about 50 bp each), which add extra sequence to the final construct.
Gibson Assembly
Gibson Assembly joins multiple DNA fragments in a single isothermal reaction (50 °C, 15–60 minutes). It requires three enzymatic activities:
- A 5′ exonuclease chews back the 5′ ends, leaving overlapping single-stranded 3′ overhangs.
- A DNA polymerase fills in the gaps.
- A DNA ligase seals the nicks.
The fragments must have 15–40 bp of overlapping sequence at their ends, which is typically added via PCR primers. Gibson Assembly can join up to 10–15 fragments simultaneously and is ideal for assembling large constructs such as synthetic genes, whole plasmids, or genome engineering cassettes. The assembly is scarless — no restriction sites are left behind.
Golden Gate Cloning
Golden Gate cloning uses Type IIS restriction enzymes, which cut outside their recognition sequence, producing defined 4 bp overhangs. These overhangs can be specified by the user and are designed to be unique and compatible.
The reaction is a one-pot digest-ligation: the Type IIS enzyme and T4 DNA ligase are added together, and the reaction is cycled between the enzyme’s optimal temperature and 37 °C. Because the recognition sites are removed after cutting, the final assembled product is no longer digestible, driving the reaction to completion.
Golden Gate is the basis of modular cloning systems such as MoClo and the Plant Toolbox. It is ideal for combinatorial assembly of genetic parts (promoters, coding sequences, terminators) and for building TALEN arrays or sgRNA libraries.
