Golden Gate Cloning

Golden Gate cloning or Golden Gate assembly ^[1] is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4 DNA ligase.^[2] This assembly is performed in vitro. Most commonly used Type IIs enzymes include BsaI, BsmBI and BbsI.

Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create non-palindromic overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences. In practice, this means that Golden Gate cloning is typically scarless. Additionally, because the final product does not have a Type II restriction enzyme recognition site, the correctly-ligated product cannot be cut again by the restriction enzyme, meaning the reaction is essentially irreversible.

A typical thermocycler protocol oscillates between 37°C (optimal for restriction enzymes) and 16°C (optimal for ligases) many times.^[3] While this technique can be used for a single insert, researchers have used Golden Gate cloning to assemble many pieces of DNA simultaneously.^[4]

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