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.

References

1. "PLOS ONE: A One Pot, One Step, Precision Cloning Method with High Throughput Capability". Plosone.org. Retrieved 20 November 2014.
2. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015.