Golden Gate Assembly

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  • Golden Gate Assembly and its derivative methods(1,2) exploit the ability of Type IIS restriction endonucleases (REases) to cleave DNA outside of the recognition sequence. The inserts and cloning vectors are designed to place the Type IIS recognition site distal to the cleavage site, such that the Type IIS REase can remove the recognition sequence from the assembly. The advantages of such an arrangement are three-fold:

    1. The overhang sequence created is not dictated by the REase, and therefore no scar sequence is introduced.
    2. The fragment-specific sequence of the overhangs allows orderly assembly of multiple fragments simultaneously.
    3. The restriction site is eliminated from the ligated product, so digestion and ligation can be carried out simultaneously.
    The net result is the ordered and seamless assembly of DNA fragments in one reaction. The accuracy of the assembly is dependent on the length of the overhang sequences. Therefore, Type IIS REases that create 4-base overhangs (such as BsaI/BsaI-HF®, BbsI, BsmBI and Esp3I) are preferred. The downside of these Type IIS REase-based methods is that the small number of overhanging bases can lead to the mis-ligation of fragments with similar overhang sequences(3). It is also necessary to verify that the Type IIS REase sites used are not present in the fragments for the assembly of the expected product. Nonetheless, Golden Gate Assembly is a robust technology that generates multiple site-directed mutations(4) and assembles multiple DNA fragments(5,6). Golden Gate Assembly has been widely used in the construction of custom-specific TALENs for in vivo gene editing(7), among other applications.

    Golden Gate Assembly Workflow

    In its simplest form, Golden Gate Assembly requires a BsaI recognition site (GGTCTC) added to both ends of a dsDNA fragment distal to the cleavage site, such that the BsaI site is eliminated by digestion with BsaI or BsaI-HF (GGTCTC 1/5). Upon cleavage, the overhanging sequences of the adjoining fragments anneal to each other. DNA ligase then seals the nicks to create a new covalently linked DNA molecule. Multiple pieces of DNA can be cleaved and ligated simultaneously.


    References:

    1. Engler, C., Kandzia, R., and Marillonnet, S. (2008) PLoS ONE 3, e3647.
    2. Sarrion-Perdigones, A., et al. (2011) PLoS ONE 6, e21622.
    3. Engler, C., et al. (2009) PLoS ONE 4, e5553.
    4. Yan, P., et al. (2012) Anal. Biochem. 430, 65-67.
    5. Scior, A., et al. (2011) BMC Biotechnol. 11, 87.
    6. Werner, S., et al. (2012) Bioeng. Bugs 3, 38-43.
    7. Sanjana, N. E., et al. (2012) Nat. Protoc. 7, 171-192.

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