Restriction Enzymes in Golden Gate Assembly

Becky Kucera explains that Golden Gate Assembly requires Type II S restriction enzymes, which differ from traditional Type II enzymes like EcoR1. Type II S enzymes have a recognition site but cut downstream from that site rather than within it.

Once DNA fragments are cut by Type II S enzymes, the recognition sequence remains separate from the pieces of interest, which are flanked by complementary four-base overhangs. These overhangs allow sequential fragments to anneal together during assembly.

When overhangs anneal, DNA ligase seals them together, but if the original sequence reforms instead, the Type II S enzyme can cut again to regenerate the productive four-base overhangs. This recycling ensures a 50% probability of productive ligation at each step.

All fragments in the assembly possess complementary four-base overhangs for guidance, while restriction enzyme sites remain separate from the final product DNA. Once fragments are incorporated into the growing assembly, they become protected from further cutting and seamlessly integrate into the destination construct.

Becky Kucera highlights ongoing research into discovering new Type II S enzymes with superior catalytic functions and improved turnover rates. She notes the remarkable progress from three to four-fragment assemblies to recent publications demonstrating 20-fragment Golden Gate Assemblies, suggesting further improvements are achievable.