This video provides a detailed explanation of Golden Gate Assembly, a versatile cloning technique used to seamlessly assemble multiple DNA fragments in a single reaction. It focuses on the unique properties of Type IIS restriction enzymes that make this technique possible and efficient.
Type IIS Enzyme Properties: Golden Gate Assembly utilizes Type IIS restriction enzymes, characterized by non-palindromic recognition sites, cleavage outside the recognition site, and the generation of variable overhangs (e.g., BsaI generates a four-nucleotide overhang). These overhangs, termed fusion sites, enable specific joining of fragments.
Vector and Fragment Preparation: The destination vector requires two outward-facing Type IIS restriction sites flanking the insert region. Fragments of interest have inward-facing sites, ensuring that after digestion, the recognition sites are removed, preventing further digestion.
Assembly Process: The assembly is a one-pot, one-step process involving DNA ligase and the Type IIS enzyme. Thermocycling alternates between digestion, annealing, and ligation steps, culminating in the complete assembly. A final digestion step removes any undigested vector.
PCR Application: PCR can be used to add Type IIS recognition sites and overhang sequences to DNA fragments, enabling their incorporation into Golden Gate Assembly.
Post-Assembly: The final plasmid is ready for transformation after enzyme inactivation at 80°C.
