Which enzyme type is used in Golden Gate assembly to enable scarless, ordered assembly?

• A. Type IIS restriction enzymes
• B. Type I restriction enzymes
• C. RNA polymerases
• D. DNA polymerases

Answer: (A)

Golden Gate assembly relies on enzymes that cut DNA at a distance from their recognition site. Type IIS restriction enzymes do exactly that: they recognize a specific sequence but nick the DNA outside of it, producing short, designed overhangs. Those overhangs can be chosen to be unique and complementary in a precise order, so multiple fragments can be stitched together in a single reaction in a defined sequence. After ligation, the recognition sites are removed from the product, leaving seamless junctions—no extra bases or scars appear, which is why it’s described as scarless and ordered assembly.

Other enzyme types don’t provide this combination. Type I restriction enzymes cut within or near their recognition sites and aren’t used for programmable, seamless assembly. RNA polymerases and DNA polymerases aren’t restriction enzymes and don’t generate the specific, designed overhangs needed to guide one-pot, directional assembly.