What is the purpose of overlapping sequences in Gibson assembly?

• A. They are ligated directly without modification
• B. They are chew-back and fill-in to create seamless junctions in a single isothermal reaction
• C. They are cut by restriction enzymes
• D. They do not participate in assembly

Answer: (B)

Overlapping sequences provide homologous ends that guide the joining of DNA fragments in Gibson assembly. An exonuclease chews back the fragment ends to reveal single-stranded overlaps that are complementary between pieces. These overlaps let the fragments anneal to one another, creating a continuous template. A DNA polymerase fills in any gaps, and a DNA ligase seals the remaining nicks, all in one isothermal reaction. This seamless junction formation is the core reason overlaps are essential in Gibson assembly. It’s not about restriction enzymes cutting anything, and the overlaps are actively used in the assembly rather than being ignored.