What is a key principle of Gibson assembly in joining DNA fragments?

• A. Fragments are ligated at matched sticky ends using ligase in multiple steps
• B. Overlapping sequences are chew-back and fill-in reactions to create a seamless construct in a single isothermal reaction
• C. Restriction enzymes cut out fragments for assembly in a stepwise fashion
• D. PCR amplifies fragments before ligation

Answer: (B)

Gibson assembly works by using overlaps between DNA fragments to build a seamless construct in one reaction. A 5' exonuclease chews back the ends of the fragments to create single-stranded overlaps. These overlaps find and anneal to each other, so the fragments line up correctly. A DNA polymerase then fills in any missing bases, and a ligase seals the nicks, yielding a continuous DNA molecule without extra sequences. Everything happens in a single isothermal step, so there’s no need for separate restriction enzyme cuts or multiple ligation cycles.

The other descriptions describe traditional cloning methods that rely on matching sticky ends created by restriction enzymes or on separate PCR and ligation steps, which Gibson assembly is designed to avoid by using the overlapping-end strategy and one-pot, isothermal conditions.