The Gibson assembly method is a relatively straightforward DNA assembly method, requiring only a few additional reagents: the 5 T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase. The DNA fragments to be assembled are synthesised to have overlapping 5 and 3' ends in the order that they are to be assembled in. These reagents are mixed together with the DNA fragments to be assembled at 50 °C and the following reactions occur:
# The T5 exonuclease chews back DNA from the 5 end of each fragment, exposing 3 overhangs on each DNA fragment.
# The complementary overhangs on adjacent DNA fragments anneal via complementary base pairing.
# The Phusion DNA polymerase fills in any gaps where the fragments anneal.
# Taq DNA ligase repairs the nicks on both DNA strands.
Because the T5 exonuclease is heat labile, it is inactivated at 50 °C after the initial chew back step. The product is thus stable, and the fragments assembled in the desired order. This one-pot protocol can assemble up to 5 different fragments accurately, while several commercial providers have kits to accurately assemble up to 15 different fragments in a two-step reaction. However, while the Gibson assembly protocol is fast and uses relatively few reagents, it requires bespoke DNA synthesis as each fragment has to be designed to contain overlapping sequences with the adjacent fragments and amplified via PCR. This reliance on PCR may also affect the fidelity of the reaction when long fragments, fragments with high GC content or repeat sequences are used.