Gibson assembly

Gibson assembly is a cloning technique that does not rely on restriction sites like traditional cloning techniques. The Gibson assembly relies on the presence of homologous regions in the ends of the DNA pieces.

The three enzymes T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase all process different parts of the DNA ends. First, the T5 exonuclease, chews back on the 5’ ends, creating single-stranded complementary overhangs between the two inserts. These overhangs anneal and the Phusion polymerase inserts the missing nucleotides between the sequence of double stranded DNA, using the homologous overhang as priming sequence. Once started, the Phusion reaction is much faster than the slow nuclease and the gaps are quickly filled. As the last step the ligase closes the strand break and fuses the two DNA fragments together. All these enzymes are active at 50 ºC. As a result, multiple pieces of DNA can be assembled in a single tube.

The Gibson assembly is used to construct large circuits from position vectors.

Read more about how to set up a Gibson reaction.