The Gibson Assembly method and Picky thermodynamic junction analysis.

<p>(a) The Gibson Assembly reagent includes three enzymes. The 5’ exonuclease erodes the 5’ ends on each dsDNA fragment, exposing single-stranded 3’ overhangs. The overhangs anneal to each other according to their compatible base-pairing. The DNA polymerase repairs gaps and the DNA ligase covalently binds the fragments to create a full-length product. (b) To design an optimal fragment set for gene assembly, the target gene is first analyzed using the Picky software to identify all its thermodynamically unique junction regions. Next, a separate Perl program takes these junction coordinates as well as some user specified design parameters such as acceptable minimum and maximum fragment lengths and the optimization goal for lower cost or fewer fragment count to finalize the optimal fragment set.