Fig. 4. Schematic representations of assembly methods for DNA synthesis.

A, Gibson assembly^25,139,140. Two duplex DNA strands 34 and 35 are selected with a complementary terminal overlap region (black). Digestion with T5 exonuclease (step a) degrades each strand of the DNA duplexes in the 5ʹ-to-3ʹ direction, yielding the sticky ends of 36 and 37, followed by the annealing of complementary sticky ends between the two DNA duplexes¹⁴³ (step b). Phusion polymerase and Taq ligase are then combined (step c) to ligate the two short DNA duplexes into a single, long DNA duplex construct 38 (refs. ^144,145). The process is repeated (step d) for gene assembly. B, Polymerase cycling assembly (PCA)^26,141. High-purity synthetic oligonucleotides 39 are designed, such that annealing of complementary overlaps generates the desired long duplex DNA construct (step a). The desired construct is then assembled in either a single step from 43 or two steps from 39 using a DNA polymerase (step b) to yield template 40. PCR amplification (steps c and d) of 41 amplifies the desired long duplex DNA construct 42.