PCA assembly of a gene from overlapping Oligo-sets. A, A schematic diagram of the PCR assembly of a synthetic gene is shown (NS5B, HCV Polymerase). After design of the gene and creation of an Oligo-set using Gene Composer, the PCR gene synthesis protocol is initiated by pooling overlapping complementary oligonucleotides which together comprise individual sub-fragments (Pools 1–5) of the gene which are amplified by PCR and analyzed by agarose gel electrophoresis. B, PCR products of sub-fragments from oligo Pools 1–5. Each product band is about 400 bp. C, The amplification products of Pool 1 (lane 1) are denatured and re-annealed to generate heteroduplexes which are treated with CEL1 nuclease to digest any DNAs with base-mismatches (lane 3, Pool 1 shows a smear of DNAs below ~400bp indicating that CEL1 was able to digest some of the heteroduplexes, in comparison to lane 2 which is CEL1 treated Pool 1 that was not denatured and re-annealed and could therefore not be digested with CEL1). D, The remaining CEL1 resistant intact (undigested) heteroduplex sub-fragments are gel purified (Pools 1–5, lanes 1–5) and pooled for final assembly by PCR (lane 6, arrow). The expected size of the full length gene is about 2,000 bp, and this material can be cloned directly into Topo activated vectors (Invitrogen).