Directed Assembly of Many DNA Fragments in a Single Reaction.

<p>(<b>A</b>) A large number of DNA fragments can be assembled in a single reaction. The order of the inserts is determined solely by the included SOCs. Denaturation and renaturation allows SOCs to link fragments that become substrates for ligase (green). Further cycles of denaturation and renaturation, without any polymerase activity, assemble the desired double-stranded construct. (<b>B</b>) PCR amplification of the assembly product using a forward primer for the first fragment and a reverse primer for the last fragment yields large amounts of correctly assembled product. (<b>C</b>) Gel electrophoresis of PCR amplification of cycled ligation assembly products. Eight ∼650 bp DNA sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107329#pone-0107329-t001" target="_blank"><b>Table 1</b></a>) were successfully assembled in a single reaction, in a pre-determined order and orientation, and subsequently amplified. The number of inserts (n) assembled into each product is displayed above lanes. The size (in kb) of assembled products is displayed below each lane. Products greater than 5 kb were successfully assembled in a defined order from eight DNA fragments.