多基因克隆方法（多DNA片段组装）

To construct the final plasmid from the six starting materials (gene 1–4, replication origin and a selectable marker), SFs are first prepared by linking every two materials together, usually suing overlap-extension PCR (OE-PCR). So as shown in this figure, every SF has its 3′-half overlapped with the 5′-half of the next SF and the 5′-half of the first SF overlaps with the 3′-half of the last SF. A mixture of these SFs was denatured at 100°C to free all single strands. When it cools back down to room temperature, annealing between the overlaps would assemble the single strands one after another into a cycle which can be further repaired into double-stranded, closed circular molecule after transformation into the cells.
http://www.plosone.org/article/i ... ournal.pone.0030267