Figure 2

Hypothetical principle of multiple DNA fragments fusion by overlap extension PCR. Stage 1: designing primers and amplifying DNA1 to DNAn with overlap sequences respectively through normal PCR; Stage 2: One chain of a fragment matched with another one. Stage 3: Single chain matching with another one extended along 5′ to 3′ at higher annealing temperature and then a series of double splicing DNAs might come into being after one cycles. Step 4: One double splicing DNA could continuously match with another one and then extend, and so forth. Finally, some full multiple splicing DNAs would always be synthetized after more several cycles in the former condition. Stage 5: Amplifying full multiple fusion DNAs at lower annealing temperature through normal PCR.