Figure 3. Assembly by ISA-PCR produces more scFv than SOE-PCR. The splicing by overlap extension PCR (SOE-PCR) and the independent strand amplification (ISA-PCR) methods were compared in terms of final scFv gene production. A) In the case of SOE-PCR, 50 ng of heavy and light variable chain amplicon were mixed with VIgKFor01 and IgGrevNheI primers and amplified for 30 cycles. For ISA-PCR, 50 ng of light chain PCR product was incubated with VIgKFor01 primer while the heavy chain PCR product was incubated with IgGrevNheI primer and 30 cycles of PCR were performed. Both single strand products were mixed and the assembly reaction performed using 30 cycles of annealing and extension. An aliquot of 10% of the assembly reaction was resolved by electrophoresis on a 1% w/v agarose gel. The amount of scFv gene produced was quantified as described in Material and Methods and the results were normalized using the mean scFv gene produced by SOE-PCR. Analysis represents the average of 3 independent assembly reactions. ** p < 0.05. B) scFv gene produced by ISA-PCR at different cycles during the assembly reaction. MW: 100bp DNA ladder.