Figure 2. Schematic representation of different scFv gene assembly methods. Heavy and light chain variable regions were amplified by PCR and used for the assembly reaction. For the splicing by overlap extension PCR (SOE-PCR), each amplicon was mixed with VIgKFor01 (VLK) and IgGrevNheI primers and amplified for 30 cycles. In the case of the Independent Strand Amplification PCR (ISA-PCR), light chain PCR product was incubated with VLK 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. Different numbers of denaturation annealing and extension cycles were tested during the assembly reaction.