Figure 2

Schematic outline of the strategy used for the construction of compact scFv library. The total RNA was extracted from peripheral blood lymphocyte (PBL) of 140 non-immunized healthy donors. cDNA was synthesized by using a mix of random hexamer and oligo dT₁₈. The locations of all PCR primers on the two variable region genes are shown. The list of all primers used for the construction of the library is given in Table 1. Three step PCR reactions were performed. The first PCR step comprises 75 reactions for amplification of V gene repertoire. The second and third PCRs link and amplify full-length scFv gene repertoire, which were cloned into pMod1 phagemid. The DNA sequence encoding the flexible linker is depicted in detail.