Figure 2

Schematic illustration of DNA fragments and primer positions. (A) 3'-end poly dG-tailed cDNA and primers used for the amplification of V genes by 5'-RACE PCR. Arrows represent the position and orientation of the primers. dC13 is the first-round PCR forward primer specific for the poly dG-tailed cDNA, and P0 is the first-round PCR reverse primer specific for the respective IgG or IgK constant regions. P1 is the second round PCR forward primer, and P2 is the second round PCR reverse primer specific for the IgG or IgK constant regions. (B) Schematic representation of homology overlaps between the Ig-cassette and DNA fragments. The Ig-cassette contains V gene-specific sequences (VT1 and VT2) external to the primer-derived sequences (VP1 and VP2). The V gene contains specific sequences (T1 and T2) for the 5'-RACE PCR-amplified V gene internal to the primer-derived sequences (P1 and P2). The mock DNA has P1 and P2 sequences at its ends. P3 and P4 PCR primers hybridize to the f1 replication origin of the Ig-cassette and are used for amplifying the Ig-expression constructs.