Simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR

A method for the facile simultaneous mutagenesis of complementary-determining regions (CDRs) in a single chain antibody (scFv) is described. Overlapping sets of oligonucleotides containing random sequences within the CDRs corresponding to the heavy chain variable region (VH) jointed to a linker peptide (J) and the light chain variable region (VL) were extended under PCR conditions to full-length genes. These gene products were then further amplified using short PCR primers containing complementary overlaps between the 3' and 5' ends of the VH-J and VL genes respectively. In a final step, the VH-J and VL gene products were mixed and assembled into scFv DNA products by overlap extension under standard PCR conditions. Sequence analyses indicated that the method is basically successful. However, some deletions were observed, which probably reflects difficulties in the automatic synthesis of long degenerate oligonucleotides.