Creation of Recombinant Antibodies: Using Degenerate Oligonucleotides to Amplify Heavy- and Light-Chain Sequences.

Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. However, Ig gene families are large and the variable region gene segments are, indeed, variable, precluding the use of polymerase chain reaction (PCR) with two simple primers to amplify the heavy and light chain gene segments. A wide variety of approaches have been taken to obviate the complexity of the variable region gene segments, including using "universal" or degenerate primers. In this method, mRNA is obtained from B cells as a source of heavy- and light-chain sequences. cDNA is prepared using purified mRNA and either oligo(dT) or a C-region 3' primer. The cDNA negative strand is complementary to the coding sequence of the heavy or light chain. This approach can aid in the design of a variable region primer or in selection of the appropriate degenerate primer for a specific variable gene subfamily.