Degenerated Primer Design to Amplify the Heavy Chain Variable Region from Immunoglobulin cDNA

The amplification of variable regions (Fv) of immunoglobulins (Ig) becomes a major challenge in cloning antibody genes either from hybridoma cell lines or splenic B cells. When we amplified the heavy-chain variable regions (VH) gene from one hybridoma cell line which were not amplified successfully under conventional protocols, a novel method was developed to design the degenerated primer of immunoglobulin cDNA and rapid amplification of cDNA ends polymerase chain reaction (RACE PCR) protocols were performed to recognize the VH gene from the hybridoma cell line. The most highly conserved region in the middle of the VH regions of the Ig cDNA was identified and a degenerated 5'primer was designed using our algorithms. The VH gene was amplified by the 3'RACE and 5'RACE. The VH sequence of CSA cells was 399 bp. The new protocol rescued the amplifications of the VH gene that had failed under conventional protocols and notably increased amplification specificity. The algorithm improved the primer design efficiency and is useful in building VH and VL gene libraries or cloning of unknown genes in gene families.