resistance of cloned 1f5 chimeric anti-cd20 antibody heavy-chain gene to dna polymerase due to a predicted hairpin structure

Background: formation of secondary structures such as dna hairpins or loops may influence molecular genetic methods and pcr-based approaches necessary for genetic engineering as well as gene regulation. materials and methods: a polymerase chain reaction with splice overlap extension (soe-pcr) was used to create fully synthetic 1f5 chimeric anti-cd20 heavy- and light-chain genes. the chimeric genes were cloned into the pcr-blunt ii-topo vector followed by cloning into the pbudce4.1 expression vector. prediction of secondary structure was performed with the vienna rnafold webserver. pcr and sequencing across the predicted secondary structure of chimeric 1f5 heavy-chain gene were performed with multiple protocols for standard and gc-rich templates. results: in an attempt to design vectors which aimed at generating mouse-human chimeric antibody against cd20 (1f5), we found that the coding sequence of 1f5 chimeric heavy-chain gene constructed by soe-pcr was resistant to polymerase during both pcr and sequencing reactions. furthermore, we were also unable to analyze some positive transformants by restriction enzyme digestion. encountering such difficulties to identify the cloned anti-cd20 chimeric heavychain gene, we found that the chimeric heavy-chain sequence is highly gc-rich and predicted to form a stable secondary structure. conclusion: in conclusion, this is the first report on several difficulties with production of chimeric 1f5 anti-cd20 antibody due to a predicted hairpin cluster which correlates with barriers to pcr, sequencing and possibly restriction analysis. our findings provide a probable note for researchers experiencing technical difficulties with construction of chimeric anti-cd20 antibody 1f5 gene vectors and also with other genes and molecular biology techniques requiring pcr-based method or restriction enzyme analysis.