Cloning Hybridoma cDNA by RACE

Abstract

V region primer PCR is usually successful in the amplification of hybridoma V genes, especially if using diverse primer sets (Wang et al. 2000; Krebber et al. 1997; Coloma et al. 1991; Gavilondo-Cowley et al. 1990; Rohatgi et al. 2008). However, there are a number of potential pitfalls in using V region PCR. Mutations within the 50 or 30 ends of the V genes may inhibit primer annealing, and so, prevent amplification. In some cases, the use of universal V region primers can introduce mutations that can reduce stability, production yield, and antigen affinity (Honegger and Pluckthun 2001; Jung et al. 2001). Another problem is the presence of other V genes within the hybridoma that are preferentially amplified. These arise for two reasons. The first is nonproductive rearrangments, which, not being mutated, are very good PCR templates (Carroll et al. 1988; Storb et al. 1980), while the second is probably caused by the fusion of more than one spleen cell to the myeloma cell line, resulting in multiple functional (as well as nonfunctional) V genes (Zack et al. 1995). In this situation, an alternative to V gene PCR is to use either traditional cDNA cloning or rapid amplification of cDNA ends (RACE) (Frohman et al. 1988). This technique relies on knowledge of a small part of gene sequence to amplify from that gene sequence to either end of the cDNA. For both cases, an oligo-dT primer containing a specific tag is used to amplify the cDNA end. In the case of the 30 end, the sequence to which it anneals is the naturally occurring poly-A tail, while in the case of the 50 end (which is that used when RACE is used to clone hybridoma V genes), a poly-A tail is added using terminal transferase. PCR specificity can be A. Bradbury B division, MS-M888, Los Alamos National Laboratory, Los Alamos, NM 87545, USA e-mail: amb@lanl.gov R. Kontermann and S. Du¨bel (eds.), Antibody Engineering Vol. 1, DOI 10.1007/978-3-642-01144-3_2, # Springer-Verlag Berlin Heidelberg 2010 15 subsequently improved by using the specific tag primer and a nested sequence specific primer (Pescatori et al. 1995).