PCR STRATEGIES FOR ISOLATION OF THE 5'-END OF AN IMMUNOGLOBULIN-ENCODING BOVINE CDNA

We have employed two in vitro amplification strategies in our attempts to characterise the variable region of bovine immunoglobulin heavy ch ains (V-H). Products derived by 5' RACE (rapid amplification of cDNA e nds) spanned the coding sequence, but diversity in the complementarity determining regions was highly restricted, indicating that a limited subset of the heavy-chain repertoire had been recovered. In contrast, unique, full-length V-H determinants were obtained with ease by an inv erse application of the polymerase chain reaction. The strength of thi s approach is considerable: it is straightforward to perform, requirin g none of the tailing procedures inherent to RACE strategies, yet it e nables the rapid isolation of uncharacterised regions of genes for whi ch limited sequence data are available. Our findings suggest strongly that the heavy-chain repertoire of cattle is highly dependent upon a v ery limited number of germline V-H and J(H)(joining region) gene famil ies.