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 chains (VH). Products derived by 5' RACE (rapid amplification of cDNA ends) 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 VH determinants were obtained with ease by an inverse application of the polymerase chain reaction. The strength of this approach is considerable: it is straightforward to perform, requiring none of the tailing procedures inherent to RACE strategies, yet it enables the rapid isolation of uncharacterized regions of genes for which limited sequence data are available. Our findings suggest strongly that the heavy-chain repertoire of cattle is highly dependent upon a very limited number of germline VH and JH (joining region) gene families.