PCR-based isolation of multigene families: lessons from the avian MHC class IIB

The amount of sequence data available today highly facilitates the access to genes from many gene families. Primers amplifying the desired genes over a range of species are readily obtained by aligning conserved gene regions, and laborious gene isolation procedures can often be replaced by quicker PCR-based approaches. However, in the case of multigene families, PCR-based approaches bear the often ignored risk of incomplete isolation of family members. This problem is most prominent in gene families with highly variable and thus unpredictable number of gene copies among species, such as in the major histocompatibility complex (MHC). In this study, we (i) report new primers for the isolation of the MHC class IIB (MHCIIB) gene family in birds and (ii) share our experience with isolating MHCIIB genes from an unprecedented number of avian species from all over the avian phylogeny. We report important and usually underappreciated problems encountered during PCR-based multigene family isolation and provide a collection of measures to help significantly improving the chance of successfully isolating complete multigene families using PCR-based approaches.