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

TL;DR

This study develops new primers and shares insights on PCR-based methods for isolating avian MHC class IIB genes, highlighting challenges and strategies for complete gene family recovery across diverse bird species.

Contribution

The paper introduces novel primers for avian MHC class IIB genes and provides practical lessons for improving PCR-based multigene family isolation.

Findings

New primers enable better isolation of avian MHCIIB genes.

PCR-based methods face challenges with gene copy variability.

Strategies to improve gene family completeness are proposed.

Abstract

The amount of sequence data available today highly facilitates the access to genes from many gene families. Universal 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 case of multigene families, PCR-based approaches bear the 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 the present 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 that may help to significantly improve the chance of successfully isolating complete multigene families using PCR-based approaches.