Simultaneous analysis of multiple PCR amplicons enhances capillary SSCP discrimination of MHC alleles

Major histocompatibility complex (MHC) genotyping still remains one of the most challenging issues for evolutionary ecologists. To date, none of the proposed methods have proven to be perfect, and all provide both important pros and cons. Although denaturing capillary electrophoresis has become a popular alternative, allele identification commonly relies upon conformational polymorphisms of two single-stranded DNA molecules at the most. Using the MHC class II (beta chain, exon 2) of the black kite (Aves: Accipitridae) as our model system, we show that the simultaneous analysis of overlapping PCR amplicons from the same target region substantially enhances allele discrimination. To cover this aim, we designed a multiplex PCR capable to generate four differentially sized and labeled amplicons from the same allele. Informative peaks to assist allele calling then fourfold those generated by the analysis of single PCR amplicons. Our approach proved successful to differentiate all the alleles (N=13) isolated from eight unrelated birds at a single optimal run temperature and electrophoretic conditions. In particular, we emphasize that this approach may constitute a straightforward and cost-effective alternative for the genotyping of single or duplicated MHC genes displaying low to moderate sets of divergent alleles.