Genetic sexing of pinnipeds: a real-time, single step qPCR technique

Existing molecular techniques to determine sex of pinnipeds rely on multiplex PCR amplification and gel electrophoresis, and are consequently expensive, time consuming, insensitive and involve handling hazardous DNA-binding dyes. We developed a qPCR high resolution melt assay that involves a single multiplex PCR with dissociation/melting-curve analysis to determine the melting point temperatures (Tm) of two PCR products. Two sets of primers were selected to amplify short regions of the SRY and ZFX/ZFY loci for molecular sex identification. Primers were designed based on alignment of a broad range of pinniped species to maximize applicability for most or all of the 33 species representing two complete superfamilies in the sub-order Caniformia. The assay was validated using 15 pinniped species totaling 122 animals of known sex. The co-amplification of short products also demonstrated improved results for sex determination of degraded samples. The use of a single step qPCR saves time and reduces the cost of running sex determination tests through reduction of labor and supply costs. This new technique will generate results more quickly and reliably, aiding in the study of population health and sex-specific dispersal and behavior patterns.