A cost-driving factor in Pyrosequencing is the need for single-stranded PCR products that are usually obtained by biotin-labeling of one primer. We designed new universal primers that allow the introduction of biotin during the specific PCR at either the forward or the reverse primer in a single reaction. When converting five human single nucleotide polymorphism assays from the standard format into the universal format, we obtained pyrograms of similar good quality. Although the universal nonhuman sequences are unlikely to form loop structures, we mostly failed to establish assays without using the proprietary software from Biotage AB. Nevertheless, our universal primer pair adds flexibility to the process of assay design due to optional strand selection and contributes to the reduction of genotyping costs.