Pyrosequencing® is a high-throughput technique for single-nucleotide polymorphism (SNP) genotyping, DNA sequencing, and SNP allele frequency and DNA methylation assays. The Pyrosequencing assay involves initially purifying PCR-generated single-strand DNA labeled with biotin as a sequencing template. The disadvantage of the synthesis of a biotinylated sequence-specific primer to assay each DNA variant lies in costs and time consumption. To overcome this problem, methodology has been developed to generate biotinylated Pyrosequencing DNA templates using a universal biotinylated primer. Using this methodology, biotinylated DNA fragments can be generated without the use of sequence-specific biotinylated primers for each DNA variant.