Pyrosequencing? technology is a powerful genotyping tool that requires the generation of single-stranded DNA. Currently, two simple, solid-phase-based methods are available for this, but they require special equipment, they are not automated, and they are relatively expensive because of the need for biotinylated polymerase chain reaction primers. In this article, an enzymatic liquid-phase method for the generation of high-quality, single-stranded DNA, and its novel use for Pyrosequencing? are described. The method has also been fully automated.