Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing

Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of fou r coupled enzymes in a single tube assay to monitor DNA synthesis in real t ime using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on dou ble-stranded PCR product. Pyrosequencing data on the human glutathione pero xidase gene (GPXI) from several individuals were analysed and three differe nt allelic variants were determined and confirmed. The possibility of furth er simplifying the sequencing and template-preparation steps is discussed.