title: Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing.
abstract: Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.
authors.affiliation: Department of Biotechnology, The Royal Institute of Technology, SE-100 44 Stockholm, Sweden.
authors.forename: T
authors.initials: T
authors.lastname: Nordström
authors.forename: M
authors.initials: M
authors.lastname: Ronaghi
authors.forename: L
authors.initials: L
authors.lastname: Forsberg
authors.forename: U
authors.initials: U
authors.lastname: de Faire
authors.forename: R
authors.initials: R
authors.lastname: Morgenstern
authors.forename: P
authors.initials: P
authors.lastname: Nyrén
mesh_terms: D000483:Alleles; D001483:Base Sequence; D001709:Biotechnology; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D014644:Genetic Variation; D005979:Glutathione Peroxidase / Q000235:genetics; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers; D004247:DNA; D005979:Glutathione Peroxidase