title: Pyrosequencing protocol requiring a unique biotinylated primer.
abstract: BACKGROUND
  DNA sequencing has markedly changed the nature of biomedical research. Large-scale sequencing projects have generated several millions of potential polymorphisms widespread in the human genome requiring validation and incorporation into screening panels. As a consequence, high-throughput analysis of these variants in different populations of interest is now the cornerstone of structural genomics. Pyrosequencing is a versatile technique allowing an easy 96-well typing format. However, every polymorphism requires a specific labeled primer to generate a single-stranded DNA fragment containing the region of interest.
  
  
  METHODS
  We describe how with an adjusted primer stoichiometry we can standardize the labeling of every amplicon with a single biotinylated universal primer (BM13S).
  
  
  RESULTS
  We circumvent the need for specific biotinylated primers for each single-nucleotide polymorphism (SNP) under study. As an example, we assessed this novel protocol by genotyping three SNPs mapping calpain-10, caveolin-1 and CYP19A1.
  
  
  CONCLUSIONS
  The present approach represents an alternative to standard pyrosequencing protocols, since it requires a single biotinylated primer that is suitable for each SNP under study.
authors.affiliation: Departamento de Genomica Estructural, Neocodex SL, Sevilla, Spain. jlroyo@neocodex.es
authors.forename: Jose Luis
authors.initials: JL
authors.lastname: Royo
authors.forename: Manuel Hidalgo
authors.initials: MH
authors.lastname: Pascual
authors.forename: Ana
authors.initials: A
authors.lastname: Salinas
authors.forename: Francisco Jose
authors.initials: FJ
authors.lastname: Tello
authors.forename: Maria Del Carmen
authors.initials: Mdel C
authors.lastname: Rivero
authors.forename: Eduardo Ferrero
authors.initials: EF
authors.lastname: Herrero
authors.forename: Luis Miguel
authors.initials: LM
authors.lastname: Real
authors.forename: Agustín
authors.initials: A
authors.lastname: Ruiz
mesh_terms: D019921:Biotinylation; D017931:DNA Primers / Q000737:chemistry*; D023281:Genomics / Q000379:methods; D006801:Humans; D008722:Methods; D009154:Mutation; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers