title: Fundamentals of pyrosequencing.
abstract: BACKGROUND
  DNA sequencing is critical to identifying many human genetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps, and has a superior limit of detection compared with Sanger sequencing. The fundamental basis of pyrosequencing is that pyrophosphate is released when a deoxyribonucleotide triphosphate is added to the end of a nascent strand of DNA. Because deoxyribonucleotide triphosphates are sequentially added to the reaction and because the pyrophosphate concentration is continuously monitored, the DNA sequence can be determined.
  
  
  OBJECTIVE
  To demonstrate the fundamental principles of pyrosequencing.
  
  
  METHODS
  Salient features of pyrosequencing are demonstrated using the free software program Pyromaker ( http://pyromaker.pathology.jhmi.edu ), through which users can input DNA sequences and other pyrosequencing parameters to generate the expected pyrosequencing results.
  
  
  CONCLUSIONS
  We demonstrate how mutant and wild-type DNA sequences result in different pyrograms. Using pyrograms of established mutations in tumors, we explain how to analyze the pyrogram peaks generated by different dispensation sequences. Further, we demonstrate some limitations of pyrosequencing, including how some complex mutations can be indistinguishable from single base mutations. Pyrosequencing is the basis of the Roche 454 next-generation sequencer and many of the same principles also apply to the Ion Torrent hydrogen ion-based next-generation sequencers.
authors.affiliation: Sol Goldman Pancreatic Cancer Research Center, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
authors.forename: Colleen T
authors.initials: CT
authors.lastname: Harrington
authors.forename: Elaine I
authors.initials: EI
authors.lastname: Lin
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Olson
authors.forename: James R
authors.initials: JR
authors.lastname: Eshleman
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D011756:Diphosphates / Q000737:chemistry; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software
chemical_list: D004273:DNA, Neoplasm; D011756:Diphosphates