Protocol

Bacterial Artificial Chromosomes

Volume 255 of the series Methods in Molecular Biology™ pp 211-219

Pyrosequencing

A Tool for DNA Sequencing Analysis
  • Elahe ElahiAffiliated withStanford Genome Technology Center, Stanford University
  • , Mostafa RonaghiAffiliated withStanford Genome Technology Center, Stanford University

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Abstract

Pyrosequencing, a bioluminometric DNA sequencing technique based on sequencing by synthesis, is emerging as a widely applicable tool for detailed characterization of nucleic acids (13). This technique relies on the real-time detection of inorganic pyrophosphate (PPi) released on successful incorporation of nucleotides during DNA synthesis. PPi is immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of generated ATP is sensed by luciferase-producing photons. Unused ATP and deoxynucleotide are degraded by the nucleotide-degrading enzyme apyrase (Fig. 1). The presence or absence of PPi, and therefore the incorporation or nonincorporation of each nucleotide added, is ultimately assessed on the basis of whether or not photons are detected. There is a minimal time lapse between these events, and the conditions of the reaction are such that iterative addition of nucleotides and PPi detection are possible. Prior to the start of the Pyrosequencing reactions, an amplicon is generated in a polymerase chain reaction (PCR) in which one of the primers was biotinylated at its 5′ terminus. The biotinylated double-stranded DNA PCR products are then linked to a solid surface coated with streptavidin and denatured. The two strands are separated, and the strand bound to the solid surface is usually used as template. After hybridization of a sequencing primer to this strand, DNA synthesis under Pyrosequencing conditions can commence. In Pyrosequencing, 1 pmol of DNA template molecules can generate the same number of ATP molecules per nucleotide incorporated, which, in turn, can generate more than 6×109 photons at a wavelength of 560 nm.