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 (1–3). 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.
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References
Ronaghi, M., Karamohamaed, S., Petterson, B., Uhlen, M., and Nyren, P. (1996) Real time DNA sequencing using detection of pyrophosphate release. Anal. Biochem. 242, 84–89.
Ronaghi, M., Uhlen, M., and Nyren, P. (1998) A sequencing method based on real time pyrophosphate. Science 281, 363–365.
Ronaghi, M. (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res. 11, 3–11.
Ronaghi, M. (2001) “Pyrosequencing for SNP genotyping,” in Single Nucleotide Polymorphisms: Methods and Protocols (Kwok, P.-Y., ed.), Humana, Totowa, NJ, pp. 189–195.
Nordstrom, T, Ronaghi, M., Forsberg, L., de Faire, U., Morgenstern, R., and Nyren P. (2000) Direct analysis of single-nucleotide polymorphism on doublestranded DNA by Pyrosequencing. Biotechnol. Appl. Biochem. 31, 107–112.
Nordstrom, T, Gharizadeh, B., Pourmand, N, Nyren, P., and Ronaghi, M. (2000) Method enabling fast partial sequencing of cDNA clones. Anal. Biochem. 292, 266–271.
Gharizadeh, B., Kalantari, M., Garcia, C. A., Johansson, B., and Nyren P. (2001) Typing of human papilloma virus by Pyrosequencing. Lab. Invest. 81, 673–679.
Garcia, C. A., Ahmadian, A., Gharizadeh, B., Lundeberg, J., Ronaghi, M., and Nyren P. (2000) Mutation detection by Pyrosequencing: sequencing of exons 5–8 of the p53 tumor suppressor gene. Gene 253, 249–257.
Monstein, H., Nikpour-Badr, S., and Jonasson, J. (2001) Rapid molecular identification and subtyping of Helicobacter pylori by Pyrosequencing of the 16S rDNA variable V1 and V3 regions. FEMS Microbiol. Lett. 199, 103–107.
Ronaghi, M., Nygren, M., Lundeberg, J., and Nyren, P. (1999) Analyses of secondary structures in DNA by Pyrosequencing. Anal. Biochem. 267, 65–71.
Ronaghi, M. (2000) Improved performance of Pyrosequencing using single-stranded DNA-binding protein. Anal. Biochem. 286, 282–288.
Ronaghi, M., Pettersson, B., Uhlen, M., and Nyren, P. (1998) PCR-introduced loop structure as primer in DNA sequencing. BioTechniques 25, 876–884.
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© 2004 Humana Press Inc., Totowa, NJ
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Elahi, E., Ronaghi, M. (2004). Pyrosequencing. In: Zhao, S., Stodolsky, M. (eds) Bacterial Artificial Chromosomes. Methods in Molecular Biology™, vol 255. Humana Press. https://doi.org/10.1385/1-59259-752-1:211
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DOI: https://doi.org/10.1385/1-59259-752-1:211
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