The History of Pyrosequencing®

  • Pål Nyrén
Part of the Methods in Molecular Biology™ book series (MIMB, volume 373)


One late afternoon in the beginning of January 1986, bicycling from the lab over the hill to the small village of Fullbourn, the idea for an alternative DNA sequencing technique came to my mind. The basic concept was to follow the activity of DNA polymerase during nucleotide incorporation into a DNA strand by analyzing the pyrophosphate released during the process. Today, the technique is used in multidisciplinary fields in academic, clinical, and industrial settings all over the word. The technique can be used for both single-base sequencing and whole-genome sequencing, depending on the format used.


Magnetic Bead Nucleotide Incorporation Pyrosequencing Method Apyrase Activity Medical Research Council Laboratory 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Nyrén, P. (2001) Method for sequencing DNA based on the detection of the relese of pyrophosphate and enzymatic nucleotide degradation. Patents: US 6 258 568BI and WO98/28440.Google Scholar
  2. 2.
    Ronaghi, M., Uhlén, M., and Nyrén, P. (1998) A sequencing method based on real-time pyrophosphate detection. Science 281, 363–365.CrossRefPubMedGoogle Scholar
  3. 3.
    Margulies, M., Egholm, M., Altman, W. E., et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380.PubMedGoogle Scholar
  4. 4.
    Runswick, M. J., Powell, S. J., Nyrén, P., and Walker, J. W. (1987) Sequence of the bovine mitochondrial phosphate carrier protein: structural relationship to ADP/ATP translocase and the brown fat mitochondria uncoupling protein. EMBO J. 6, 1367–1373.PubMedGoogle Scholar
  5. 5.
    Nyrén, P. and Lundin, A. (1985) Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis. Anal. Biochem. 151, 504–509.CrossRefPubMedGoogle Scholar
  6. 6.
    Nyrén, P., Nore, B. F., and Baltscheffsky, M. (1986) Studies on photosynthetic inorganic pyrophosphate formation in Rhodospirillum rubrum chromatophores. Biochim. Biophys. Acta 851, 276–282.CrossRefGoogle Scholar
  7. 7.
    Nyrén, P., Nore, B. F., and Baltscheffsky, M. (1986) Inorganic pyrophosphate synthesis after a short light flash in chromatophores from Rhodospirillum rubrum. Photobiochem. Photobiophys. 11, 189–196.Google Scholar
  8. 8.
    Nyrén, P. (1987) Enzymatic method for continuous monitoring of DNA-polymerase activity. Anal. Biochem. 167, 235–238.CrossRefPubMedGoogle Scholar
  9. 9.
    Melamede, R. J. (1985) Automatable process for sequencing nucleotide. US Patent 4863849.Google Scholar
  10. 10.
    Ståhl, S., Hultman, T., Moks, T., and Uhlén, M. (1988) Solid phase DNA sequencing using the biotin-avidin system. Nucleic Acids Res. 16, 3025–3038.CrossRefPubMedGoogle Scholar
  11. 11.
    Nyrén, P. (1994) Apyrase immobilized on paramagnetic beads used to improve detection limits in bioluminometric ATP monitoring. J. Biolumin. Chemilumin. 9, 29–34.CrossRefPubMedGoogle Scholar
  12. 12.
    Nyrén, P., Pettersson, B., and Uhlén, M. (1993) Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay. Anal. Biochem. 208, 171–175.CrossRefPubMedGoogle Scholar
  13. 13.
    Nyrén, P., Karamouhamed, S., and Ronaghi, M. (1997) Detection of single-base changes using a bioluminometric primer extension assay. Anal. Biochem. 244, 367–373.CrossRefPubMedGoogle Scholar
  14. 14.
    Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlén, M., and Nyrén, P. (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal. Biochem. 242, 84–89.CrossRefPubMedGoogle Scholar
  15. 15.
    Nyrén, P. (1994) A method for the detection of cells, cell lysis or cell-lysing activity, Swedish patent application.Google Scholar
  16. 16.
    Nyrén, P. and Edwin, V. (1994) Inorganic pyrophosphate-based detections: Detection and enumeration of cells. Anal. Biochem. 220, 39–45.CrossRefPubMedGoogle Scholar
  17. 17.
    Nyrén, P. and Edwin, V. (1994) Inorganic pyrophosphate-based detections: Detection and quantification of cell lysis and cell-lysing activity. Anal. Biochem. 220, 46–52.CrossRefPubMedGoogle Scholar
  18. 18.
    Karamohamed, S., Nordström, T., and Nyrén, P. (1999) A real-time bioluminometric method for detection of nucleoside diphosphate kinase activity. Biotechniques 26, 728–734.PubMedGoogle Scholar
  19. 19.
    Karamouhamed, S., Nilsson, J., Nourizad, K., Ronaghi, M., Pettersson, B., and Nyrén, P. (1999) Production, purification, and real-time functional analysis of recombinant Saccharomyces cervisiae MET3 adenosine triphosphate sulfurylase expressed in Escherichia coli. Protein Expr. Purif. 15, 381–388.CrossRefGoogle Scholar
  20. 20.
    Ronaghi, M. (2000) Improved performance of Pyrosequencing using single-stranded DNA-binding protein. Anal. Biochem. 286, 282–288.CrossRefPubMedGoogle Scholar
  21. 21.
    Nordström, T., Gharizadeh, B., Pourmand, N., Nyrén, P., and Ronaghi. M. (2001) Method enabling fast partial sequencing of cDNA clones. Anal. Biochem. 292, 266–271.CrossRefPubMedGoogle Scholar
  22. 22.
    Nordström, T., Ronaghi, M., and Nyrén, P. (1999) Automation of a novel DNA sequencing method. In: Bioluminescence and Chemiluminescence: Perspective for the 21st Century, (Roda, A., Pazzagli, M., Kricka, L. J., and Stanley, P. E., eds.), John Wiley and Sons, Hoboken, NJ, pp. 528–531.Google Scholar
  23. 23.
    Gharizadeh, B., Nordström, T., Ahmadian, A., Ronaghi, M., and Nyrén, P. (2002) Long read Pyrosequencing using pure 2′-deoxyadenosine-5′-O′-(1-thiotriphosphate) Sp-isomer. Anal. Biochem. 301, 82–90.CrossRefPubMedGoogle Scholar
  24. 24.
    Eriksson, J., Gharizadeh, B., Nourizad, N., and Nyrén, P. (2004) 7-deaza-2′-deoxyadenosine-5′-triphosphate as an alternative nucleotide for the Pyrosequencing technology. Nucleosides Nucleotides Nucleic Acids 23, 1583–1594.CrossRefPubMedGoogle Scholar
  25. 25.
    Gharizadeh, B., Eriksson, J., Nourizad, N., Nordström, T., and Nyrén, P. (2004) Improvements in Pyrosequencing technology by employing Sequenase polymerase. Anal. Biochem. 330, 272–280.CrossRefPubMedGoogle Scholar
  26. 26.
    Eriksson, J., Nordström, T., and Nyrén, P. (2003) Method enabling firefly luciferase based bioluminometric assays at elevated temperature. Anal. Biochem. 314, 158–161.CrossRefPubMedGoogle Scholar
  27. 27.
    Eriksson, J., Gharizadeh, B., Nordström, T., and Nyrén, P. (2004) Pyrosequencing technology at elevated temperature. Electrophoresis 25, 20–27.CrossRefPubMedGoogle Scholar
  28. 28.
    Nordström, T., Ronaghi, M., Morgenstern, R., Ekström, L., de Faire, U., and Nyrén, P. (2000) Direct analysis of single nucleotide polymorphism on double-stranded DNA. Biotechnol. Appl. Biochem. 31, 107–112.CrossRefPubMedGoogle Scholar
  29. 29.
    Nordström, T., Nourizad, K., Ronaghi, M., and Nyrén, P. (2000) Method enabling Pyrosequencing on double-stranded DNA. Anal. Biochem. 282, 186–193.CrossRefPubMedGoogle Scholar
  30. 30.
    Nordström, T., Alderborn, A., and Nyrén, P. (2002) Method for one-step preparation of double-stranded DNA template applicable for use with Pyrosequencing technology. J. Biochem. Biophys. Methods 52, 71–82.CrossRefPubMedGoogle Scholar
  31. 31.
    Garcia, C. A., Ahmadian, A., Garizadeh, B., Lundeberg, J., Ronaghi, M., and Nyrén, P. (2000) Mutation detection by Pyrosequencing: sequencing of exons 5 to 8 of the p53 tumor suppressor gene. Gene 253, 249–257.CrossRefPubMedGoogle Scholar
  32. 32.
    Nourizad, N., Gharizadeh, B., and Nyrén, P. (2003) Method for clone checking. Electrophoresis 24, 1712–1715.CrossRefPubMedGoogle Scholar
  33. 33.
    Gharizadeh, B., Ghaderi, M., Donnelly, D., Wallin, K-L., and Nyrén, P. (2003) Multiple-primer DNA sequencing method. Electrophoresis 24, 1145–1151.CrossRefPubMedGoogle Scholar
  34. 34.
    Gharizadeh, B., Ohlin, A., Mölling, P., et al. (2003) Multiple group-specific sequencing primers for reliable and rapid DNA sequencing. Mol. Cell. Probe 17, 203–210.CrossRefGoogle Scholar
  35. 35.
    Gharizadeh, B., Oggionni, M., Zheng, B., et al. (2005) Type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by Pyrosequencing technology. J. Mol. Diagn. 7, 198–205.CrossRefPubMedGoogle Scholar
  36. 36.
    Leamon, J. H., Lee, W. L., Tartaro, K. R., et al. (2003) A massively parallel PicoTiterPlate based platform for discrete picoliter-scale polymerase chain reaction. Electrophoresis 24, 3769–3777.CrossRefPubMedGoogle Scholar
  37. 37.
    Ahmadian, A., Gharizadeh, B., Gustafsson, A., et al. (2000) Single nucleotide polymorphism analysis by Pyrosequencing. Anal. Biochem. 280, 103–110.CrossRefPubMedGoogle Scholar
  38. 38.
    Gruber, J. D., Colligan, P. B., and Wolford, J. K. (2002) Estimation of single nucleotide polymorphism allele frequency in DNA pools by using Pyrosequencing. Hum. Genet. 110, 395–401.CrossRefPubMedGoogle Scholar
  39. 39.
    Uhlmann, K., Brinckmann, A., Toliat, M. R., Ritter, H., and Nurnberg, P. (2002) Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis. Electrophoresis 23, 4072–4079.CrossRefPubMedGoogle Scholar
  40. 40.
    Ahmadian, A., Lundeberg J., Nyrén, P., Uhlén, M., and Ronaghi, M. (2000) Analysis of the p53 tumor supressor gene by Pyrosequencing. Biotechniques 28, 140–147.PubMedGoogle Scholar
  41. 41.
    Goriely, A, McVean, G. A., Rojmyr, M., Ingemarsson, B., and Wilkie, A.O. (2003) Evidence for selective advantage of pathogenic FGFR2 mutations in the male germ line. Science 301, 643–646.CrossRefPubMedGoogle Scholar
  42. 42.
    Andreasson, H., Asp, A., Alderborn, A., Gyllensten, U., and Allen, M. (2002) Mitochondrial sequence analysis for forensic identification using Pyrosequencing technology. Biotechniques 32, 124–133.PubMedGoogle Scholar
  43. 43.
    Allen, M. and Andreasson, H. (2005) Mitochondrial D-loop and coding sequence analysis using Pyrosequencing. Methods Mol. Biol. 297, 179–196.PubMedGoogle Scholar
  44. 44.
    Cebula, T. A., Brown, E. W., Jackson, S. A., Mammel, M. K., Mukherjee, A., and LeClerc, J. E. (2005) Molecular applications for identifying microbial pathogens in the post-9/11 era. Expert Rev. Mol. Diagn. 5, 431–445.CrossRefPubMedGoogle Scholar
  45. 45.
    Gharizadeh, B., Norberg, E., Löffler, J., et al. (2004) Identification of medically important fungi by Pyrosequencing technology. Mycoses 47, 29–33.CrossRefPubMedGoogle Scholar
  46. 46.
    Gharizadeh, B., Kalantari, M., Garcia, C., Johansson, B., and Nyrén, P. (2001) Typing of human papillomavirus (HPV) by Pyrosequencing. Laboratory Investigation 81, 673–679.CrossRefPubMedGoogle Scholar
  47. 47.
    Milan, D., Jeon, J. T., Looft, C., et al. (2000) A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle. Science 19, 1248–1251.CrossRefGoogle Scholar
  48. 48.
    Mochida, K., Yamazaki, Y., and Ogihara, Y. (2003) Discrimination of homoeologous gene expression in hexaploid wheat by SNP analysis of contigs grouped from a large number of expressed sequence tags. Mol. Genet. Genomics 270, 371–377.CrossRefPubMedGoogle Scholar
  49. 49.
    Svantesson, S., Westermark, P. O., Hellgren Kotaleski, J., Gharizadeh, B., Lansner, A., and Nyrén, P. (2004) A mathematical model of the Pyrosequencing reaction system. Biophys. Chemist. 110, 129–145.Google Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Pål Nyrén
    • 1
  1. 1.Department of BiotechnologyAlbaNova University CenterStockholmSweden

Personalised recommendations