Pyrosequencing: history, biochemistry and future

Clin Chim Acta. 2006 Jan;363(1-2):83-94. doi: 10.1016/j.cccn.2005.04.038. Epub 2005 Sep 13.


Background: Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle.

Methods: The technique is built on a 4-enzyme real-time monitoring of DNA synthesis by bioluminescence using a cascade that upon nucleotide incorporation ends in a detectable light signal (bioluminescence). The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA-strand. Thereby, the signal can be quantitatively connected to the number of bases added. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis and genotyping. Mutation detection and single-nucleotide polymorphism genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. In order to expand the field for pyrosequencing, the read length needs to be improved.

Conclusions: Th pyrosequencing system is based on an enzymatic system. There are different current and future applications of this technique.

Publication types

  • Review

MeSH terms

  • Chemistry, Clinical / methods*
  • Chemistry, Clinical / trends
  • DNA / analysis*
  • DNA / genetics
  • DNA / metabolism
  • DNA-Directed DNA Polymerase / metabolism
  • Diagnosis
  • Genotype
  • Humans
  • Luciferases / metabolism
  • Luminescent Proteins / chemistry
  • Organometallic Compounds / chemistry
  • Organophosphorus Compounds / chemistry
  • Polymorphism, Single Nucleotide / genetics
  • Sequence Analysis, DNA / methods*
  • Sequence Analysis, DNA / trends
  • Sulfate Adenylyltransferase / metabolism
  • Templates, Genetic


  • Luminescent Proteins
  • Organometallic Compounds
  • Organophosphorus Compounds
  • Cascade Blue
  • DNA
  • Luciferases
  • Sulfate Adenylyltransferase
  • DNA-Directed DNA Polymerase