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Pyrosequencing is component of molecular pathology.


  • Sequence-by-synthesis
  • Relies on light detection during synthesis.

Simplified workflow:

  • A single strand of the DNA is immobilized by beads in a chamber.
  • The starting complementary primer is present.
  • The complementary strand is produced enzymatically by DNA polymerase.
  • Sequential dispensation of nucleotides (one base pair is analyzed at a time).
  • When the correct nucleotide is added, inorganic pyrophosphate is released.
  • The pyrophosphate is converted into ATP which oxidizes luciferin (and light emits).
  • The light emission is directly proportional to the number of nucleotides added.
  • Light ist recorded by a scanner and displayed as a pyrogram peak.


  • Order of nucleotide dispensation is important:
  • Cyclic dispensation ("AGCT" repeated)
    • Detects unknown targets.
    • May result in mutant and wild-type allele being out of phase.
  • Opimized dispensation (nucleotides dispensed as expected)
    • Type of mutation has to be known.
    • "Cleaner" pyrogram (easy to interpret).
    • Suboptimal dispensations may not show different pyrogram between simple hotspot and complex mutations.


  • Superior detection limit compared to Sanger sequencing
  • Cost-effective.
  • Quantitative analyis.


  • Shorter read lengths (usu. 100bp, max 400bp)

See also


External links