Pyrosequencing

Pyrosequencing is component of molecular pathology.

Methodology

• 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.

Dispensation

• 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.

Advantages

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

Disadvantages

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

See also

• Molecular pathology.

External links

• - a tool to generate expected pyrosequencing results.