Pyrosequencing™ was initially designed for SNP genotyping, a mini sequencing purpose, and has become a golden standard for CpG methylation analyses. Pyrosequencing is a DNA sequencing technique that is based on the detection of released pyrophosphate (PPi) during DNA syntheses. In a cascade of enzymatic reactions, visible light is generated that is proportional to the number of incorporated nucleotides. The cascade starts with a nucleic acid polymerization reaction in which inorganic PPi is released as a result of nucleotide incorporation by polymerase. The released PPi is subsequently converted to ATP by ATP sulfurylase, which provides the energy to luciferase to oxidize luciferin and generate light. Because the added nucleotide is known, the sequence of the template can be determined. The nucleic acid molecule can be either RNA or DNA. (Genome Res, 2001, 11(1)). Since the light intensity is proportional to the amount of nucleotides added, it also can quantify the allele frequency.
This technique is unique in that it presents an analyzed mutation in the context of the neighboring genetic.  Due to this, it is a foolproof means of guaranteeing that the assay worked correctly.  This demonstrates a significant flexibility in primer placement. Therefore it is easy to design a Pyrosequencing™ assay to analyze virtually any genetic marker; Pyrosequencing assays are mutation-tolerant. Unlike hybridization-based assays, Pyrosequencing generates a correct sequence regardless of the appearance of a new, unexpected mutation; it provides real-time quantitative monitoring and is label-free. This method has a medium throughput of up to 5K genotyping per day. Qiagen provides the complete solution that comes with the auto dispenser/reactor/scanner system PyroMark MD, reagent, vacuum separation workstation, and software.
References
1. Chen D C, Saarela J, Nuotio I, Jokiaho A, Peltonen L, Palotie A. J Mol Diagn. 2003 Nov;5(4):243-9. Comparison of GenFlex Tag array and Pyrosequencing in SNP genotyping.
2. Pourmand N, Elahi E, Davis RW, Ronaghi M. Nucleic Acids Res. 2002 Apr 1;30(7):e31. Multiplex Pyrosequencing.
3. McNeely T. Pharmacogenomics. 2003 Mar;4(2):217-21. Pyrosequencing AB.
4. Godde R, Akkad D A, Arning L, Dekomien G, Herchenbach J, Kunstmann E, Meins M, Wieczorek S, Epplen JT, Hoffjan S. Electrophoresis. 2006 Mar;27(5-6):939-46. Electrophoresis of DNA in human genetic diagnostics - state-of-the-art, alternatives and future prospects.