The Principle of Pyrosequencing

The purpose of pyrosequencing is to determine the base sequence of a short DNA strand by synthesising the complementary strand along the template. This process can be divided into several steps. 

a single stranded DNA template, usually PCR amplified and purified by chromatography, gel filtration or electrophoretic techniques, is immobilised onto a surface. A suitable primer is then hybridised to this single strand (Fig. 5.26). This ensemble is incubated with four enzymes (DNA polymerase, ATP sulfyrase, luciferase and apyrase) and two substrates (adenosine 5 phosphosulfate (APS) and luciferin). 

one of the four deoxynucleotide triphosphates (dNTPs), dATP, dGTP, dCTP or dlTP, is added to the reaction mixture. If the nucleotide is complementary to the next base in the strand, then DNA polymerase catalyses its incorporation. This reaction is accompanied by the release of a pyrophosphate (PPi) molecule (Fig. 5.27). The amount of PPi released is equimolar to the amount of nucleotide incorporated. For example, if three nucleotides are incorporated, then three molecules of PPi are released. 

PPi then triggers a cascade of enzyme-catalysed reactions (Fig. 5.28). The substrate APS reacts with the PPi and is catalysed by ATP sulfurylase, to form adenosine triphosphate (ATP). The enzyme luciferase catalyses the reaction of ATP with the substrate luciferin, generating oxyluciferin and visible light. This light can be detected by a charged coupled device (CCD) camera as a peak in a so-called pyrogram . The signal obtained is directly proportional to the amount of dNTP incorporated. 

Any excess nucleotide dNTP and any excess ATP are degraded by the nucleotide degrading enzyme apyrase to their respective mono-and diphosphates (Fig. 5.29). When degradation is complete, the next dNTP can be added. 

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