pyrosequencing


pyrosequencing

a real-time DNA sequencing technique based on monitoring DNA synthesis by BIOLUMINESCENCE using four enzymes: DNA POLYMERASE, ATP sulfurylase, LUCIFERASE and apyrase. The NUCLEOTIDES are tested one at a time for incorporation into the nascent DNA strand during synthesis. Each incorporation event, by DNA polymerase, generates pyrophosphate (PPi) as a by-product. The PPi is converted, by ATP sulfurylase, to ATP and this drives the conversion of luciferin to oxyluciferin by luciferase, with the emission of a light pulse. The light produced is detected by a charge coupled device camera and displayed as a peak in a light profile (pyrogram(tm)). Peak height relates to the number of nucleotides incorporated. If not incorporated the nucleotide is degraded by apyrase and no light is emitted. The DNA sequence can be determined from the pyrogram, knowing the sequence of nucleotides added.