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Pyrosequencing is a DNA sequencing method based on the principle of sequence-to-synthesis. It involves sequencing a single strand of DNA by synthesizing its complimentary strand through sequential addition nucleotides, one base at a time, and detecting the incorporated base at each step. It utilizes the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase along with adenosine 5´ phosphosulfate (APS) and luciferin as substrates. As DNA polymerase catalyze the addition of nucleotides to DNA, pyrophosphate molecules are released in equimolar amount of incorporated nucleotides. Pyrophosphate reacts with APS in the presence of ATP sulfurylase to produce ATP which converts luciferin to oxyluciferin with the help of luciferase. This reaction produces visible light which is detected to generate a pyrogram showing peaks; the height of each peak being proportional to the number of nucleotides incorporated. Unincorporated nucleotides and ATP are degraded by apyrase. It has proved to be a highly suitable method for single nucleotide polymorphism genotyping, mutation detection, DNA methylation analysis and for sequencing short stretches of DNA . It is also used for both de novo and confirmatory sequencing procedures. (Credit: Muhammad Atif Khan)
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