DNA sequencing is one of the most important platforms for the study of biological systems today, so Reportergene decided to devote its 50th post to a curious technology rebound of reporter genes. Sequence determination is most commonly performed using dideoxy chain termination technology. Recently, pyrosequencing has emerged as a new sequencing methodology. Pyrosequencing is a DNA sequencing technique that is based on the detection of released pyrophosphate (PPi) during DNA synthesis. 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 in a pyrogram.
The technique and methodology of Pyrosequencing from Methods in Molecular Biology, is available for free at the Journal of Visualized Experiment, the "youtube" of biologist we previously reviewed.