Pyrosequencing

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Pyrosequencing (also known as 454-sequencing) is a method of DNA Sequencing. It involves using an enzyme called luciferase, amongst others, to determine the nucleotide sequence based upon the light emitted when a correct dNTP binds to a template.

The 454 method of sequencing has increased the speed and efficiency of DNA sequencing. It incorporates a laminar flow method of delivering reagents and washing away unused reagents along with etched fibre optic slides to help measure the light output of the reactions occurring. It is a massively parallel process reducing the cost of sequencing greatly. 

The 454 method of sequencing intially involves a primer being added to a single stranded template. This is then incubated with substrates of adenosine 5’ phosphosulfate and luciferin, as well as the enzymes of DNA polymerase, ATP sulphurylase, luciferase and apyrase.
DNA polymerase adds complementary dioxyribonucleotide triphosphates (dNTP) to the template strand; each time a dNTP is added, a pyrophosphate (PPi) is released. The released PPi is converted into ATP, by a sulphurylase enzyme, which then helps to drive the conversion of luciferin to oxyluciferin. The latter conversion is mediated by luciferase and results in the production of light.
The light produced is automatically detected and can be visualised as peaks on a graph; the height of each peak is proportional to the number of dNTPs added.  [1]

454 sequencing has been shown to give "99.5% accuracy at 200 bases" and is capable of giving ~20 megabases of 110 base-reads taking around 8 hours and looks to increase as the system becomes more refined.

The downsides are that the system still has a relatively low throughput. This increases the cost per base of the system [2].   

Alternatives to this system include the Solexa system [3].

References



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