Pyrosequencing

Pyrosequencing (Figure) is a DNA sequencing technology based on the sequencing-by-synthesis principle. The technique is built on a fourenzyme real-time monitoring of DNA synthesis by bioluminescence using a cascade that upon nucleotide incorporation ends in a detectable light signal (bioluminescence). The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA-strand.
Thereby, the signal can be quantitatively connected to the number of bases added. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis and genotyping. The technology is promising for the identification of antimicrobial resistance and bacterial strain identification.

Figure : Principle of pyrosequencing. Known deoxynucleotide triphosphates (dATP, dCTP, dGTP, TTP=dNTPs) are added sequentially. The three enzymes shown will lead to light emission if the added nucleotide is complementary to the next base of the template and therefore is incorporated into the growing DNA chain so that pyrophosphate (PPi) is released. The 4th enzyme, apyrase, degrades remaining ATP and remaining dNTP before addition of the next type of dNTP. APS=adenosine phosphosulfate.

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