Figure 1: Principle of pyrosequencing technology
The primer for the sequencing step is hybridized to a single-stranded DNA template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates. Deoxyribonucleotide triphosphate (dNTP) is added, one at a time, to the pyrosequencing reaction. The incorporation of a nucleotide is accompanied by release of pyrophosphate (PPi). The ATP sulfurylase quantitatively converts PPi to ATP. The signal light produced by the luciferase-catalyzed reaction in presence of ATP is detected by a charge coupled device (CCD) camera and integrated as a peak in a Pyrogram. The nucleotide degrading Apyrase enzyme continuously degrades ATP excess and unincorporated dNTPs. The process continues with addition of the next dNTP and the nucleotide sequence of the complementary DNA strand is inferred from the signal peaks of the pyrogram.
The primer for the sequencing step is hybridized to a single-stranded DNA template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates. Deoxyribonucleotide triphosphate (dNTP) is added, one at a time, to the pyrosequencing reaction. The incorporation of a nucleotide is accompanied by release of pyrophosphate (PPi). The ATP sulfurylase quantitatively converts PPi to ATP. The signal light produced by the luciferase-catalyzed reaction in presence of ATP is detected by a charge coupled device (CCD) camera and integrated as a peak in a Pyrogram. The nucleotide degrading Apyrase enzyme continuously degrades ATP excess and unincorporated dNTPs. The process continues with addition of the next dNTP and the nucleotide sequence of the complementary DNA strand is inferred from the signal peaks of the pyrogram.