The Principle of Pyrosequencing
The Principle of Pyrosequencing
The Principle of Pyrosequencing
Cascade Reaction
Chemistry
P Y R O S E Q U E N C I N G – O N E H O U R TO E X P L I C I T S E Q U E N C E D ATA
light
time
Step 1
A sequencing primer is hybridized to a single stranded, PCR amplified, DNA
template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, Step 2
luciferase and apyrase, and the substrates, adenosine 5´ phosphosulfate Polymerase
(DNA)n + dNTP (DNA)n+1 + PPi
(APS) and luciferin.
Step 2
The first of four deoxyribonucleotide triphosphates (dNTP) is added to the Step 3
reaction. DNA polymerase catalyzes the incorporation of the deoxyribo-
nucleotide triphosphate into the DNA strand, if it is complementary to the Sulfurylase
base in the template strand. Each incorporation event is accompanied by light
release of pyrophosphate (PPi) in a quantity equimolar to the amount of APS+PPi ATP
incorporated nucleotide.
luciferin oxyluciferin
Step 3 Luciferase
Step 4 ATP
Apyrase
ADP + AMP + phosphate
Apyrase, a nucleotide degrading enzyme, continuously degrades ATP and
unincorporated dNTPs. This switches off the light and regenerates the
reaction solution. The next dNTP is then added. Step 5
nucleotide sequence
G C – A GG CC T
Step 5
Addition of dNTPs is performed one at a time. It should be noted that
deoxyadenosine alfa-thio triphosphate (dATPaS) is used as a substitute for
the natural deoxyadenosine triphosphate (dATP) since it is efficiently used
by the DNA polymerase, but not recognized by the luciferase.