Pyrosequencing of the DNA is sequencing technique which was one of the suitable method for DNA sequencing. It is very useful for the part of genomics and proteomics which will results into the knowledge of the DNA sequencing.

1. Course :- Genomics
Topic:- Pyrosequencing

2. Pyrosequencing of DNA
Principle of Pyrosequencing:-
• Pyrosequencing technique is based on sequencing-by-synthesis principle and on the detection
of released Pyrophosphate (PPi) during DNA synthesis.
• It employs a series of four enzymes to accurately detect nucleic acid sequences during the
synthesis.
• In Pyrosequencing the sequencing primer is hybridized to a single-stranded DNA biotin labeled
template and mixed with the enzymes;
• DNA polymerase, ATP sulfurylase, luciferase and apyrase and the substrates adenosine 5’
phosphosulfate (APS) and luciferin.
• Cycles of four deoxynucleotide triphosphates (dNTPs) are separately added to the reaction
mixture iteratively.
• The cascade starts with a nucleic acid polymerization reaction in which inorganic PPi is
released as a result of nucleotide incorporation by polymerase.
• Each nucleotide incorporation event is followed by release of inorganic Pyrophosphate (PPi)
in a quantity equimolar to the amount of incorporated nucleotide.

3. • The released PPi is quantitatively converted to ATP by ATP sulfurylase in the presence of APS.
• The generated ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin,
producing visible light in amounts that are proportional to the amount of ATPs.
• The light in the luciferase-catalyzed reaction with a maximum of 560 nanometer wavelength is
then detected by a photon detection device such as A Charge Coupled Device (CCD) camera or
photomultiplier.
• Apyrase is a nucleotide-degrading enzyme, which continuously degrades ATP and non-
incorporated dNTPs in the reaction mixture.
• The generated light is observed as a peak signal in the pyrogram (corresponding to
electropherogram in dideoxy sequencing) proportional to the number of nucleotides
incorporated (a triple dGTP incorporation generates a triple higher peak).
• During this synthesis process, the DNA strand is extended by complementary nucleotides and
the DNA sequence is demonstrated by the pyrogram on a screen.

4. Cyclic Nucleotide Dispensation
dATP>dGTP>dCTP>dTTP
AATAG
TTATCGTA
dNTP dNDP dNMP
PI PI
PPI
ATP Sulfurylase
APS ATP+SO4
-2
ATP AMPADP
Apyrase
Luciferin
Luciferase
PPI
Luciferase + Oxyluciferin + AMP + CO2 + h

5. h
CCD camera for
photomultiplier
Sequenced DNA
Pyrogram

6. Steps Involved in Pyrosequencing
Step 1
Polymerase chain reaction is performed by using a reverse Biotin-labelled on the 5’ end to produce the PCR product.
Step 2
The biotinylated PCR product Is bound to the Streptavidin (White/open polygon) coated onto the beads(gray half circles).

7. Step 3
The bead immobilized by using the magnet . The DNA is denatured and the top strand will washed away.
Magnet
Washing
Magnet

8. Step 4
A complementary primer (5’-AAGGCT-3’) has been annealed, and If the complementary deoxynucleotide triphosphate (dNTPs) is
added (dCTP), the DNA polymerase (gray oval) will incorporate into nascent elongation strand at position of open box. (Each
bead is coated with innumerable PCR product, represented here by single molecule.)
Step 5
dNTPs sequentially dispensed into the chamber containing the template with the primer and polymerase bound. When the
correct complementary dNTP is injected and added by polymerase, inorganic phosphate is released during condensation
reaction.
(DNA)n + dNTP (DNA)n+1 + PPi + H +
Magnet

9. C
dCTPPPi + H +
PPi + APS
ATP Sulfurylase
ATP + SO4
-2
Equation 1
Equation 2
Luciferin + O2 + ATP
Luciferase
Oxyluciferin + CO2 + AMP + PPi + h
CCD camera for
photomultiplier
Sequenced DNA
Reactions involved in Pyrosequencing

10. Analysis of Pyrosequencing Graph
• The graphical presentation of pyrosequencing is the result of the
production of light which is catch by the CCD photo multimeter which
is then transferred to graphical presentation.
• Peak shown in the graph is the result of the incorporation of the right
dNTP at the complementary chain.
• Double peak or high peak is the result of the incorporation of similar
or same subsequent addition of dNTP at complementary chain.
• Height of the peak is directly proportion to the subsequent addition of
homopolymer dNTPs.

11. Materials specification for Pyrosequencing
• In pyrosequencing the natural deoxyadenosine triphosphate (dATP) results in the false signal because, like ribose adenosine
5’-triphosphate (rATP), it is a substrate of luciferase.
• the dATP analog, deoxyadenosine - thio triphosphate is used In lieu of dATP.
• This analog - thio triphosphate produce much high peak than the normal dNTPs so it should be observed during the reading
of pyrosequencing.