Pyrosequencing is a sequencing method that detects light signals from enzymatic reactions triggered by nucleotide additions during DNA synthesis. It was developed in 1996 and allows high-throughput sequencing. There are solid and liquid phase variants, with the latter using an additional enzyme to eliminate washing steps. The process involves preparing DNA fragments, attaching to beads, amplification by PCR, and sequencing by flowing nucleotides over wells containing DNA-coated beads and enzymes, detecting light signals with each nucleotide incorporation.

1. z
PYROSEQUENCING

2. zWHAT IS PYROSEQUENCING??
“Pyrosequencing is a method of
sequencing that relies on light
emitted in an enzymatic reaction
set in motion by the release of
pyrophosphate when a base is
added during the sequencing
process”.

3. z
HISTORY
 The technique was developed by Mostafa Ronaghi and Pål Nyrén at
the Royal Institute of Technology in Stockholm in 1996.
 A second solution-based method for Pyrosequencing was described
in 1998 (In this alternative method, an additional enzyme apyrase is
introduced.
 A third microfluidic variant of the Pyrosequencing method was
described in 2005[5] by Jonathan Rothberg.
 This allowed for high-throughput DNA sequencing and an
automated instrument was introduced to the market. This became
the first next generation sequencing instrument starting a new era
in genomics research

4. zVARIANTS OF PYROSEQUENCING
There are two different pyrosequencing strategies that are currently
available:
Solid-phase pyrosequencing: Utilizes
immobilized DNA in the three-enzyme system described previously. In this
system a washing step is performed to remove the excess substrate after
each nucleotide addition.

5. z
Liquid-phase pyrosequencing: In liquid-
phase pyrosequencing (apyrase, a nucleotide-degrading enzyme from
potato, is introduced to make a four-enzyme system. Addition of this
enzyme has eliminated the need for solid support and intermediate washing
thereby enabling the pyrosequencing reaction to be performed in a single
tube

6. z
FOR METHOD
Reaction mixture contains
 DNA sample to be sequenced
 Primers
 Deoxynucleotides
 DNA polymerase
 Sulfurylase

7. z

8. z
1. ENZYME COCKTAIL. { DNA polymerase, ATP-
Sulfurylase, }
2. Single dNTPs.
3. APS (Adenosine 5’-phosphosulfate).
.
5. DNA Adaptors.

9. z
Which is then cut
into 300-800 bp
fragments
We Start with Double
Stranded Genomic DNA
(Our Sample)…

10. z
Then we add
small
to our
fragments…
Then our double stranded
DNA is denatured to a
single strand.

11. z
Then we take tiny
micro beads
coated with DNA
primers
complementary to
one of the adaptors
on the DNA
strand…
Then single stranded DNA
binds complementarily
with the primer on bead.
Under conditions that
favour one DNA strand
per bead

12. z
PCR is run for
amplification, so
that enough
identical DNA
strands are
available to
analyse.

13. z
Then beads are loaded
into tiny wells with room
for a single bead per well.
Each well consists of a
different amplified DNA
fragment.

14. z
Along with a
single bead…
The well contains enzyme
beads covered with 2
types of enzymes,
&

15. z
Pyrosequencing
Technique:
First a primer is allowed to
attach to the DNA…
Then DNA Polymerase
begins to add
nucleotides…
a single type of dNTP is
flowed across the wells

16. z
The Thymine can
base pair with
Adenine allowing
the DNA
Polymerase to
attach thymine on
the DNA strand…
In the process phosphate
groups are snipped off the
dNTPs in the form of a
pyrophosphate ion
One of the enzyme on
the bead uses this
pyrophosphate ion to
generate ATP molecule
and the other enzyme
uses energy in ATP to
produce a flash of light

17. z
The light is
detected by a
camera and
recorded. And the
nucleotides are
washed out of the
wells…
And a new nucleotide set
is added into the wells.
But because Adenine
doesn’t pair with Guanine
so no nucleotide will be
added thus no light is
emitted.
The next set of
nucleotides bears a
cytosine which is
incorporated, after
which light is emitted
too.

18. z
The next step
involves a set of
dGTPs which are
incorporated…
3 nucleotides are incorporated
and each pyrophosphate ion
results in emission of a
photon.
3 times as much as light is
emitted from this
incorporation, indicating that 3
Cytosine bases appear
consecutively in the template
strand.

19. z
The 4 dNTPs are
flowed
sequentially in
the wells.
This grid represents a
moment of time in which a
single set of dNTPs has
been flowed across the
wells.

20. z
The data from a
single well can be
depicted on a
chart, showing the
level of light
emitted from
incorporation of
each nucleotide.
Then Software packages
assemble these fragments
of sequences into longer
pieces.
And in this way determine
the overall sequence of
the genome.