2. Pyrosequencing
Pyrosequencing is the second important type
of DNA sequencing methodology that is in use
today.
Pyrosequencing does not require
electrophoresis or any other fragment separation
procedure and so is more rapid than chain
termination sequencing.
3. Pyrosequencing
Itis only able to generate up to 150 bp in a single
experiment, and at first glance might appear to be
less useful than the chain termination method,
especially if the objective is to sequence a
genome.
The advantage with pyrosequencing is that it can
be automated in a massively parallel manner that
enables hundreds of thousands of sequences to
be obtained at once, perhaps as much as 1000
Mb in a single run.
4. Pyrosequencing
Sequence is therefore produced much more
quickly than is possible by the chain termination
method.
This explains why pyrosequencing is gradually
taking over as the method of choice for genome
projects.
5. Pyrosequencing
Pyrosequencing, like the chain termination
method, requires a preparation of identical single-
stranded DNA molecules as the starting material.
Watch this!
http://www.youtube.com/watch?v=kYAGFrbGl6E
6. Pyrosequencing involves detection of pulses
of chemiluminescence
1. Step 1, A sequencing primer is hydridized to a
single-stranded DNA fragment that serves as a
template.
Mixtures incubated with the enzymes; DNA
polymerase, ATP sulfurylase, Luciferase,
& apyrase.
PLUS Substrates which are, coenzymes
adenosine 5’ phosphsulfate (APS), &
luciferin
7. Pyrosequencing involves detection of pulses
of chemiluminescence
2. The first deoxyribonucleotide
triphosphate (dNTP) is added to the
reaction.
DNA polymerase catalyzes the incorporation of
the deoxyribonucleotide triphosphate into the
DNA strand, if it is complementary to the base
in the template strand
8. Pyrosequencing involves detection of pulses
of chemiluminescence
Each incorporation event is accompanied by
release of pyrophosphate (PPi) in a quantity
equimolar to the amount of incorporated
nucleotide.
9. Pyrosequencing involves detection of pulses
of chemiluminescence
3. ATP sulfurylase converts PPi to ATP in the
presence of adenosine 5’ phosphosulfate (APS).
10. Pyrosequencing involves detection of pulses
of chemiluminescence
ATP drives the luciferase-mediated
conversion of luciferin to oxyluciferin that
generates visible light in amounts that are
proportional to the amount of ATP.
11. Pyrosequencing involves detection of pulses
of chemiluminescence
Thelight produced in the luciferase-catalyzed
reaction is detected by a charge coupled
device (CCD) chip and seen as a peak in the
raw data output (Pyrogram).
The height of each peak (light signal) is
proportional to the number of nucleotides
incorporated.
12. Pyrosequencing involves detection of pulses
of chemiluminescence
4. Apyrase, a nucleotide-degrading enzyme,
continuously degrades unincorporated
nucleotides & ATP.
When degradation is complete, another
nucleotide is added.
13. Pyrosequencing involves detection of pulses
of chemiluminescence
5. Addition of dNTPs is performed sequentially.
As the process continues, the complementary
DNA strand is built up and the nucleotide
sequence is determined from the signal peaks in
the pyrogram trace.
15. Pyrosequencing involves detection of pulses
of chemiluminescence
Of course, if all four deoxynucleotides were
added at once, then flashes of light would be seen
all the time and no useful sequence information
would be obtained This is why we add a
nucleotidase enzyme (Apyrase), so degrade
any unincorporated nucleotides (dNTP).
16. Massively parallel pyrosequencing
The high throughput version of pyrosequencing
usually begins with genomic DNA.
The DNA is broken into fragments between
300 and 500 bp in length and each fragment is
ligated to a pair of adaptors, one adaptor to
either end.
17. Massively parallel pyrosequencing
These adaptors play two important roles: First,
they enable the DNA fragments to be attached to
small metallic beads.
This is because one of the adaptors has a biotin
label attached to its 5′ end, and the beads are
coated with streptavidin, to which biotin binds
with great affinity
19. Massively parallel pyrosequencing
DNA fragments therefore become attached to
the beads via biotin-streptavidin linkages.
The ratio of DNA fragments to beads is set so
that, on average, just one fragment becomes
attached to each bead.
20. Massively parallel pyrosequencing
Each DNA fragment will now be amplified by PCR
so that enough copies are made for sequencing.
The adaptors now play their second role as they
provide the annealing sites for the primers for this
PCR.
The same pair of primers can therefore be used for
all the fragments, even though the fragments
themselves have many different sequences.
21. Massively parallel pyrosequencing
If the PCR is carried out immediately then all we will
obtain is a mixture of all the products, which will not
enable us to obtain the individual sequences of each one.
To solve this problem, PCR is carried out in an oil
emulsion, each bead residing in its own aqueous droplet
within the emulsion.
Each droplet contains all the reagents needed for PCR,
and is physically separated from all the other droplets by
the barrier provided by the oil component of emulsion.
23. Massively parallel pyrosequencing
AfterPCR, the aqueous droplets are transferred
into wells on a plastic strip so there is one
droplet and hence one PCR product per well, and
the pyrosequencing reactions are carried out in
each well.
24. Additional reference to the text book
Mardis
E.R. Annual Review of Genomics and
Human Genetics 9:387-403 (2008)
Editor's Notes
Binding with beads can provide high-throughput DNA extraction
