Pyrosequencing
Summary
TLDRThis video tutorial from 'Somos Biology' delves into the chemistry behind pyrosequencing, a high-throughput sequencing technique. It explains the process of DNA fragmentation, adapter addition, and solid surface attachment using beads. The core of pyrosequencing is the release of pyrophosphate during DNA polymerization, which is converted into ATP and then into light via luciferin and luciferase. Light intensity, detected by sensors, indicates the presence and quantity of nucleotides, allowing for the sequencing of DNA fragments. The tutorial covers both solid and liquid phase pyrosequencing, highlighting the importance of accurate nucleotide detection for genomic analysis.
Takeaways
- π Pyrosequencing is a high-throughput sequencing method, also known as modern next-generation sequencing, that is fast and produces data simultaneously for multiple fragments.
- π¬ The process involves several common steps across sequencing technologies, such as the preparation of DNA fragments to be sequenced, which includes fragmenting large DNA into small, double-stranded pieces.
- 𧬠Adapter DNA sequences are added to the ends of the DNA fragments to facilitate attachment to solid surfaces, such as beads, which are crucial for the sequencing process.
- 𧲠The solid surface attachment is essential because the sequencing process cannot be conducted in a liquid solution; it requires a stable, solid platform.
- π‘ Pyrosequencing relies on the release of pyrophosphate during DNA polymerization, which is an energetic molecule that can be converted into ATP.
- β¨ The conversion of pyrophosphate into ATP is facilitated by the enzyme sulfurylase and the molecule ammonium persulfate, leading to the production of light through the action of luciferin and luciferase.
- π Light sensors, such as CCD or CMOS sensors, detect the light produced during the sequencing reaction, allowing for the determination of the DNA sequence based on the intensity of the light.
- π The sequencing process is iterative, adding one nucleotide at a time and detecting the light produced, which indicates the presence of specific nucleotides in the DNA sequence.
- π The intensity of the light produced can vary depending on the number of consecutive identical nucleotides present, helping to distinguish between different DNA sequences.
- β»οΈ In liquid-phase pyrosequencing, instead of washing the wells after each cycle, an enzyme called apyrase is used to break down unincorporated nucleotides and minimize errors.
- π The video tutorial aims to provide a deeper understanding of the chemistry behind pyrosequencing, highlighting its significance in modern genomic research.
Q & A
What is pyrosequencing and how does it differ from other modern sequencing technologies?
-Pyrosequencing, also known as high-throughput sequencing, is a method of DNA sequencing that is part of modern generation sequencing processes. It is known for its speed and simultaneous data production for multiple fragments, which can be combined to understand the complete genome. It differs from other technologies like Illumina or Ion Torrent in the specific method it uses to detect the incorporation of nucleotides, which is through the release of pyrophosphate and the subsequent production of light.
What is the significance of the term 'pyro' in pyrosequencing?
-The term 'pyro' in pyrosequencing comes from the release of pyrophosphate (PPi) during the DNA polymerization process. Pyrophosphate is a high-energy molecule that is released every time a nucleotide is added to the growing DNA chain.
Can you describe the initial steps involved in the preparation of DNA for pyrosequencing?
-The initial steps in the preparation of DNA for pyrosequencing include fragmenting the genomic DNA into small, double-stranded DNA fragments. These fragments have adapter DNA sequences added to their ends, which allow them to be attached to solid surfaces, such as beads, for the sequencing process.
Why is it necessary to attach the DNA to a solid surface in pyrosequencing?
-Attaching the DNA to a solid surface, such as beads, is crucial for the pyrosequencing process because it ensures that the DNA remains in place during the sequencing reactions. This immobilization allows for the sequential addition of nucleotides and the detection of light produced by the reactions to determine the DNA sequence.
What role do adapters play in the pyrosequencing process?
-Adapters play a key role in pyrosequencing by providing a complementary sequence to the single-stranded DNA on the beads. This allows the target DNA, with the adapter sequence, to bind to the beads, effectively immobilizing the DNA for the sequencing process.
How does the detection of light relate to the sequencing process in pyrosequencing?
-In pyrosequencing, the detection of light is directly related to the sequencing process. Each time a nucleotide is incorporated into the growing DNA chain, pyrophosphate is released, which is then converted into ATP. ATP subsequently reacts with luciferin in the presence of the enzyme luciferase to produce light. The intensity of this light is detected and used to determine which nucleotide was added.
What is the purpose of the enzyme sulfurylase and the molecule ammonium persulfate in pyrosequencing?
-Sulfurylase and ammonium persulfate are used in pyrosequencing to convert the released pyrophosphate (PPi) into adenosine triphosphate (ATP). This ATP is then used in the subsequent reaction with luciferin and luciferase to produce light, which is detected to infer the incorporation of a nucleotide.
How does the intensity of the light produced during pyrosequencing relate to the number of nucleotides added?
-The intensity of the light produced during pyrosequencing is proportional to the number of nucleotides added. A higher intensity indicates a greater number of nucleotides being incorporated at that position in the DNA sequence, which helps in determining the exact sequence of nucleotides.
What is the difference between solid surface and liquid phase pyrosequencing?
-In solid surface pyrosequencing, the DNA is attached to beads and remains in place during the process, allowing for washing steps between the addition of each nucleotide. In liquid phase pyrosequencing, the DNA is not attached to beads and floats in the solution, requiring the use of enzymes like apyrase to break down unincorporated nucleotides and prevent interference.
Why is it important to measure the intensity of light in pyrosequencing?
-Measuring the intensity of light in pyrosequencing is important because it allows for the determination of the number of identical nucleotides in a row. Different intensities can indicate single, double, or multiple nucleotide incorporations, providing a clearer picture of the DNA sequence.
How does the use of apyrase in liquid phase pyrosequencing help prevent errors?
-Apyrase is used in liquid phase pyrosequencing to break down unincorporated nucleotides, preventing them from causing false signals or errors in the sequencing process. This ensures that only the light produced by the incorporation of nucleotides during the sequencing reaction is detected.
Outlines
π Introduction to Pyrosequencing
This paragraph introduces the topic of pyrosequencing, a high-throughput sequencing technique that is part of modern sequencing processes. It highlights the speed and simultaneous data production for multiple fragments, allowing for a comprehensive understanding of the genome. The paragraph also mentions other sequencing technologies such as Illumina and Ion Torrent, emphasizing the commonality in the preparation of DNA fragments for sequencing. The unique aspect of pyrosequencing lies in its detection method, which involves the production of light as nucleotides are sequenced, setting it apart from other methods that may use fluorescence or hydrogen ions.
𧬠The Chemistry of DNA Sequencing in Pyrosequencing
This section delves into the chemical process of DNA sequencing in pyrosequencing. It explains that pyrophosphate is released during DNA polymerization, which is a key aspect of the technique's name. The paragraph describes the energetic nature of pyrophosphate and its conversion into ATP with the help of the enzyme sulfurylase and ammonium persulfate. The generated ATP then converts luciferin into light in the presence of the enzyme luciferase, which is detected by sensors to determine the DNA sequence. The process is detailed step by step, from the addition of nucleotides to the detection of light, which indicates the presence of specific nucleotides in the sequence.
π οΈ Pyrosequencing Process and Detection Mechanism
The final paragraph outlines the step-by-step process of pyrosequencing, including the addition of nucleotides one at a time and the detection of light to confirm their incorporation into the DNA sequence. It discusses the importance of the intensity of light, which varies depending on the number of consecutive nucleotides present. The paragraph also touches on the differences between solid-surface and liquid-phase pyrosequencing, explaining the use of beads in solid-phase to anchor the DNA and the use of enzymes like apyrase to prevent interference in liquid-phase sequencing. The process concludes with the analysis of light intensity data to determine the exact DNA sequence, emphasizing the accuracy and precision of pyrosequencing.
Mindmap
Keywords
π‘Pyrosequencing
π‘Sequencing
π‘Adapter DNA
π‘Beads
π‘Sequencing Wells
π‘Pyrophosphate
π‘ATP
π‘Luciferin and Luciferase
π‘Light Sensors
π‘Intensity of Light
Highlights
Introduction to pyrosequencing, a high-throughput sequencing technique that is part of modern generation sequencing.
Explanation of how pyrosequencing is fast and produces data simultaneously for many fragments, allowing for complete genome sequencing.
Overview of modern sequencing technologies including pyrosequencing, Illumina, and Ion Torrent sequencing, and their common initial steps.
Description of the preparation of DNA fragments for sequencing, including the addition of adapter DNA sequences.
Process of separating double-stranded DNA into single strands and attaching adapters for solid surface attachment.
Importance of fixing DNA onto a solid surface, such as beads, for the sequencing process.
Mechanism by which adapter sequences allow DNA to bind to beads, facilitating the sequencing process.
Loading of bead-bound DNA into sequencing wells for the commencement of the chemical sequencing process.
Chemical basis of pyrosequencing, focusing on the release of pyrophosphate during DNA polymerization.
Conversion of pyrophosphate into ATP with the help of the enzyme sulfurylase and ammonium persulfate.
Role of ATP in converting luciferin into light through the action of luciferase enzyme.
Detection of light production as an indicator of nucleotide incorporation during sequencing.
Use of light sensors, such as CCD or CMOS, to detect light and provide data for DNA sequence determination.
Process of adding each nucleotide one at a time and detecting light to confirm incorporation into the DNA sequence.
Differentiation between solid surface and liquid phase pyrosequencing, and their respective methods for avoiding error.
Use of the enzyme apyrase to break down unincorporated nucleotides in liquid phase pyrosequencing.
Importance of measuring light intensity to determine the number of consecutive nucleotides in the DNA sequence.
Final process of washing wells or using apyrase in liquid phase to minimize errors in pyrosequencing.
Conclusion summarizing the key points of pyrosequencing and its practical applications in DNA sequencing.
Transcripts
welcome back friends welcome to another
video from somos biology and in this
video tutorial we'll be talking about
pyrosequencing
I have a different video it's an
animation of pyrosequencing in my
channel but not any demonstrative video
like that
so here I'll be talking about the
chemistry behind the pyro sequencing
process which is very interesting but
pyro sequencing is also known as
high-throughput sequencing which is also
kind of modern generation sequencing
process and in this sequencing process
it's very very fast and it produces the
data simultaneously for many different
fragments at a time and we can combine
the data together to get the idea of the
complete genome sequencing now this pyro
sequencing now there are many different
modernist sequencing technologies like
pyro sequencing Illumina sequencing for
5 for sequencing Ion Torrent sequencing
in all the sequencing there are some
very basic common things that are
present every every in every sequencing
process that we know very common things
for example the preparation of the DNA
fragment which is to be sequenced now
the thing different differs is how the
DNA is sequenced right that means the
sequencing of the DNA means you have to
know the ID of each of the nucleotide
base that is present one after another
that is the actual sequencing the
meaning of sequencing and that meaning
of sequencing is organized and we can
get the idea only by checking for
different complementary DNA strands and
DNA sequences there but in all these
other needs generation sequencing
approaches there is a specific process
known as the preparation of target DNA
for DNA sequencing and that preparation
is very same the only thing differs if
the exact sequencing process some uses
DNA fluorescence technology to detect
some uses the production of hydrogen
ions to detect which is Ion Torrent
sequencing some detects the production
of as I told you the fluorescence which
is 4 5 4 sequencing and some uses the
production of light as a source to know
the sequencing in this case of
while sequencing it is the light that is
going to tell us whether the sequencing
is occurring or not so but the first
stage first few stage are the same the
stages this is the genomic DNA genomic
DNA the complete DNA sequence large DNA
sequence what we need to do we need to
fragment eyes this DNA because this is
big so we'll break the DNA down into
small fragments so we get
double-stranded DNA fragments like that
once we have this double stranded DNA
fragments we add what is known as
adapter DNA sequence this is known as
adapter DNA sequence okay to the end of
of all this double stranded breakdown
portion of the breakdown DNA of the
genomic DNA so we are the adapters after
adding the adapters we separate the
double-stranded DNA into a single strand
so now we get a single stranded DNA
because you separate both the strands so
this is the ultimate condition that we
get we get a single stranded DNA adapter
attached to one of this end so get this
so this is the preparation of the DNA
that we are talking about once we have
this DNA attached with one adapter at
the end then we take this DNA and we
want this DNA to be fixed permanently
into a solid surface that's very very
important because you cannot run this
whole process in liquid solution it is
not possible we need to attach it to a
solid surface the solid surface that we
are talking about is known as beads we
have the bead and the bead is surrounded
by single-stranded DNA sequences the be
discovered by single-stranded DNA
sequences all around
okay now this DNA we prepare this
adapter remember the reason we add
adapter is to fix this target DNA to the
bit because beat carries a
single-stranded DNA the sequence of it
is complementary to the adapter sequence
so now we add this adapter containing
the target DNA and attach it to each of
the beads they can easily pair as you
can see it here they can
easily bind and now the target DNA
remember target DNA is only the black
portion here the target DNA is not fixed
now we can run this process so the DNA
will not go away from this place so once
we prepare the beads now the exact
process of DNA sequencing to be done now
we take the beads we load them into what
we known as sequencing wells okay
small grooves where we can put this
beads we put the beads here and they
contain some volume areas so that we add
all the enzymes and all the chemicals
that is required buffer solutions and
washing solutions for the process for
the reaction to occur so we load the
beads we load them here in different
wells so once everything is done now the
final process of DNA sequencing will
take place and this is the chemical
process of DNA sequencing now the
chemical process of DNA sequencing
relies on a very simple fact that every
time DNA polymerization take place pyro
sequel pyrophosphate is released okay
now if you look at the idea of DNA
sequencing this is a growing chain let
us say this is the three prime hydroxyl
group okay let us say this is the
template TLM okay now the new upcoming
nucleotide sequence
carries 3 phosphate groups together this
is the nucleotide with three different
phosphate groups now this hydroxyl group
it has a lone pair of electron which can
attack the alpha phosphate as a result
it will kick this two different
phosphate groups out it is known as a
pyro phosphate and there is the name
pyro sequencing comes from this
pyrophosphate now pyrophosphate is very
energetic molecule it contains a lot of
energy any molecule with lot of
phosphate groups attached contains
higher amount of energy remember that so
pyrophosphate is very energetic molecule
once the pyrophosphate is released let
us look at here now the step by step
details we delete this part everything
now whatever we looking they are
occurring at inside the wells okay so we
know this is the basic thing this is the
process and we know there are DNA's
added there say let me draw it here once
make you understand say this is the
adapter portion the rest of the DNA I
draw only one for clear understanding
now here this is the condition now this
is the three prime hydroxyl remember
that is already present okay so now we
are only doing the polymerization stages
now we know chemically that once
polymerization stage will perform it
will generate the inorganic the
pyrophosphate PP I now the pyrophosphate
can produce ATP it can convert it into
the adenosine triphosphate with the help
of an enzyme and a chemical molecule the
molecule that we require is known as a
PS for ammonium persulfate and the
enzyme that is responsible for doing
that is sulfury lays sulfury lays ok
sulfuring is enzyme using ammonium
persulphate to convert pyrophosphate
into adenosine triphosphate so
ultimately adenosine triphosphate is
generated from PP I so once ATP is
generated ATP now converts luciferin
into luciferase now the thing is
normally in this wales ppi is normally
generated once we add every nucleotide
sequence ppi is generated after that
what we had we have sulfury less as well
as aps so what it does it will convert
we pies into ATP so now we have ATP's
present then we are Luciferian
okay and Luciferian
is converted to light in presence of ATP
in when we add the enzyme luciferase
okay so in this reaction this is a
chemical reaction as you know
biochemical reaction and in every stage
we need to add many enzymes and also
some substrate for conversion of this
substrate into products so we add first
the sulfuryl is an aps to convert them
into ATP then we also need to add
Luciferian as well as luciferase to
produce light but the actual thing if
you look at the simpler form this is the
chemical form the simpler form is every
time a nucleotide sequence is added
light is produced this is the mechanism
how its producing but every time a
nucleotide sequence is attached light is
released and there are light sensors
that can detect the production of light
that could be CCD sensors CMOS sensors
common light sensors that that are
present in your digital camera or mobile
phone so the light can be sensed with
the sensors okay and the sensor will
give the output the data the output data
with which we can understand what
sequence what DNA sequence we are
dealing with now remember every time
it's this whole process is going on we
run this whole process for one
nucleotide at a time okay we added for
adenosine first now let's say for
guanine and at every single time and
after each of the round what we do let's
say I add any and we go for that engine
completely go for that in the cytosine
thymine so we do this so once we had
adenine then we check for whether there
is presence of adenine let us say here
there is a thymine one time in residue
we are checking for adenine swiat
adenine adenine pairs with it it
generates light so the light is detected
by the sensor output is provided so we
know that yes adenine is properly added
or attached if there is no timing
present
araignee will not attach note light is
produced no sensors and suitability
nothing can be seen okay this is the
idea now the intensity of light can also
be measured remember because you know we
need to know exactly whether it's
adenine or guanine or sub stuff we know
that because we add each nucleotide at a
time
we only add adenine in each of the wells
then once the whole process is done we
check for the light production we sense
it then again we wash that whole whale
off now remember sometimes in these
cases sometimes you also run it without
the beads sometimes we also run it in
the in a solution see in case of in the
solutions what happens in this case if
you even wash off the wells the DNA
fragments will not come off because they
are fixed there they are attached to the
beads but this pyrosequencing can be of
two different type this solid or solid
surface or liquid surface this is the
solid surface that we are talking about
when the bead is attached the DNA's
attached to the beads so the DNA will
not come off but in some cases where
it's a liquid surface pyrosequencing
then this DNA sequences are not present
and attached to the beads they are just
floating into the into the solution in
that conditions we cannot wash the
whales because if you once the wells in
that condition it will take the target
DNA away that we don't definitely do not
want so in those cases instead of
washing we add another enzyme called a
PI raise DNA nucleotide up iris like
adenosine Epirus time in a Pyrus now
this up iris enzymes will break down at
any time in your guanine cytosine they
will break down all these all these
nucleotides so that they will not
interfere and they will not give us any
blank results or any wrong or error
results this is the idea but this is
very simple the intensity of light is
very very important if one timing is
there one adenine attach intensity will
be lower but if consecutive three
timings are present see Iranians will
have attached so the intensity of the
light will also increase okay so we can
measure the intensity of the light from
from this graph using the CPU the data
we get
and by that we understand whether we
have one adenine or two adenine or three
Iranian or what exact sequence we have
right after another so we run it for
each of the nucleotide let's say we run
it for adenine first then we have for
guanine thymine cytosine each at a time
and every time the process is done we
wash it off but if it's in the liquid
phase state then in those case we cannot
wash it
instead of that we need to use app iris
to break them down okay to minimize the
error so this is the idea of
pyrosequencing I hope this video helps
you to understand pyrosequencing if you
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