Pyrosequencing

Summary

This 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

• 🌟 Pyrosequencing is a high-throughput sequencing method, also known as modern next-generation sequencing, that is fast and produces data simultaneously for multiple fragments.
• 🔬 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.
• 🧬 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.
• 🧲 The solid surface attachment is essential because the sequencing process cannot be conducted in a liquid solution; it requires a stable, solid platform.
• 💡 Pyrosequencing relies on the release of pyrophosphate during DNA polymerization, which is an energetic molecule that can be converted into ATP.
• ✨ 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.
• 🔍 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.
• 🔄 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.
• 📉 The intensity of the light produced can vary depending on the number of consecutive identical nucleotides present, helping to distinguish between different DNA sequences.
• ♻️ 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.
• 📚 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

00:00

🌟 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.

05:00

🧬 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.

10:01

🛠️ 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.

Keywords

💡Pyrosequencing

Pyrosequencing, also known as high-throughput sequencing, is a method of DNA sequencing that is highlighted in the video for its speed and simultaneous data production for multiple fragments. It is one of the modern sequencing technologies and is integral to the discussion, as it forms the main theme of the tutorial. The script explains the chemistry behind pyrosequencing, emphasizing its efficiency in genome sequencing.

💡Sequencing

Sequencing in the context of the video refers to the process of determining the precise order of nucleotides within a DNA molecule. It is a fundamental concept in the script, as it is the ultimate goal of pyrosequencing. The video explains how sequencing is achieved by identifying each nucleotide base in succession, which is crucial for understanding the genetic information contained within a DNA sample.

💡Adapter DNA

Adapter DNA is a specific sequence added to the ends of DNA fragments during the preparation for sequencing. In the script, it is mentioned as a necessary step to facilitate the attachment of the target DNA to beads, which is a critical part of the pyrosequencing process. Adapters ensure that the DNA fragments can be anchored to a solid surface, allowing for the subsequent sequencing reactions to occur.

💡Beads

Beads in the script are solid surfaces to which the DNA fragments, equipped with adapter sequences, are attached. They play a crucial role in the pyrosequencing process by providing a stable platform for the DNA to remain fixed during the sequencing reactions. The beads are covered with single-stranded DNA that is complementary to the adapters, allowing for the specific binding of the target DNA.

💡Sequencing Wells

Sequencing wells are the small grooves or chambers where the bead-bound DNA is loaded for the sequencing process. The script describes how these wells contain the necessary enzymes, chemicals, and buffer solutions for the sequencing reaction to take place. They are essential for the compartmentalization and execution of the sequencing process.

💡Pyrophosphate

Pyrophosphate (PPi) is a molecule released during DNA polymerization in the pyrosequencing process. The script explains that the release of this energetic molecule is a key event in pyrosequencing, as it leads to the production of ATP, which is then used in a subsequent reaction to produce light. The term 'pyrosequencing' is derived from this molecule.

💡ATP

Adenosine triphosphate (ATP) is a molecule that stores and transfers energy within cells. In the context of the video, ATP is generated from pyrophosphate and is crucial for the pyrosequencing process. The script describes how ATP is used to convert luciferin into a form that emits light in the presence of the enzyme luciferase, which is a key step in detecting the incorporation of nucleotides during sequencing.

💡Luciferin and Luciferase

Luciferin and luciferase are components of the light-producing reaction in pyrosequencing. The script explains that when ATP is present, luciferin is converted into a light-emitting form by the enzyme luciferase. This light production is directly related to the incorporation of nucleotides and is detected by sensors to determine the DNA sequence.

💡Light Sensors

Light sensors, such as CCD or CMOS sensors, are used in pyrosequencing to detect the light produced when nucleotides are incorporated into the growing DNA chain. The script mentions that these sensors can be found in digital cameras or mobile phones and are essential for capturing the light signals that correspond to the DNA sequence being determined.

💡Intensity of Light

The intensity of light produced during pyrosequencing is a measure of the amount of a particular nucleotide present at a given position in the DNA sequence. The script explains that a higher intensity indicates more consecutive nucleotides of the same type, which is important for accurate sequencing. This concept is used to distinguish between single and multiple occurrences of the same nucleotide in a sequence.

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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