Nanopore sequencing technology

Summary

This video tutorial delves into the innovative technique of nanopore DNA sequencing, which utilizes a minuscule pore and electric current to identify nucleotide sequences. By detecting changes in current as each nucleotide passes through the pore, a unique pattern is created for each base, allowing for DNA sequence determination. The video explains both natural and artificial nanopores, the importance of pore diameter, and methods to drive DNA through the pore, such as electrophoresis or guide proteins. It highlights the potential and challenges of this developing field in genetics.

Takeaways

• 🧬 Nanopore DNA sequencing is a technique that uses an electric current to determine the sequence of nucleotides in DNA.
• 🚀 The concept of nanopore sequencing has been around since 1955 but continues to be developed and refined.
• 🔍 The process involves a very small pore, with a diameter close to 1 nanometer, through which the DNA must pass.
• 💧 The pore can be made from natural materials or artificially created, such as on a silica plate.
• 🌐 The pore is lined with conducting molecules, such as iron particles, to ensure it can carry the electric current needed for sequencing.
• 🔋 The sequencing relies on detecting changes in current magnitude as different nucleotides pass through the nanopore.
• 🔬 Each type of nucleotide (adenine, guanine, cytosine, thymine) alters the current magnitude in a unique way, creating a characteristic pattern.
• 🔎 By comparing the detected patterns with known sequences, the unknown DNA sequence can be determined.
• 🧵 DNA is driven through the nanopore using methods like electrophoresis or with the help of guide proteins that interact with the DNA.
• 🔬 Nanopore sequencing is a promising technique with potential applications in various fields, though it also faces some challenges.
• 📚 The field of nanopore sequencing is evolving, with ongoing research and development to improve its accuracy and applications.

Q & A

What is nanopore DNA sequencing?

Nanopore DNA sequencing is a method that uses an electric current to determine the sequence of nucleotides in a DNA strand. It involves a very small pore through which the DNA passes, and changes in the electric current as each nucleotide passes through the pore are detected to identify the type of nucleotide.

How does the detection of changes in electric current help in identifying nucleotides?

Each type of nucleotide alters the magnitude of the electric current differently when it passes through the nanopore. By detecting these characteristic changes in current, the specific nucleotide can be identified.

When was the concept of nanopore sequencing first developed?

The concept of nanopore sequencing was first developed in 1955, making it an older idea that is still being refined and used in certain applications today.

What is the significance of the pore diameter in nanopore sequencing?

The pore diameter is crucial because it needs to be small enough for the nucleotides to pass through one at a time. Changes in the diameter can affect the voltage and current used in the process, influencing the accuracy of the sequencing.

Can natural or artificial materials be used to create the nanopore?

Yes, both natural materials, such as protein channels extracted from cells, and artificial materials like a silica plate with a created pore, can be used to form the nanopore for sequencing.

Why is it necessary to embed iron particles in the pore wall?

Iron particles are embedded in the pore wall to ensure that the internal diameter is close to the desired size, such as 1 nanometer, and to create a conducting pathway for the electric current during sequencing.

What role does the conducting solution play in nanopore sequencing?

The conducting solution is essential as it allows the electric current to flow through the nanopore and the surrounding environment, which is necessary for detecting changes in current as nucleotides pass through.

How is the DNA strand driven through the nanopore during sequencing?

There are two main methods to drive the DNA through the nanopore: electrophoresis, which uses an electric current to pull the DNA, and guide proteins, which can interact with the DNA and guide it through the pore.

What are guide proteins, and how do they assist in nanopore sequencing?

Guide proteins are proteins that interact with the DNA and help to guide it into the nanopore. They are particularly useful in naturally occurring nanopores or membrane channels, assisting in the accurate sequencing of the DNA.

How does knowing the characteristic current magnitude changes for each nucleotide base help in identifying an unknown DNA sequence?

By comparing the detected changes in current magnitude to known patterns for each nucleotide base, the sequence of an unknown DNA strand can be determined as the changes match the characteristic patterns of adenine, guanine, cytosine, and thymine.

Why is nanopore sequencing considered a developing field?

Nanopore sequencing is a developing field because it is still being refined and improved. There is ongoing research to overcome difficulties and enhance its accuracy and applicability in various sequencing scenarios.

Outlines

00:00

🌟 Introduction to Nanopore DNA Sequencing

This paragraph introduces the concept of nanopore DNA sequencing, a technique that utilizes electric current and voltage to determine the sequence of nucleotides in DNA. The method involves a very small pore through which nucleotides pass, and changes in the current are detected as each nucleotide passes through. This allows for the identification of the specific nucleotide. The idea dates back to 1955 and has evolved to be used in certain cases despite some challenges. The pore's diameter is critical to the process, as even slight variations can affect the voltage and current used. Both natural and artificial pores can be employed, with the latter involving a silica plate with a pore surrounded by iron particles to ensure conductivity. The paragraph sets the stage for understanding the intricacies of nanopore sequencing.

05:00

🔋 The Nanopore Sequencing Process

This section delves into the practical application of nanopore sequencing, explaining how a nanopore is placed in a conducting solution and a voltage is applied across it. The current's magnitude, which is influenced by the pore's size and shape, is a key parameter. DNA strands are then threaded through the pore one nucleotide at a time, with each type of nucleotide altering the current's magnitude differently. This creates a unique pattern for each base, which can be detected and used to identify unknown sequences. The paragraph also discusses the necessity of driving the DNA through the pore, either through electrophoresis or with the help of guide proteins that interact with the DNA. These methods ensure that the DNA is pulled through the nanopore for sequencing.

10:00

🚀 Driving DNA Through the Nanopore

The final paragraph addresses the question of how DNA is driven through the nanopore for sequencing. Two solutions are presented: electrophoresis, which uses electric current to pull the DNA through the pore, and the use of guide proteins that interact with and guide the DNA into the pore. The paragraph highlights the importance of these methods in ensuring that the DNA is successfully sequenced through the nanopore. It concludes by emphasizing the ongoing development in the field of nanopore sequencing and encourages further study through research publications. The video tutorial aims to provide a foundational understanding that can be built upon with more in-depth research.

Keywords

💡Nanopore Sequencing

Nanopore sequencing is a DNA sequencing method that involves detecting changes in electric current as individual nucleotides pass through a nanopore. It is central to the video's theme, illustrating the technique's unique approach to DNA analysis. The script describes how this method uses a small pore and electric current to identify the sequence of DNA bases.

💡Electric Current

Electric current is the flow of electric charge and is essential in nanopore sequencing for detecting the passage of DNA nucleotides. The video explains how a change in current magnitude is indicative of the type of nucleotide passing through the nanopore, thus revealing the DNA sequence.

💡Nucleotide

A nucleotide is the basic building block of DNA, consisting of a base, a sugar molecule, and a phosphate molecule. In the context of the video, the unique alteration of electric current caused by each type of nucleotide is key to determining the DNA sequence.

💡Pore Diameter

The pore diameter is critical in nanopore sequencing as it affects the voltage and current used in the process. The script mentions that the diameter is initially around 1 nanometer, which is sufficient for a nucleotide to pass through, and that changes in diameter can alter the sequencing process.

💡Transmembrane Protein Channels

Transmembrane protein channels are proteins that span across cell membranes and contain hydrophilic regions allowing molecules to pass through. The video describes how these channels can be isolated and used as natural nanopores for sequencing DNA.

💡Silica Plate

A silica plate is an artificial method used to create nanopores. The script explains that a pore is created in the silica plate, which can then be lined with iron particles to achieve the desired diameter for nanopore sequencing.

💡Conductivity

Conductivity refers to the ability of a material to conduct electric current. In the video, the importance of using conducting materials in the nanopore and the solution used in the sequencing process is emphasized to ensure the flow of current necessary for detection.

💡Electrophoresis

Electrophoresis is a technique used to separate molecules based on their size and charge by applying an electric field. The video mentions electrophoresis as a method to drive the DNA through the nanopore during sequencing.

💡Guide Proteins

Guide proteins are proteins that assist in directing certain molecules into channels, such as transmembrane protein channels. The script describes their potential use in nanopore sequencing to help guide the DNA through the nanopore.

💡Characteristic Pattern

A characteristic pattern, in the context of the video, refers to the unique change in electric current magnitude for each type of nucleotide as it passes through the nanopore. This pattern is used to identify the nucleotide and, consequently, the DNA sequence.

💡Detectors

Detectors in the video refer to the instruments used to measure the change in electric current magnitude as nucleotides pass through the nanopore. These detectors are crucial for identifying the DNA sequence based on the characteristic patterns of current change.

Highlights

Introduction to nanopore DNA sequencing, a unique method using electric current and voltage to detect nucleotide sequences.

The concept of nanopore sequencing involves passing nucleotides through a very small pore and detecting current changes.

The technique's development dates back to 1955, highlighting its long-standing yet evolving nature in the field of DNA sequencing.

Challenges and limitations of nanopore sequencing are acknowledged, suggesting its use in specific cases rather than universally.

The importance of pore diameter in nanopore sequencing, with implications for voltage and current adjustments.

The use of natural materials or artificial methods to create nanopores, including protein channels and silica plates.

Embedding iron particles within the pore walls to achieve the desired diameter for nucleotide passage.

The necessity for the nanopore and solution to be conductive to support the flow of electric current in the sequencing process.

The process of placing the nanopore in a conducting solution and applying voltage to create an electric current ring.

The role of current magnitude as a key parameter in detecting the type of nucleotide passing through the nanopore.

The characteristic pattern of current change for each nucleotide base, which is essential for identifying unknown sequences.

The method of using known sequences to establish patterns for identifying nucleotide bases during sequencing.

Techniques for driving the DNA molecule through the nanopore, including electrophoresis and guide proteins.

The practical application of nanopore sequencing in identifying the sequence of DNA strands, such as AGGCTC.

The innovative aspect of nanopore sequencing that allows for real-time, single-molecule DNA analysis.

The potential of nanopore sequencing in advancing DNA analysis, with a call to explore further research publications.

A closing note encouraging viewers to like and subscribe for more educational content on DNA sequencing and related topics.

Transcripts

welcome back guys in this video tutorial

we'll be talking about nanopore DNA

sequencing we have talked about many

varieties of DNA sequencing processes

but this is a kind of different right so

let's talk about the nanopore DNA

sequencing where we use electric current

we use the voltage to finally get

through a particular very small

Poe so we use a small Poe and we allow

the nucleotide sequences to pass through

that small pore by applying some

current and we detect the alteration of

current when a nucleotide is passing

through that Poe and by doing that we

get the idea of the nucleotide we are

dealing with right this is the overall

idea of nanoport sequencing this idea

was uh developed since 1955 it's

underdeveloping idea from 1955 it's huge

I mean it's older idea but still some

cases promising some not we can use it

in a particular orientation or in some

cases only not in most of the cases

because there are some difficulties with

this process but still this is a kind of

interesting idea I like this technique

very much it's kind of difficult and

different the idea here I've told you we

need a Poe that's why you call nanop Poe

sequencing and obviously it is a nano

Poe that means the poe diameter is very

very small small it's in 1 nanometer

close to 1 nanometer at the very

beginning when this technique was

developed we use that 1 nanom range but

now it can be changed we have seen that

that those diameter of the

poe is remarkably important for this

process so if you change that diameter

in fraction it will vary the voltage and

current that we use that's why that

diameter is very very important 1

nanometer diameter is sufficient

for the nucleotide to pass because the

nucleotide is further thinner right so

in this case let's imagine what we need

we need a

small

diameter P so the diameter is 1

nanometer for example and this pore we

can use natural material as a pore

extracted from the cell or we can

produce this Spore on our own right we

can use both this stuff previously we

just took protein molecules

transmembrane protein channels remember

there are protein channels Barrel like

channels right those channels if you

imagine in cell

membrane we have seen certain protein

channels

embedded right something like that now

that channel contains a hydrophilic

region inside small pore inside through

which molecules can pass so we can

isolate those kind of protein ch

channels we can use that as this nanop

four

molecule or what we can do is we we can

use a silica

plate nowadays the artificial method is

we we are using a silica plate silica

you know it's a silica plate and in that

silica plate we just create a pore it

doesn't matter whether the pore is I

mean we don't create a huge P though but

still that doesn't matter that we need

to create 1 nanometer or something we

create a small P out there

once we create that Poe it can be larger

than this 1 nanometer it's fine then

what we do we start

adding

iron we start implementing and adding

irons throughout this poor wall to

finally cover up it in such a way that

the internal diameter becomes close to 1

Nom or whatever diameter we want to ACH

right so we we take our iron particles

iron particles and we start embedding

them

in those wall of those pores and kind of

masking that right so now what we have

we have a silica plate in a Poe and that

PO is surrounded by this iron particles

overall right and we know irons are

conductors now in this whole process of

nanop for sequencing we'll see as the

sequences relies on electric

current in this case whatever thing we

use should should be able to carry some

current or voltage it should be carrier

of current so we use all those things

conducting molecules throughout this

place even if uh the fluid that we use

this process the solution that should be

conducting

also so this is natural this is

artificial we can use both of this but

the goal is to create this pore this

nanoe and in both this case we create

the nanop so once we have the nanop the

idea here is to then place this nanopore

in a solution

we place it in a

solution let's take the example for this

artificial model

here and we place it in a

solution and once you place it in

solution that solution also consisting

of conducting

molecule it is also

conducting

okay now what we do we apply a voltage

throughout this Nano for we apply a

voltage so this ring it acts like a ring

of electric it's a current ring right

and a voltage is applied throughout this

place throughout this

pole now this voltage that is generated

right or the current that is generated

here the magnet magude of current that

is generated here is the most important

parameter now the magnitude of current

depends on the size of this pole as well

as the shape of this PO okay so now

let's see we prepare this after that

what we do we add the nucleotide there

we add a nucleotide sequence the DNA

this is a DNA for

example we want to sequence this DNA

strands consecutively and let's say it

has a a g g c t c for example this is

the sequence some sequence we want to

detect we don't know the sequence though

now we add it now the idea is this DNA

to pass through this

nanop once this DNA will move through

this nanoe remember one of this

nucleotide at a time will be in contact

with this pore to pass because it will

pass like a thread just imagine a needle

and a thread thread is the DNA needle

the small pore of needle here is acting

as a nanop and the the thread is passing

through that needle once it is passing

through the needle one at a time each

bases each nucleotide base will be in

contact with this magnitude of current

okay and this the type of nucleotide

sequence will change or alter the

magnitude of current okay for example

once they in contact with adenine

it will change the

magnitude differently when it is in

contact with guanine it will change the

magnitude differently than the change

observed in case of AD c t whatever all

of these

bases they change the magnitude of

current they alter the magnitude of

current

differently so there is a characteristic

pattern of magnitude change of current

for each of these bases that we can

detect

so what we do in first place we use our

known sequence to finally get those

patterns once we know the pattern let's

say the pattern or the change of

magnitude for guanin we know that arine

know C and thine both of them we know

that then when we add an unknown

sequence and let's say we see the change

in

magnitude and that is matching with

guanin so we can tell that base is

definitely guanin after that

again if it is again guanin so the

magnitude is same guanin the magnitude

is now different and it's matching with

the magnitude of cytosine we can call it

a cyto right so we know this magnitude

for example Guan in this this this this

for example at the very beginning so if

you test this data at the very beginning

what we see we are seeing the magnitude

change it is matching with adinin so we

can say the first nucleotide here is

Adin in then we see something like this

guanin again we see something like this

see guanin again we see something like

this cytosin then we see something like

this thyine then again

cytosin by knowing this magnitudes

that's what we can detect using electric

detectors we have the detectors to

detect the change in magnitude and we

also know the signature magnitude of

each of these bases so once we know when

with that we know the unknown base that

is passing through that pole and that's

how we know the sequence of the

DNA it's easy but interesting now the

question the one more question still

there is that we add DNA molecule there

what is the guarantee that DNA will be

passing through this Poe who will drive

this DNA it's the thread if we want to

put a thread in the needle we need to

drive that so who will drive this

again solution there are two different

solution for that one is simple

electroforesis right so we can use this

beautiful technique that we have using a

lot

electroforesis that means we are again

using electric current we are using

current to take this DNA through this

pore so we are embedding this pore in

the agos gel in such a way so that the

DNA is passing through this Poe while it

is Electro foring so it's a force that

will drag this DNA thread through this

nanopore okay other hand what we can do

is we can use certain

proteins that protein

will interact with the DNA and can drag

it or guide this DNA to interact with

this four usually we've seen these cases

in case of the naturally occurring Nano

force or the membrane transmembrane

proteins or channel proteins there are

some other proteins associated with

channel proteins in some cases which are

called guide proteins which helps to

guide certain

molecules the entrance of the channel

and that's the thing we can use in this

case also to drag the DNA through this

nanoor right so these are the things

these are the two process that we can

use to drag our desired DNA molecule to

be to be sequenced through this nanopore

right

so in a sense this is nanoport

sequencing and I hope you understand

this video Once you know this video it

will be very easier to know all these

other process this is a developing field

so if you want to study more you can

watch uh you can you can go and uh go

for different uh research Publications

they are good so if you like the video

hit the like button subscribe to my

channel to get more and more videos like

this and I hope that's helpful thank you