Nanopore sequencing technology
Summary
TLDRThis 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
- 𧏠Nanopore DNA sequencing is a technique that uses an electric current to determine the sequence of nucleotides in DNA.
- đ The concept of nanopore sequencing has been around since 1955 but continues to be developed and refined.
- đ The process involves a very small pore, with a diameter close to 1 nanometer, through which the DNA must pass.
- đ§ The pore can be made from natural materials or artificially created, such as on a silica plate.
- đ The pore is lined with conducting molecules, such as iron particles, to ensure it can carry the electric current needed for sequencing.
- đ The sequencing relies on detecting changes in current magnitude as different nucleotides pass through the nanopore.
- đŹ Each type of nucleotide (adenine, guanine, cytosine, thymine) alters the current magnitude in a unique way, creating a characteristic pattern.
- đ By comparing the detected patterns with known sequences, the unknown DNA sequence can be determined.
- 𧔠DNA is driven through the nanopore using methods like electrophoresis or with the help of guide proteins that interact with the DNA.
- đŹ Nanopore sequencing is a promising technique with potential applications in various fields, though it also faces some challenges.
- đ 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
đ 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.
đ 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.
đ 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.
Mindmap
Keywords
đĄNanopore Sequencing
đĄElectric Current
đĄNucleotide
đĄPore Diameter
đĄTransmembrane Protein Channels
đĄSilica Plate
đĄConductivity
đĄElectrophoresis
đĄGuide Proteins
đĄCharacteristic Pattern
đĄDetectors
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
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