This course introduces you to the basic biology of modern genomics and the experimental tools that we use to measure it. We'll introduce the Central Dogma of Molecular Biology and cover how next-generation sequencing can be used to measure DNA, RNA, and epigenetic patterns. You'll also get an introduction to the key concepts in computing and data science that you'll need to understand how data from next-generation sequencing experiments are generated and analyzed. This is the first course in the Genomic Data Science Specialization.
Polymerase Chain Reaction
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Do curso por Johns Hopkins University
Introduction to Genomic Technologies
1162 classificações
Na lição
Measurement Technology
In this module, you'll learn about polymerase chain reaction, next generation sequencing, and applications of sequencing.
Conheça os instrutores
Steven Salzberg, PhDProfessor
Biomedical Engineering, Computer Science, and Biostatistics
Jeff Leek, PhDAssociate Professor, Biostatistics
Bloomberg School of Public Health
In this lecture, we're going to talk about polymerase chain reaction.
A remarkable and very powerful way to copy DNA and make, in fact,
as many copies of DNA as you want to, with a really simple but powerful technique.
So how do we make copies of DNA?
We'd like to just feed our DNA to some sort of machine like a copier,
like I'm showing here, and make copies of it.
And we use this in various aspects about technology and DNA sequencing,
in genomics, all the time.
There are many, many reasons why we need to make copies of DNA.
For example, when we're doing RNA sequencing,
we need to turn our RNA into DNA and make lots of copies of that.
When we're sequencing someone's genome, we don't just sequence a single cell or
a single molecule.
All the technology we use for sequencing requires us to have many many identical
copies of the DNA molecules before we get started.
So, DNA copying is a really important, sort of, basic tool that we need for
many of the things we want to do.
So how do we do it?
So polymerase chain reaction uses a couple of simple properties of DNA and
turns it into this wonderful method for DNA replication or copying.
So recall first that DNA sticks to itself, so DNA's always double-stranded.
So here we have two strands of DNA.
We have A, C, G, and Ts on the top, and the complementary strands on the bottom.
And remember that the DNA is directional.
And the direction that it goes,
we always talk about it going from the 5' direction to the 3' direction.
So the beginning of the DNA sequence will be the 5' end and
the end will be the 3' end.
So, the fact that DNA will stick to itself is a very important property that we're
going to use in PCR.
So, another thing we need is something called primers.
So what's a primer?
So primer is simply a short sequence, usually they are 15 or 20 bases long.
They can be a little shorter, or a little longer, but it's a sequence of
DNA bases that's complementary to the DNA that we want to copy.
So, here I'm showing an example with two primers, one in green, and one in blue.
So along the top strand, the forward strand, we call that, there's a primer you
see at the beginning which is in green, which is the reverse complement that is
the matching bases of the top strand of DNA, right at the beginning.
And on the other strand we have a different primer going from the other end
that is shown in blue.
That's the reverse complement of the reverse strand going
on the reverse strand.
So it goes 3' to 5'.
So these primers, if I were just to mix these primers with this DNA sequence,
they would stick to it, because they're complementary to it.
So how does PCR work?
So we start with some DNA and some primers.
And let's not worry how we get those primers.
But just if you're curious,
you can actually easily generate any primer you want of any length.
You can just order it from a company these days.
And, in fact, you can order a mixture of all possible combinations of DNA bases,
of say eight bases or even a little longer so you have primers even for unknown DNA.
So what do we do with that?
Well, now we're ready to start the process of PCR.
We're going to heat up the mixture gently.
What happens when you heat up DNA is it melts or
rather [NOISE] the two strands separate from one another, they fall apart.
So you see, I've just moved them apart physically on the screen here.
So if the mixture is hot, then the primers won't stick to the DNA and
the two strands of the DNA won't stick to each other, either.
Then we cool it down, or anneal it and cool it down gently.
And the primers will stick to the DNA,
and they tend to actually stick to the DNA before the two strands find themselves.
Because the primers are small and they can float around a little bit more easily.
So we cool it down.
And we let the primers stick to our DNA.
And if we kept cooling, eventually the DNA would cool back to itself.
But we don't wait that long, so we also add another mix.
We need something now to copy our DNA.
So we need a copier molecule.
Fortunately, nature has provided us with a very good copier molecule
called DNA polymerase.
It's what all of our cells use to copy their own DNA.
So we can synthesize that and make large quantities of it and
add that to the mixture as well.
So, DNA polymerase acts in the following, very straightforward way.
It looks for a place where the DNA is partly single stranded and partly
double stranded and it grabs on to the sequence right there and starts to copy.
So this DNA polymerase here will notice both of these two primers are now attached
to sequences that are single stranded, except where the primers have stuck.
So, the DNA polymerase will go and it will find those sites,
and it will start to fill in the missing sequence starting at the primer.
So that's the property of DNA polymerase that we really need,
and there are many polymerases.
Every living organism on the planet actually has one.
So, we don't actually use the human polymerase for this process,
but it doesn't matter.
You can use, in theory, any DNA polymerase to do the copying.
Although, we do need to specialized one for PCR.
So the result after doing this is that, if I show you here,
after one round of copying, I'll have completely filled in the sequence across
the top from my green primer.
As you see here, and then another polymerase, assuming I added lots of
polymerase molecules, another polymerase will have filled in the sequence
of the other strand across the bottom, starting with the blue primer.
Now, I also needed a mixture of As, Cs, Gs, and Ts, the raw material to make DNA.
So, I didn't say that yet, but in addition to adding my DNA polymerase,
I'm going to add raw As, Cs, Gs, and Ts in large quantities.
So that the DNA polymerase can incorporate them into the new double-stranded
DNA that it's creating.
So after one round of PCR, if we let things cool down, these two strands will
stick together, and we've now created two strands where we only had one before.
So we can just repeat that whole process.
And if we repeat the whole process, we get four strands.
And a very important property of PCR, the reason it's called a chain reaction,
is that with each round, we double the amount of DNA we had before.
So, very quickly, after just a few rounds, you go from just one molecule to many,
many molecules.
Or many, many copies.
So, typically we'd repeat this for 30 cycles or more.
And if you do the math, 2 to the 30th is about two billion,
so you can take one molecule and turn it into billions of molecules
after just a few dozen cycles of polymerase chain reaction.
So let's look at a little cartoon of how this works, just to drive home the point.
So we start with melting or denaturing the DNA at 94 degrees Celsius.
So here you see a double strand of DNA with some polymerase.
You'll notice some little arrows floating around.
And primers are shown as the little short fragments of DNA that are floating in
the solution here.
And as we heat it up,
you'll see the two strands of DNA in the middle start to denature.
They're falling apart.
So, you see they're melting apart there, and
eventually they're completely separated.
Now, an important part of PCR is you have to denature for long enough for
the two strands to fall apart.
But it doesn't take long at all.
We're talking only a few minutes to do that.
Then you cool it down to 54 degrees, and
at that temperature the primers will stick to the DNA, as you see here.
And the polymerase can then find these double-stranded pieces and
start to fill it in.
So here you see the two polymerases in this picture are starting to
fill in the existing strand and create double-stranded DNA.
So they'll just walk along until they get to the end of the molecule, and
then they'll fall off.
So now we've created two complete copies, and
we do that at a slightly higher temperature of 72 degrees.
And then we simply repeat the whole process to make another copy.
So in summary, the PCR recipe is the following ingredients.
You need some DNA that you want to copy,
it could be any DNA at all from any living organism.
You need primers, you need DNA polymerase,
a special copier molecule, and you need lots of A's, C's, G's, and T's.
And then the way you execute the recipe is you melt it at 94 degrees.
You cool it down to 54 degrees, then warm it back up to 72 degrees.
And then simply repeat.
So all you have to do is mix those ingredients in a single mixture and
go through this process of heating and cooling, and
the rest of the reaction takes care of itself.
So that's why it's called polymerase chain reaction.
It uses the DNA polymerase to cause this chain reaction,
this explosion in the number of copies of your DNA.
And it all happens fully automatically just using the properties of DNA itself,
which hybridizes to itself, and of DNA polymerase, which copies DNA.
So this is such a clever and powerful idea, and was so revolutionary and
had such a great impact on the field, that not that long after it was was discovered,
the Nobel Prize in Chemistry was awarded for invention of DNA to Kary Mullis.
And this is just a picture from the Nobel site describing his Nobel lecture.
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