slide 11:

In this section we're going to talk about PCR. PCR is definitely one of the most
important tools in molecular biology and is heavily used for diagnostics nowadays.
But to really understand PCR we have to take a step back. We have to look at the
enzymes that enable PCR. So in a way it's the DNA

polymerases. There are different types of DNA polymerase, some involved in

replication of organisms, and these kind of tend to be harnessed for molecular
biology for PCR. Repair, which there are some biotechnological applications
emerging from them, but also reverse transcription. So these are DNA polymerases
that can synthesize DNA from RNA. So for instance, when you have your COVID test,
COVID being an RNA virus, you need to convert that information from RNA to DNA and
then amplify it. So that involves the reverse transcription of the information to
DNA and then the amplification, which is often done by a separate enzyme. DNA
polymerases are divided in different families, but they have a common mechanism,
which I'm going to describe in a second. They also have common structural elements.
So you see here kind of the three subdomains of the polymerase domain, the fingers,
the palm, and the thumb, and that's common to most polymerases. So you can really
see what's often described as a right-hand topology because you have sort of the
palm where the active site sits here with fingers controlling the rate of
nucleotide incorporation and the thumb keeping hold of that nascent duplex. It also
controls exactly whether the DNA stays in the active site or get moved to
additional domains, but that's beyond the scope of this course. Different families
have different domains, and that can be harnessed for application. And in that
sense, maybe this is the key message of biotechnology. You learn biology, you learn
how biology operates. Biotechnology means taking that knowledge and making
applications on top of them.

And that will become clear as we go further into PCR. In terms of mechanism, all
polymerases rely on exactly the same mechanism. So they take these nucleotide
triphosphates, and you can look at them as an activated monomer. So if you think
that the beta and gamma phosphates are a living group activating the phosphate,
what the catalytic site of the enzyme does is activate the hydroxyl to permit an
attack onto the alpha phosphate. And this is also sort of metal ion catalyzed. The
result is that pyrophosphate is removed from the active site, further breaking down
into phosphate, and the substrate gets incorporated into the nascent chain. You
will see here that the incorporation is always at the 3' end of the molecule, and
therefore you have a synthesis always directional, so always 5 to 3, because it's
the 3' end that extends. And that's one of the reasons why we also always describe
DNA in a 5-to-3 orientation. Once we come to specific enzymes, I mentioned how
there are seven families.

I'm going to show examples of three of them. So the A family polymerase, which can
have roles in replication or repair, but best known for biotechnology is TAC. So
the Thermos Aquaticus DNA polymerase 1. is by far the most used enzyme in sort of
biotechnology PCR. What he allows, kind of what allows it to be used in PCR is this
thermostability. So there are more thermostable enzymes than TAC, and that can
bring advantages. But TAC is thermostable enough for it to be used in
biotechnology. It has a very high affinity to DNA, and that translates into two
things. First, they're very good detection, so you can detect even single copies.
And hence, if you're looking at forensic science, TAC is a very good tool in
forensic science for detection of DNA trace. The second consequence of this higher
affinity to DNA is that once it binds, it's capable of putting multiple
incorporations. So for TAC in particular, it tends to put about 20, 25
incorporations per binding event, comes off, rebinds, another set of incorporations
and so on. And that's how you get the extension. It is a high-fidelity enzyme, so
that's your error rate per incorporation. In the real world, that would be roughly
equivalent to writing two pages of text with only two typos. But the speed is also
quite important. So TAC is capable of synthesizing one kilobase of DNA per minute,
which would translate right in those two pages in about 10 seconds. What the A
family domain, sort of family-specific domain in TAC allows is this 5 to 3
exonuclease. So it's almost a sort of read-ahead or remove blockages downstream as
you amplify. And I'll give you, I'll come back to that, because that is the basis
of TACMAN assays. For those of you who are going to go on to do lab work, it's also
interesting to know that TAC and other A-family polymerases always leave an A or G
overhang. And that can have consequence depending on what the next step in the
reaction is.

As a counterpoint, these are the B-family polymerases, which again can also have a
role in replication or repair. And one of the more common ones is PFU, or
pyrochoccus furiosus DNA polymerase. So if you compare its stability to TAC, you
can immediately see that it's much more stable. So certainly much better suited for
very long PCRs, but it's less processive. So it has a lower affinity for DNA, and a
TICH binding event incorporates fewer bases. partly because of that partly because
the family specific three to five exonuclease domain it has a much improved
fidelity and in fact that can be engineered even further so at present commercially
available enzymes have error rates roughly tenfold lower than that and that's
essential for application because it has a lower binding affinity to dna it also
means it's not as good for when template is limiting and that's kind of that's a
consequence of that in terms of application it kind of is worth remembering the b
family polymerase is leave a blunt end on your dna so again so if you think in
terms of how you're going to clone it later if you don't have a cut site within the
the fragment you've just amplified you then have to think how you're going to clone
it. And PFU-amplified DNA can be cloned by the blunt end. Yeah, I didn't highlight
here, but this is there are two domains in that space. In fact, sorry, here is your
N-terminal domain and then the exonuclease domain. Reverse transcriptase,

as I mentioned, are enzymes that can start from RNA template, but then synthesize
DNA. And that's very important for RNA detection. That is in comparison to TAC and
PFU, who take DNA template and make DNA product. An additional class of polymerase
that is worth mentioning are the terminal transferases, or TDT. So these enzymes,
they have no requirement for template. So they don't have to have a template. They
only need a DNA with a free 3' end, and they start synthesizing a random sequence
depending on the deoxyribonucleotides available. That will come when we start
talking about applications. In terms of the RT enzymes, you can still see here kind
of the finger, palm, thumb domains. and in particular the MMLV is the workhorse in
molecular biology. It has been thermostabilized so even though it started as a
mesophilic enzyme it can now tolerate higher temperatures and that's very good for
biotechnological applications because it means you can run the reaction at higher
temperature and that has a consequence of removing secondary structure from your
RNA, which are now easier to get through. The natural form does have an RNA's age
function, which means it synthesizes DNA, but at the same time, these domains start
degrading the original RNA, but that can be engineered out. So then enzymes are
capable of synthesizing DNA without destroying the RNA message. but when it comes
to PCR

we go back to just the DNA polymerase or rather a DNA dependent DNA polymerase and
you know if you have a primer in DNA you will extend towards the 3 prime so here is
your extension if you were to put a second primer binding the complementary strand
but facing the other way so here is your 5 primes just to mark them out they would
extend this way in a single cycle that doesn't look powerful but here's where pcr
becomes powerful so in i'm highlighting here your binding sites for the two

primers in a initial cycle yes you're going to have these sequences which
essentially extend too far. So you can see here your blue prime extending beyond
the red end and a red prime extending beyond the blue end. So it still doesn't look
useful. But now you have twice as many copies, even if they are too long. When you
again repeat them, you can again start overshooting in some places, but you start
also getting nearly correct fragments. So here you can see that one strand is
already the correct fragment. We're only amplifying from red to blue, but the other
is still too long. From cycle three, you start getting molecules which are
precisely from red to blue and nothing else. And this is the power of this
logarithmic process Because as you repeat it further and further, this correct,
kind of perfectly from red to blue molecule, it starts to take over the population.
And after 30 cycles, you have over a million times more molecules of the expected
correct fragment. I have put on the slides a link. this is not always easy to see
how PCR gets started but it's an important concept to be able to understand what
you can do with it so I recommend you look into more detail

there are many different PCR techniques so for instance the inverse PCR touchdown
PCR and gradient PCR and these modifications of the technique get developed to
address a certain need. So in the case of inverse PCR, you can imagine you have,
let's say, you have your plasmid, and you want to amplify a small fragment. That's
PCR. But sometimes to make it clear that you're not amplifying the small part, but
in fact you're amplifying kind of the entire vector. so your PCR here is trying to
amplify the entire vector, you call it IPCR. There's a historical name behind the I
for inverse, but we're not going to go into. Touchdown a gradient, look at the
reaction conditions, and they try to change how the reaction conditions change or
are set up. Touchdown to enhance specificity, so you decrease your annealing
temperature as you go between cycles and gradient is a way of optimizing the
reaction so you carry out separate reactions each at a set temperature set
annealing temperature and you find which one works best the ones we're going to
cover more in more detail and are more important for biotech are RT-PCR and Q-PCR
So, of course, as I mentioned already, if you have RNA, you can't replicate RNA. So
you have to convert it to DNA and then amplify it. So that's the RT-PCR. QPCR is a
way of trying to monitor in real-time monitoring the amplification. And that gives
you an insight into how many copies you have of each.

I mentioned here digital PCR because that's coming up but we're not going to
discuss it in this course when it comes to real time PCR so you have your PCR, you
have your amplification and one of the ways to do it is to add a dye so cybergreen
or ethidium bromide is used to be used they are fluorescent molecules but they have
very low fluorescence once they intercalate into DNA they can have up to a thousand
times more fluorescence and that's the key thing because at the correct
concentration when you don't have dna so at the beginning of pcr you have no signal
or you have very low signal as pcr takes place and you start to have multiple
copies of your fragment made you start to get more signal because you have now more
dna for these fluorophores to intercalate and to generate signal.

And the ideal result is this. So in all cases, you can see here these are relative
fluorescent units. You always start with a low signal. As PCR starts to take hold,
you see the signal come up, become really truly an amplification step, and then you
start to saturate you start to lose the amplification if you do the quantitative
aspect you can really sort of in theory you can really see how fast these molecules
amplify and if you mark down the number of cycles until we it was required to get
into the amplification stage so this is the kind of the definition of the threshold
cycle you can start to make assumptions as to how many copies you have of a certain
of a certain molecule in a certain sample and this can be important so if you want
if you want to discover not necessarily just what virus you have but what viral
load you have this becomes an interest an interesting technique but qpcr is not
only done with the fluorescent dye there's an alternative way of doing it and this
is through the use of

TAC. So I mentioned before how TAC can have this exonuclease activity and you can
try and exploit it for the diagnostics. So the experimental setup is somewhat like
this. You have your DNA that you want to amplify. You want to check how many copies
you have. You have your primers shown in red and you want to carry out the
amplification reaction. What you then add is a second internal primer that you see
here shown in blue. But you put in it a fluorophore and a quencher. So because
fluorophore and quencher are too close in this molecule because they're bound to
the oligo, you see no signal from the fluorophore. However, and this is how TAC was
exploited for this, as the polymerase is extending the DNA and encounters that
oligo, this 5 to 3 exonuclease activity, it starts to break down the oligo. And
that results in fluorophore molecules escaping away from the quencher, which are
now separate in solution. But it also means that you can now detect that
fluorophore. So as the cycles go, you start to get accumulation of the fluorophore,
and you start to generate the data. the data in principle looks exactly like this
one because this will be a fluorescent measure and you can see what happens the
difference is that when you have a fluorophore that intercalates you detect every
dna that you synthesize whether it's right or not in the case of the tachman assay
Because you have the oligobinding at a specific position, if the DNA being
amplified does not have that sequence, you don't see it. So you can have a greater
specificity in assay.