Lectures okay for you all?
Okay, please.
I'm already eliminating a lot of details.
Okay, since this is a lecture and it's recorded, I'll say it here.
Yes, the details of the bio part is not tested.
I'm concentrating, you can observe that I'm concentrating on the chemistry.
So that will be what is tested.
And I think the tutorial questions will give you an idea.
The questions that I asked are chemistry related.
Okay, so all those like as I mentioned in the last lecture, how the experiments were
developed, you know, that are covered in the history section. Of course, you don't
have to remember the details of the story.
Okay?
, let's get started.
So, in the last lecture, we discussed protein synthesis.
And we said that the interest in protein synthesis arose after the discovery of how
DNA replication occurred.
So, knowing how DNA replication occurs, the next question is naturally that how this
protein made in the human system or in cells.
, so we covered the whole stuff on how protein was synthesized.
Then we said that the way protein is synthesized is that the DNA would first make the
RNA.
, and the process is known as transcription.
Therefore, after which the RNA in eukaryotes, the firstly the RNA that is being made
is the pre-messenger RNA.
, or the pre-mRNA where M stands for messenger.
And then after that, it will be processed then to give you the mature RNA which then
leaves the nucleus and enters the cytoplasm where it will hook onto a ribosome to
facilitate the synthesis of proteins.
And we said that the synthesis of proteins is known as translation.
So, how does this nucleic acid know what kind of amino acids to put in to make up a
protein?
So, this work was done by Khorana and Nuremberg.
So, in their work to decipher the entire genetic code, they did it by making triplet
nucleotides and then match them to the amino acids.
, so today we will look at how Khorana synthesized the triplet nucleotides.
, so before that, this is how they deciphered and showed that there will be three
nucleobases that makes up amino acid. And this triplet is known as a codon.
, so today we will look at the second application that came out from the discovery of
DNA replication.
So, let's discuss the oligonucleotide synthesis.
The discovery of how DNA is synthesized by nature inspired chemists to accomplish
this synthetically.
So, one such chemist was Khorana, who in his efforts to decipher the genetic code,
developed a method to synthesize simple oligonucleotides.
The original Khorana's method involved the direct formation of the phosphate ester
by attaching a three prime hydroxyl group on an activated phosphate group.
This phosphate ester chemistry mirrors the enzymatic reaction catalyzed by DNA
polymerase, as it also synthesizes the chain from the five prime end to the three prime
end.
So, this is the chain that Khorana has with the three prime hydroxyl group free. And
so, the three prime hydroxyl group will then attack a nucleotide.
So, in his method, the synthesis also occurs from five prime to three prime end, just
like the way the DNA polymerase synthesizes the nucleotides or the oligonucleotides
in nature.
The difference is that the three prime hydroxyl group of, the difference is that if you
look at the monomer, the three prime hydroxyl group is protected as an acetate ester.
Why do we need to protect it? This is to prevent the monomer from reacting with itself.
After each coupling sodium hydroxide is used to hydrolyze both the acetate and the
ester, and the phosphotriester to the phosphodiester.
Firstly, you have a phosphotriester here.
So, sodium hydroxide would hydrolyze this acetate group.
It would hydrolyze this functionality to this ester in the phosphotriester, and therefore
we get to a phosphodiester, which is exactly what you see in nature in the
oligonucleotide chain.
So, a major disadvantage of the Khorana’s method, or this chemistry, is that it uses
hindered secondary alcohol, or secondary hydroxyl group, as a nucleophile.
And thus, the use of each coupling step is not high, which makes purification a problem.
So, since a phosphate reacts sluggishly, and inefficiently with a secondary alcohol,
would a phosphite perform better?
So, to understand the difference in reactivity between a phosphate and a phosphite,
we would need to consider the effects of the lone pairs of electrons, because that's
the only place where it's different between a phosphate and a phosphite.
The oxygen lone pairs donates into the empty orbital on phosphorous, thus competing
with the incoming nucleophile. So, that's why it is kind of not so reactive.
So, since, now, so if we look at a phosphite, a protonated phosphite has one less
oxygen substituent than a protonated phosphate, as shown here.
So, the protonated phosphite is much more electrophilic, and the reaction with a
nucleophile would thus proceed faster.
Thus, the key step in the mechanism of the phosphite ester is the formation of the
protonated phosphonium intermediate.
Under acidic condition, phosphite would be converted to the phosphonium
intermediate, which undergoes nucleophilic substitution 10 to the power of 12 times
faster than a phosphate.
And the mechanism that I've drawn here shows you how to synthesize a phosphite. A
phosphite can be made from phosphorochloride.
Under acidic conditions, the acid will protonate the phosphorous, giving you a
phosphonium ion, and then the acetate counter-ion will then abstract the hydrogen.
This trigonal bipyramidal structure, this intermediate is not very stable, and the
chlorine can leave because the chlorine is a good leaving group.
It leaves as a chloride ion to generate this phosphonium ion, which can be
deprotonated to give you the phosphite.
So, now, Robert Letsinger, shown here, was the first to recognize that a phosphite is
more reactive than the corresponding phosphate acetylating agent.
This led to his team developing the phosphite method for oligonucleotide synthesis.
Thus, here, what we have is a 2-chlorophenylphospho-dichloro-dich, let's call it
Compound 2.
This compound reacted rapidly with the protected thiamidine derivative 1. This is
thiamidine here. Okay, so it reacted rapidly with the protected thiamidine 1. You can
see the protection on the 5-prime hydroxyl group.
So, and this reaction occurs at low temperature.
If you look here, 1 refers to the reagents and conditions here.
It occurs even at minus 78 degrees. So, at minus 78 degrees, the reaction occurs very
rapidly. That means this Compound 2 is very reactive, ?
So, the reaction occurs to give the putative phosphorochloride intermediate 3, which
was then reacted with a 3-prime protector.
So, this is the 3-prime hydroxyl group, and this 3-prime hydroxyl group is protected, ?
So, it's called a 3-prime protected thiamidine derivative 4 to give the fully protected,
to give the fully protected here derivative here intermediate.
Let's call this, this is an intermediate, , which was not isolated, ? So, this is not isolated.
It's not very stable, but it's then treated in situ with iodine, as shown here, number 2.
So, it's treated with iodine, shown here, in water and THF, , and in the presence of a
base.
So, this intermediate is treated with iodine, water, in the presence of a base, such as
2,6-lutidin, ?
Now, what happens in this condition? Oxidation will occur to the phosphide to give
you the fully protected dinucleotide phosphate 5.
The major advantage of this chemistry was the significant reduction in time.
Time for coupling due to the highly reactive nature of the nucleoside
phosphonochloridate 2 intermediate, ?
So, this reaction was very fast, , and so was this reaction, ? It could occur at low
temperature.
So, the major advantage of this chemistry was the significant reduction in time
required for coupling due to the highly reactive nature of the nucleoside
phosphomonochloridate intermediate, as shown here, that's compound number 3.
Now, LetSinger, made another major contribution to the field when he introduced solid
phase chemistry to oligonucleotide synthesis.
So, if you look at this, , the sugar here is protected as a protecting group, , the 3 prime
sugar.
So, instead of using a protected nucleotide derivative 4, the protecting group was
replaced with a solid support, as shown here, , and this solid support is an insoluble
polystyrene bead.
So, one nucleotide is joined to the nucleotide on the solid support.
So, you have a nucleotide here and it's joined to the nucleotide on the solid support,
followed by oxidation, as shown here, and this is the oxidation that converts the
phosphide to the phosphate, and then followed by deprotection of the sugar, right?
So, this protection group that is shown in blue is now removed, so you get a free
hydroxyl group, ?
So, after each addition, when you reach to this stage, right, that means it is one chain
longer than what it started with, right?
So, when you first start off with the nucleotide that's on the solid support also has a
free 5 prime hydroxyl group and it reacts with the monomer.
So, now you have this and now it's ready to react with another nucleotide, , and then
the reaction continues and continues and the chain grows longer and longer, okay?
So, at the end of the synthesis, the oligonucleotide chain is then cleaved from the
insoluble polymer support and purified.
So, hence, in solid phase chemistry, what we observe is that no purification occurs
until the fully assembled oligonucleotide was released from the solid support.
Now, this does make the synthesis less laborious.
Now, Marvin H. Caruthers was a member in Letsinger's research group.
So, when he began his own independent research, one of the things that he did was
to refine the phosphide method.
He developed a stable compound shown here which is a phosphoramidite.
So, he developed stable phosphoramidite building blocks.
A phosphoamidide is a derivative of phosphide where one of the OR groups is replaced
by an amino group.
So, to generate the phosphoramidite, Caruthers exchanged the chloride leaving group
with an amine, ?
So, I think I showed you the results already.
So, you can propose a mechanism for this reaction, ? And here it is. And if you look at
it, this mechanism is very similar to this mechanism, ?
It uses the same chemistry. So, just to quickly run through it.
So, you start off with this, with the chloride leaving group. And then you treat it with
an acid. The phosphorus gets protonated. It's a good electrophile.
The amine is a nucleophile, attacks the phosphorus, creates a trigonal bipyramidal
intermediate which is not very stable.
So, of course, the acetate from the acid can deprotonate the amine to give this. The
chlorine is a good leaving group.
So, the chlorine leaves as a chloride and then returns to deprotonate the acid to give
you the phosphoramidite.
So, the synthesis of a phosphoramidite is very close to the synthesis of the phosphide
that we saw in the earlier slide which I showed you, okay?
So, exchanging the chloride for an amine significantly changed the properties of the
molecules.
So, since phosphoramidite are stable and they could be made, this means that they
could be made in advance.
And they could be isolated as stable solid and stored until needed.
So, the mechanism of the phosphoramidite coupling is similar to the phosphide
coupling.
It involves first the protonation of the phosphoramidite with a mild acid to generate
the activated phosphonium salt which subsequently reacts with the 5 prime hydroxyl
of another nucleotide or nucleoside.
Then removal of the acid here, this is an acidic proton.
Removal of the acidic proton on the 5 prime hydroxyl by the neighbouring base, this
is an amine, this is an sp3 amine, so it's basic.
And so, it is just in close proximity to this acidic proton.
So, this base can abstract the proton to generate this ammonium compound. So, this
is now a good leaving group because it can leave as a stable amine.
So, deprotonation of the phospho intermediate gives the resulting phosphide which is
extremely sensitive to water.
So, this is extremely sensitive to water.
However, oxidation to the corresponding phosphate provides a stable linkage. So, the
phosphite is unstable.
So, that was the problem Carruthers saw when he was in Letsinger's group.
So, when he started his own research, he developed the phosphoramidite chemistry
and phosphoramidite building blocks because phosphonamidites were stable. They
can be made and they can be stored.
In contrast to phosphites, they are not very stable and once you make them, you have
to use them immediately. So, that's more problematic. It's always easier to handle or
to work with stable molecules.
So, Carruthers also applied the phosphoramidite method onto solid-phase synthesis of
oligonucleotides.
Today, this combination of solid-phase synthesis and phosphoramidite building blocks
is the basis of modern oligonucleotide synthesis.
So, let us now discuss how oligonucleotides are synthesized today.
If we look at a nucleoside, as shown here, we will notice that they have several reactive
groups like the primary hydroxyl group, the secondary hydroxyl group, and this amino
group here.
So, since attaching a nucleotide to a growing oligonucleotide chain involves a couple
of steps, these reactive sites must be protected so that they do not participate
unnecessarily in the oligonucleotide synthesis because if they do, then you get a lot
of side products and a very, very messy reaction.
So, different protecting groups are used to protect the amino and the hydroxyl groups.
The reason is so that they can be selectively deprotected at a specific time.
Thus, a modern phosphoramidite building block has three essential features.
An acid-labile dimethoxy trityl protecting group that is on the 5 prime hydroxyl group.
That is a relatively stable cyanoethyl phosphoramidite.
And a protecting group on the nucleophilic amino groups that's on adenine, cytosine,
and guanine.
Thymine requires no protection because it does not have a hydroxyl group or an amino
group on it.
Adenine and cytosine are protected as benzoyl groups, whilst guanine is protected as
a dimethyl formamidine.
DMT, dimethoxytrityl, is commonly used to protect the hydroxyl group in nucleotides.
Why? Because under acidic conditions, the DMT group are more easily removed than
trityl groups.
So trityl groups have only three phenyl groups. There's no methoxy on it.
So dimethoxytrityl, they have two methoxy groups on it.
Versus trityl, which is just three phenyl rings without the methoxy groups.
So the methoxy groups will make it easier to remove the protecting groups under
acidic conditions.
Why is DMT more easily removed than trityl?
This is because the dimethoxy groups stabilize the cation.
So once they leave, what you get is a cation, C plus here.
And the methoxy groups can, the electrons can move into the ring and to stabilize the
plus charge.
So what's generated as a byproduct is more stable and so that the reaction proceeds
faster.
That's why dimethoxy groups are removed more easily than trityl groups.
And if you look at this reaction, is it an SN1 or an SN2?
This reaction, the removal of the dimethoxy group, how does it proceed?
I'm already giving you a hint, SN1 or SN2. SN1, right?
So first, the acid under, remember, dimethoxy groups are acid labile.
So under acid conditions, when I say when there's an acid, as I said before, always
look for the base.
So the base is the oxygen here.
So under acidic conditions, this oxygen will be protonated. So now it's a good leaving
group. So this will then kick it off to generate the carbocation.
So the dimethoxy groups are removed through SN1 reaction with tri-, and what acid
do we use? We use trichloroacetic acid. Why do we use trichloroacetic acid?
Because this pKa is about 0.8, which makes it about 100,000 times more acidic than
acetic acid.
So in addition to the dimethoxy trityl cation imparts a deep orange colour to the
reaction mixture.
So this is the second reason why we use dimethoxy trityl
It colours the mixture, and this colouring has an absorbance at 498 nanometres, which
can be used to, so you can use spectroscopic methods to estimate the overall yield of
this deprotection reaction.
, so these are the two reasons why we use dimethoxy trityl for the protection of the
5-prime hydroxyl group.
Okay, now let's go through the step-by-step procedure of oligonucleotide synthesis
using the phosphoramidite synthesis, or phosphoramidite method.
So the first step is to attach the protected, this is your protected deoxy nucleotide to
a solid support via an ester linkage at the 3-prime hydroxyl group, so this is your 3prime hydroxyl group.
, so this is the 3-prime hydroxyl group of your protected deoxy nucleotide.
So after that, the DMT protecting group is removed by treatment with dichloroacetic
acid in dichloromethane as a solvent.
Following the tritylation, the solid supported nucleotide is ready to couple to a
protected deoxy nucleoside, containing a phosphoramidite group at the 3-prime
position.
The reaction is performed in polar, a protic solvent such as acetonitrile, because
acetonitrile is a good solvent for nucleophilic displacement reactions.
And another reagent that is present for this reaction is this compound known as
tetrazole, which is approximately of the same acidity as acetic acid, it has a pKa of 4.9.
So with the coupling step accomplished, what we have here is now a phosphite.
The phosphite is then oxidized to a phosphate by treatment with iodine in aqueous
tetrahydrofuran in the presence of 2,6-dimethylpyridine.
Then the cycle, what we have now is this.
So if you look at this, this is very similar to this compound, but now on the solid
support you have two nucleosides instead of one.
So then with this, what you do now is to remove the DMT, and once you remove the
DMT, you have the 3-old hydroxyl group on the 5-prime hydroxyl, then it reacts with
another protected phosphoramidite, and then the reaction continues and goes on, and
then the oligonucleotide chain grows longer and longer.
So the synthesis continues until the desired sequence has been constructed.
So, the final step is the removal of the protecting groups on the bases.
So, and how is this done? This is done via amide hydrolysis.
The elimination of the cyanoethyl group and the cleavage of the ester bond holding
the nucleotide to the solid support, they are removed under this condition with amines.
So all these are done at the same time.
So the protection of the cyanoethyl groups and those on the bases, they are all
removed in one reaction using amines or aqueous amines.
So why do we use ammonium hydroxide? So ammonia in water is ammonium
hydroxide. Why don't we just use sodium hydroxide?
So the advantage of using ammonium hydroxide over sodium hydroxide is the ease of
removal because ammonia is a gas, it's volatile, it can be removed very easily, whereas
sodium hydroxide is a solid and you have to treat it, it's harder to remove.
For a gas you can just blow it off.
So now let's look more closely at the reaction between the phosphoramidite and the
tetrazole.
So I have not mentioned, why do we need tetrazole in the reaction?
So when the appropriate nucleoside phosphoramidite, as shown here, is mixed with
the tetrazole in acetonitrile, the deoxy nucleoside phosphoramidite becomes
protonated to form the phosphonium ion, which is a strong electrophile.
And so the byproduct is the tetrazole anion.
Now this tetrazole anion that is generated can react with this phosphorus, because it's
a plus charge and this is a nucleophile, to form a pentavalent intermediate.
Protonation of the isopropyl group, because this is acidic, it's like acetic acid, right?
pKa is 4.9, so this acidic proton here and you have a base here.
So once you have an acid and a base, the acid and a base will react.
So this acid will then protonate the isopropyl amine to form a second molecule of
tetrazole, , intermediate, cation intermediate, and then you have the protonated
ammonium salt, okay?
So this is a good leaving group, so it leaves to generate this intermediate, right?
Now finally, deprotonation of the phosphonium.
So this amine is basic, this hydrogen is acidic, because it's attached to a phosphorus
that has a plus charge.
So the base can abstract the hydrogen, so resulting in the deprotonation of the
phosphonium ion to generate a highly reactive phosphate suitable for oligonucleotide
synthesis.
Now, so if you look at this, why did we go through such a long process to just to
convert this, , phosphoramidite to this phosphoramidite?
If you look at these two, why is this stable and why is this less stable?
Everything else is the same, the phosphorus is attached to two oxygen and a nitrogen, ,
everything else is the same except here, , this is stable, but this is not stable, why less
stable, ?
This is more electrophilic, more reactive, why is that so?
The reason is because, look at this, this is an sp3 nitrogen, this is an sp2 nitrogen.
Sp2 nitrogens, they are less electron donating than the sp3 nitrogens.
So this will make the phosphate here, this phosphate more electrophilic than the
starting phosphate.
So having a more electrophilic phosphate, , the reaction can proceed more readily.
So the distinctive feature of the phosphoramidite method is the ability to convert a
relatively stable deoxyribonucleotide phosphoramidite to a highly reactive phosphite,
which enables the coupling reaction to give almost quantitative yield within a few
minutes, so that's how reactive it is.
Now, let's discuss the mechanism of the oxidation, , of the phosphite to a robust
phosphate, which uses iodine in aqueous tetrahydrofuran.
The reaction mixture includes 2,6-dimethylpyridine, and what is it used for? It serves
as a proton sponge, , to soak up the acids.
The phosphites are easily oxidized under relatively mild conditions, ?
So if you can see here, , so you can, the reaction occurs mildly, you don't have to heat
it, it occurs at room temperature.
So the phosphorus here, right, acts as, will react with the iodine, right?
So the phosphorus acts as a nucleophile because it has lone pairs of electrons.
It will abstract this iodine, and the two electrons on the iodine-iodine bond goes to
this iodine, so you generate an iodide, and you get to this phosphonium salt, where
the phosphorus has a plus charge, and remember the reaction occurs in water, there's
water there, the water serves as a reactant.
So the water acts as a nucleophile, reacts with the phosphorus to form this
intermediate.
The 2,6-dimethylpyridine acts as a proton sponge, so then these are protons, these
are acidic protons, so it abstracts the two acidic protons to generate this, ?
So once the second proton is abstracted, the O- will form a double bond with the
phosphorus, so that's how you get to the phosphate, right?
So lastly, let's see how we can eliminate the cyanoethyl group.
So this is one of the groups that has to be removed at the end of the reaction, ?
So we already said that at the end of the reaction, all the protecting groups on the
bases, , and the cyanoethyl groups, they are all removed using ammonium in water,
or ammonium hydroxide, ?
So the reaction condition is concentrated ammonium hydroxide with a slight heating
to about 65 degrees.
So this scheme shows how the cyanoethyl group is eliminated.
The ammonium hydroxide is a base, will abstract the acidic proton on the carbon next
to the nitrile group.
A nitrile group is an electronegative group, ? Its electronegativity is close to that of a
nitro group, right? So that's how electronegative it is.
So the proton that is on the carbon that's next to the nitrile is acidic.
So in the presence of ammonium hydroxide, the proton would be removed to generate
a carbanion.
This is a leaving group (phosphate group), and so with this carbanion, it can kick off
the leaving group, , and generate the phosphate anion, which is the product that you
want.
The reaction occurs via E1cB reaction.
Now, so let's move on to the third application that came out of the discovery of DNA
replication, ?
So the discovery of how DNA replication occurs has also not only led to the
development of oligonucleotide synthesis.
The discovery of DNA replication has also revolutionized genetics.
For example, this discovery by Manselson and Stahl, , which showed that heat can
cause DNA strands to separate, , is now the basis of an important technique which
molecular biologists now frequently use in the laboratory, which is known as the
polymerase chain reaction, or in short, PCR.
Now, PCR is commonly used today for genetic testing, ?
It is also used in COVID testing. It was very, very helpful during the COVID period, ?
It's also used for forensic testing and paternity testing, ?
So if somebody claims that this child belongs to this rich man, then this rich man say,
are you sure this child belongs to me, ?
But what the rich man and the child needs is some blood, and then we just extract
the DNA and we do PCR, and then after that, we can sequence the DNA to check
whether the two of them match.
And then if they match, okay, yeah, this child really belongs to this rich man.
And that's what happens in HSA.
A lot of rich people, not only in Singapore, but all over Asia, you know, like in Indonesia,
you got those rich man. They will send their samples over to HSA to sequence, ?
Now, so let's discuss how about the polymerase chain reaction.
So imagine you are or there is a forensic scientist, and he's out collecting evidence at
a crime scene.
It often happens that, you know, the samples that he collects is only present in very
small amounts, maybe a strand of hair, you know, which he thinks may be suspicious
that may give him evidence of this crime.
Okay, so if he collects only a few strands of hair or so, so that means obviously that
the amount of DNA he's going to get from his sample is going to be a very, very tiny
amount, ?
So to carry out characterization, he needs more of the DNA, right?
So how is he going to get more of this DNA?
So he can do it with PCR reaction, and PCR was invented by Kerry Mullis in 1986.
So today, PCR is a fast and inexpensive technique used to produce multiple copies of
the DNA sequence.
So starting with as little as one picogram, so one picogram is 10 to the power minus
12 grams of DNA with a chain length of about 10,000 nucleotides.
Okay, PCR can possibly make several micrograms of this DNA in a few hours.
So how does PCR work?
So PCR is based on the Le Chatelier's principle of using excess reagents to drive a
thermodynamic equilibrium.
So in a PCR reaction, it consists essentially of two stages.
The first one is the equilibration of the double-stranded DNA with two sets of five
prime primers, both of which are present in excess.
So in Le Chatelier's principle, we say that a reaction of between A and B, we want to
push it to completion.
We put one of them, A or B, in excess. So that's how PCR works, ?
DNA is the limiting agent because, of course, you don't have too much of it, so that's
the limiting.
And to push the reaction forward, we just put excess of the primers.
, so that's the first step.
And the second step is the extension of the daughter chain using DNA polymerase, ?
So like DNA replication, PCR requires a DNA polymerase enzyme to make new strands
of DNA from the existing strands in the template.
So the DNA polymerase that is typically used in PCR is the Taq polymerase, ?
And the Taq polymerase was first isolated from a heat-tolerant bacterium known as
Thermos aquaticus, ?
And this bacteria was found in the hot springs in Yellowstone National Park in America.
Now, it is very heat-stable, and it is most reactive at around 70 degrees Celsius.
So this heat stability of the Taq polymerase makes it ideal for PCR, and PCR is able to
catalyze the incorporation of nucleotides into duplex DNA in the 5 prime to 3 prime
direction.
However, like other DNA polymerase, Taq polymerase cannot initiate DNA synthesis.
It needs a short sequence of nucleotides known as primers as a starting point.
So the two primers that are used in the PCR reaction are often designed, they are
known as PCR primers.
They can be designed, and then they are designed so that they target, so that they
flank, , the target regions to be copied.
And how do we get these primers?
We can synthesize them using oligonucleotide synthesis phosphoramidite chemistry,
which we have just covered.
Now, in the first step of PCR, the reaction mixture containing the template DNA, two
sets of 5 prime primers are present in excess.
Then you also put into, you also have the building blocks, right, the nucleotide building
blocks, the Taq polymerase, magnesium chloride.
Remember, the magnesium helps to coordinate to the triphosphate.
Remember, these DNTPs, they have a triphosphate, and the magnesium coordinates
to it to polarize the bond to make the reaction faster.
And of course, you have the buffer solution.
So all these reagents are placed into this vessel, which we call an Eppendorf tube
Then this Eppendorf tube containing all these reagents is then placed into the sample
chamber of a PCR machine, which is also known as a thermal cycler.
The thermal cycler is then programmed such that the PCR can occur repeatedly.
So in the first step, the PCR, in the first step of PCR, the reaction mixture is heated to
95 degrees Celsius.
So at this high temperature, the heat of the reaction causes the double-stranded DNA
to denature.
So the hydrogen bonds will break, and then the two strands will break apart into two
single strands, after which the temperature is lowered to 55 degrees, where the
primers anneal by hydrogen bonding to the complementary sequence, on each target
strand.
So you see the primers being attached already.
Then in the third step, the reaction mixture is heated to 72 degrees, and that's the
temperature that the Taq polymerase works very well.
So at this temperature, the Taq polymerase would catalyze the addition of the
nucleotides to the two primed DNA strands.
So when the replication of each strand is completed, what we have is now two copies
of the original DNA in the reaction mixture.
Then the reaction is repeated again and again, and it goes on and over and over again,
and as it does so, the DNA copies, it increases exponentially.
So in a typical PCR, the cycle of denature, anneal and polymerization is repeated about
30 times, with each of the three steps taking about one minute.
Thus, depending on the length of the DNA sequence being copied, the entire PCR
would usually take about two to four hours to be completed.
The results of the PCR reaction is usually visualized by gel electrophoresis, as shown
here, where the fragments of the DNA are separated according to their size.
So in the gel electrophoresis, one of the lanes would contain the standard or a DNA
ladder, and this DNA ladder contains different nucleotides of different base pair length.
So having this lane, you will know that this nucleotide will be 600 base pair, this band
will represent a nucleotide that's 400 base pair, this band will represent a nucleotide
that's 300 base pair.
So this acts as a marker, and this is a sample that is obtained from the PCR. So this
PCR has a band here, this means that the DNA strand that is made in the PCR is 400
base pairs long.
So that's how the gel electrophoresis works.
And so since DNA are microscopic, so for DNA to show up on the gel electrophoresis,
you need a sizable amount of the DNA.
So this DNA, the sizable amount is obtained through your PCR reactions, or else you
can't see it.
Okay, so far so good?
Okay, besides using PCR to produce, so I've just described to you how we can use
PCR to amplify the amount of DNA that we have.
So besides using PCR to produce large quantities of DNA sequences, another major
application of PCR today is in the detection of COVID-19 virus, and that's what was
happening during the COVID period.
We were not trying to make large amounts of the COVID DNA using PCR, but we were
trying to use it to detect whether the sample that was obtained from people will
contain the COVID-19 virus.
So before describing how PCR is used, let me briefly describe the COVID-19 virus,
which is also known as the SARS-CoV-2.
So the COVID-19 coronavirus is a RNA virus, which means that this genome is made
up of RNA, not DNA.
On its own, it can't produce itself. That is why it relies on infiltrating healthy cells to
multiply and survive.
Now, viruses travel light, right? They only pack their RNA within a coat, as shown here
And this coat is known as a capsid, C-A-P-S-I-D, and this capsid comprises of two
things, a fatty acid envelope, fatty acid. That's why the virus does not like soap, right,
because soap will wash off fats, right?
That's why you are told to wash your hands more frequently with soap
That's because the capsid comprises of a fatty acid envelope and the viral proteins
that is known as the spike protein.
Now, these proteins give the virus its distinctive shape and helps the virus to latch on
to the whole cell and then climb inside them.
So once inside the whole cell, the virus will uncoat itself, and when it uncoat itself, it
will spill its DNA into the whole cell's cytoplasm.
Now, this lonely single-strand RNA has two things that it has to do.
One, it must replicate itself in entirety as each copy constitutes the RNA of a new virus.
Since the COVID-19 virus is an RNA virus, this means it replicates itself by making
RNA.
But imagine, if the host cell is a human being, the host cell uses polymerase to
replicate DNA and to transcribe DNA into RNA.
This means that the whole human cell lacks the polymerase to replicate RNA because
we can only replicate DNA. We cannot replicate RNA.
So how are copies of the virus' RNA to be replicated?
Well, the virus genome carries a gene coding for RNA-to-RNA polymerase.
And since the COVID-19 virus is an RNA virus, this means that it is ribosome-friendly.
Remember, during translation, the messenger RNA would interact with the ribosome.
So now here in the cytoplasm of the human cell, there are lots of ribosomes, and the
ribosomes are friendly towards the RNA of the virus.
So there are millions of ribosomes in a single human cell.
So the lonely RNA strand can insert itself into the host cell's ribosome, hijacking it to
translate its genetic material to synthesize a working viral RNA-RNA polymerase.
So once the viral polymerase is made, it can replicate multiple copies of the full-length
viral RNA.
The virus' polymerase comes. The virus, although they are really small, they are very
advanced
The virus' polymerase comes with proofreading ability that allows it to identify and
correct errors during replication.
So the second thing that the lone RNA needs to do is to generate multiple partial
copies of itself. Why do I say partial copies? Because this RNA would contain introns
and exons.
So partial copies is because it will remove the introns, leaving the exons.
And this, the exons will then serve, remember we said that exons contain the coding
sequence.
The exons now will serve as the instruction for the host cell ribosome to produce a
repertoire of viral proteins needed to assemble a new viral offspring, which will then
leave the whole cell to infect a new host cell, or a new host.
So amongst the proteins that the virus forces the ribosomes to manufacture are some
proteins that will actually bite the hand that feeds them, okay?
So this virus is not very grateful, okay, they are nasty, okay?
So these proteins that are formed will then home in onto the ribosome in the act of
reading one of the host cell's messenger RNA.
So it is like the ribosome is reading a host cell's messenger RNA, the virus protein will
home in onto it, so and then onto the RNA and then so that to block the ribosome
from reading the host cell's messenger RNA.
And it's so insistent, on blocking that the ribosome finally drops off. So and then so
when the ribosome drops off, what happens? That means the host cell is unable to
make its own protein.
So in that way, the virus forces the host cell's system to make its protein but prevents
the host cell from making its own protein.
So at the end of the day, the host cell's immune system is compromised.
And so that affects the host cell's line of defence, so that's why the host cell or the
person, the host will fall ill.
so the virus hijacks the infected host cell's protein assembly to produce its own protein.
So it will end up stifling the infected host cell's line of defence.
Okay, we mentioned earlier that PCR is used to synthesize large amounts of the desired
DNA sequence.
However, to test for COVID-19, the aim is not to build large quantities of DNA but to
detect if the COVID-19 virus is in the biological sample in the fastest possible manner.
So hence, instead of the traditional PCR that is used to make, to amplify protein, the
PCR that is used is known as real-time PCR or quantitative PCR, which in short is
known as qPCR.
However, real-time PCR or qPCR employs the same principle as in the PCR that is used
for the amplification of proteins. so how is this possible?
So the only difference between qPCR and normal PCR is that in qPCR, we do not view
the DNA that is being made using a gel electrophoresis because the amount will be
very little.
But instead, in qPCR, a fluorescent dye is attached to the DNA as shown here. This
fluorescent dye is attached to the DNA sequence that is being amplified.
So the glow of this fluorescent dye shows how much DNA is present in the sample.
So imagine, so how is qPCR applied to COVID-19 testing?
Just give you an example. So a sample of the person's mucus is taken and the nucleic
acid is extracted.
So after, if the person, okay, so the nucleic acid is extracted. So this is the sample that
you have now. So the black things are the person's DNA.
So if the person is infected with COVID-19 virus, the sample will contain a mix of the
person's genetic material, which is denoted in black, and the virus RNA, which is
denoted in red.
Now, since PCR can only amplify DNA and the COVID-19 virus has only RNA, so the
RNA must first be converted into a double-stranded DNA.
And how can this be done? This can be done by an enzyme known as reverse
transcriptase.
So let's look at this picture here. You've seen this.
This is the central dogma of molecular biology where the RNA is used to make DNA,
which is using an enzyme known as reverse transcriptase, and the process is known
as reverse transcription.
Okay, so the reverse transcriptase is an enzyme used to generate complementary DNA
from an RNA template.
And it was first discovered in retroviruses, but now in more recent times, they have
also been found in some bacteria.
Okay, so as mentioned, the process of synthesizing DNA from an RNA template is
known as reverse transcription.
So this slide shows the Singapore's home team development of the COVID-19 PCR
test kit.
So at the very early stages of the COVID-19 pandemic, the public, the complete
genome of the COVID-19 was available in the public domain.
That means they already know what's the sequence.
And that is very, very important because by knowing the RNA sequence, it helps in
the development of the vaccine and also in the test kits.
So the Chinese people were very kind.
They placed this RNA sequence in the public domain. And by knowing the RNA
sequence, the primers required for the PCR could be designed.
The design of the primer is critical to ensure that the test kit is able to detect the
COVID-19 with high specificity and sensitivity.
To achieve this, the primer have to anneal to targeted regions which contain unique
sequences that belong to the COVID-19.
You do not want to make primers that target the whole cell. So you compare the
sequence on the COVID-19 with those that is found in humans.
And you see where the differences are, because you want to target only those in the
COVID-19.
So in those regions that are unique to the COVID-19, those will be the places where
you can use to design the primers.
In addition to that, the home team scientists have also incorporated degenerate
primers into their design.
Why do they have to have degenerate primers or what is degenerate primer?
A degenerate primer is a primer that in some positions you have a number of possible
bases.
So what is the purpose of this degenerate primer?
That means they are foreseeing that the virus will mutate.
So they were saying that they will guess that or they do an intelligent guess that
possibly at this site mutation will occur.
Probably at this point that they have that this site is a T or this site is a U, it's an RNA.
So this site is a U.
So maybe it will mutate and becomes an A or a G or a C.
So the degenerate primers will be primers that have at this position not only T but
also U, C and A. So that in the event that mutation occurs at this site, their kit is still
usable.
So that's the purpose of degenerate primers.
Okay, so let's continue the story.
So after the reverse transcription, so now what you have in your sample is the DNA
of the person and the DNA of the virus mixed together.
So now what we do now is to add in the primers and other chemicals, chemical
reagents that is needed for the PCR.
And now in addition to that, we also add probes that have fluorescent dyes on them.
Now these fluorescent dyes will mark out any viral DNA.
So if the virus is in the mucus sample, copies of the COVID-19 DNA will be made
during the PCR.
Since the fluorescent probe only recognizes the COVID-19 DNA, the PCR will start to
fluoresce. So the more fluorescence observed after each cycle means that more viral
DNA is present.
The PCR machine would display the information as the PCR progresses.
So here we see the fluorescence increasing exponentially as the PCR progresses,
indicating the presence of the viral DNA in the PCR mixture.
So when the PCR is done like this, the results are obtained as the PCR progresses.
This is known as real-time PCR.
Okay, so that's my lecture.
I'm going to show you a video which hopefully will give you a better understanding of
fluorescent-probe-based qPCR.
I took it from a company, Sigma, which sells these probes.
So let me get this done.
In a pro-based qPCR reaction, you have a table, which contains the target sequence
that you work against.
You also need primers, dNTPs, and DNA polymerase at your Tuesday.
In addition, you will need a probe labeled both in a quarter-volume and quarter-volume.
Typically, probes are sold with several custom models because they are specific to the
target sequence.
DNA generation is the first step in the PCR reaction.
The thermosulfur heats up to about 95 degrees Celsius,
which causes the double-stranded DNA helix to melt all of them into two singlestranded DNA templates.
During the helix, the temperature cools between 45 to 65 degrees Celsius.
Probes slated with both a quarter-volume and a wet-shovel pool and sequence specific
fibers
anneal to the single-stranded DNA template.
During this cycle, DNA polymerase attaches to the prime template and begins to
evaporate,
documenting nucleotides.
Finally, during extension, the temperature slightly rises to 65 to 75 degrees Celsius.
During this phase, DNA polymerase extends the sequence specific fiber with the
incorporation of nucleotides
that are complementary to the DNA template.
The DNA polymerase then displaces the overall pool from the probe, resulting in
fluorescence.
The fluorescence accumulates as cycling in PCR continues and is measured at the end
of each PCR cycle.
The intensity of the fluorescence generated by the ordinal curve above background
level, the CQ value,
is measured and used to quantitate the amount of newly generated double-stranded
DNA strands.
After repeating the denaturation, annealing, and extension sequence approximately
35 to 40 times,
you are ready to begin the analysis.
The CQ values can be used to quantitate stellar limits of DNA, establish a standard
curve for gene expression sequence,
or further analysis.
Probe-based QPCR is a great option for its specific polymerization.
Okay, so this is the probe-based QPCR.
Sometimes you may encounter another kind of Q-based PCR, which is the dye PCR,
but that one has,
you can get false positives.
That means what is not true, it actually shows up as the truth.
Alright, so that's why I, okay, so this is probe-based.
Now, I want to just, in case you were just wondering, how did Singapore tackle the
COVID-19 issue?
Okay, let me show you this.
There will always be about 30 to 50 bottles of my chip.
Do you know how many stacks you've taken?
It will be about 80 to 20,000.
The sample is brought by hand, although we have a pneumatic tube system for most
of our samples.
These are high-risk cases, and we don't want to risk the sample getting stuck in a
pneumatic tube.
It's not just about the contamination and the PCR, it's about the basic elements of
laboratory practice,
which is making sure you've got the right patient and the right sample.
You get the sample from the wrong patient at this stage, it's a complete disaster.
These fast, 10-minute test kits detect antibodies to the virus, and they take time, like
10, 14 days, to appear in anybody's blood.
That's too slow, we can't wait that long.
So this is the first stage of the PCR process, which is the extraction.
In the safety cabinets, the air can only flow in this direction, which means that any
virus in there is kept inside to ensure the safety of our operators.
He's mixing up the samples just to make sure that any human tissue that's adherent
to the swab goes into the liquid, because the liquid is where we extract from.
This is the room where we prepare the master mix. It's in a completely separate room.
He's not gowned up just to protect himself, he's protecting the master mix, because
contamination at this stage could ruin the whole run.
This is the patient samples, this is the extracted RNA, these are the strips that used
to be put into a PCR machine.
Then I'll be adding the master mix first, then the sample into each individual virus.
The reaction depends on the heat, the samples are heated up as the temperature
comes down, and that's the cycling process.
In a simple way, you could say we put in a specific Lego brick that combines with a
specific virus Lego brick, and once the bricks bind, then they can build up into a bigger
tower of Lego, and that's what we will detect.
Once the result is out, they will stand or write the results here. The detector will write
the detected TEG.
Now to do one test will take only three hours, but when we're testing two, three, four
hundred samples, they don't all come at the same time.
We have to factor in the fact that the sample needs to be taken from the patient,
brought to the laboratory, and that itself can be a delay of one to two hours.
We have been working more shifts than usual. The day we work actually eight-hour
shifts, and the night we work twelve-hour shifts.
The worst is that by four a.m. when we're already like very zoned out, but we still
have to concentrate very hard.
Before telling life, of course we really cannot afford to make any mistakes.
This is our daily PCR testing workload going back to the 4th of February.
As you can see, the workload has gone up to a maximum of 750 on the 23rd of March.
When we have the staff, then we have to get more equipment. And when we get more
equipment, we mean more staff.
And each member of staff takes two weeks to train. So it's like Spiderman crawling up
the wall, except very slowly.
Right now, 800 with the staff and equipment we have is the maximum we can do.
My colleagues and I believe that this wall is not just fought in the hospitals alone.
We hope to flatten the curve. So it's really important for everyone to stay home and
help us.
Okay, so that was the documentary that was made during the COVID pandemic.
, that's all for today. And if you have any questions, I'll be most happy to answer.
If not, to the Chinese, Happy New Year.