Welcome back, in the last session we have
discussed the gene cloning technique utilizing
recombinant DNA technology ok; the r DNA technology.
Now, we will discuss a completely in vitro
method of amplifying a piece of DNA. Now what
is PCR? PCR is a means to amplify a particular
piece of DNA as I already told you. Amplify
means making numerous copies ok, numerous
copies means making numerous clones.
PCR can make billions of copies of a target
sequence of DNA in a few hours that is extremely
so, it is very useful that PCR can make the
target sequence of DNA in a few hours. It
was invented in 1984 by Dr. Mullis and when
he was very young he discovered this process
of making copies of DNA fragments and he received
the Nobel Prize very quickly. I think within
the next 5 year within 5 years he after discovery
he got the Nobel Prize and now every molecular
biology lab will have one or many more PCR
machines, because this is fully automated
now and PCR machines and to in order to make
copies of the DNA. Its applications is vast
and it is an integral part as I told you of
molecular biology lab.
Now what is how do you do this amplification?
As I told you it is a laboratory version;
that means, it is a in vitro, it better to
write that it is a in vitro version of DNA
replication in cells. Last time I told you
that in cells recombinant DNA technology,
how that can be utilized via the cells to
make copies of the DNA and to make enough
of the substantial amount of proteins that
you require.
In PCR, because it is in vitro entirely in
vitro you make copies of the DNA, but you
do not get the proteins out of it, because
the DNA is in the test tube. It is not inside
the living system so, although you make millions
and millions of copies, but it is not living
because it is not in the living system. So,
you do not get the proteins. It is just a
way to make the DNA remember that, in r DNA
technology you can make the copies of the
DNA, you can make your proteins also in the
process because it is being amplified inside
the bacterial cells ok.
The following what are the requirements in
PCR? First of all, in PCR you need the DNA
that you want to amplify that is number one.
Then it requires a heat stable DNA polymerase
like Taq polymerase. What is the heat stable
DNA polymerase? Remember polymerase is an
enzyme and if it is DNA polymerase; that means
it makes DNA oligo nucleotide poly oligo nucleotide
by doing this 5 prime to 3 prime connection
I told you and so, it is a polymerase DNA
polymerase an enzyme, but interestingly this
is an enzyme which works at a higher temperature.
It is a heat stable DNA polymerase that is
very interesting, because usually our proteins
work in the biological system that we have
at 37 degree it is the temperature optimum
temperature. But if you if the temperature
goes higher, the enzyme slowly loses the activity.
So, if the temperature is above 50; above
50 usually the enzymes lose the activity ok,
but it is a heat stable DNA polymerase like
the Taq polymerase.
Now, these enzymes are isolated from volcanic
regions or hot spring where the temperature
is very high. So, if some bacteria grows there
so, those bacteria must be having and producing
enzymes which are which work at a higher temperature.
So, somebody must have thought that in the
extreme conditions you can get enzymes which
work under extreme conditions. Like heat is
one example similarly, at very cold temperatures
how the enzymes work so, you get the cryo
the enzymes which work under cryo conditions
ok. So, this is we are talking about a heat
stable DNA polymerase.
Then the all the four oligo nucleotide triphosphates;
these are mandatory requirements for synthesis
of DNA. Remember again we are doing it in
vitro, in vitro you have to supply all the
ingredients; that means, all the four nucleotide
triphosphates are required, that you have
to add, you have to add a buffer and you have
to definitely require magnesium, because this
polymerase when it works the triphosphate
remember the 3 prime O H attacks the 5 prime
triphosphate and then the pyrophosphate is
released. But the negative charges are taken
care of by chelation to the magnesium and
in addition to this two short single stranded
DNA molecules that serves as primers.
Again, just to brush up your knowledge again
I told you that DNA polymerase works only
when there is a short duplex present in the
system, double strand present in the system
ok. And this short double strand is provided
in the form of what are called primers. We
have seen the use of primers in Sanger’s
dideoxy sequencing method. Remember RNA polymerase
does not require any primer. RNA polymerase
can work only on a single on a single strand,
but DNA polymerase requires a small segment
of double strand in order to make the complementary
strand ok.
So, that is why you need two short single
stranded DNA molecule that serve as primers.
Now let us discuss it. So, what is our problem?
Our problem is defined that we have a piece
of DNA, suppose we have one single strand
present in my test tube ok, now what I need
to do? I need to; I need to take this in a
Eppendorf and then add primers.
Now, what are these primers? The primers are
one which binds to one end; that means, this
is 5 prime to 3 prime and the other primer
binds to the other end of the complementary
strand. That means, it binds to the 3 prime
end when a both prime both bind primers bind
in the 3, prime end; that means, both are
binding. This is binding at the right most
corner and this is binding at the leftmost
corner ok, because that has to be the primer
has to bind from the 5 prime to 3 prime and
then the DNA synthesis takes place by attack
of the 3 prime O H because here 3 prime OH
is free.
And here also 3 prime O H is free and then
if you supply all these oligonucleotide this
nucleotide triphosphate pardon me, this is
not oligonucleotide this is a nucleotide triphosphates.
So, one by one depending on the sequence here
they will be taken up and the reaction will
proceed and you get the DNA piece ok. So,
you add the primer and then you also add a
heat stable, not a simple DNA polymerase a
heat stable polymerase which works whose optimum
activity is usually between 70 to 80 degree
centigrade.
So, Taq polymerase I think works very well
at between this temperature, but there is
a particular temperature usually 72 degrees
maintained by the machine. So, which falls
within this range so, that has got that works
at this temperature. Now you so, basically
what you have this piece of test tube or Eppendorf
and then you take the buffer, the magnesium,
the DNA you have added the 4 oligonucleotide
triphosphates and you have added a heat stable
DNA polymerase and you have added the primers.
So, initially the primers will not will not
join, because it was basically the DNA was
present as the double sorry the DNA was colored
blue here so, may I should make that blue.
So, initially the DNA was 
initially the DNA was this, they are tied
up with each other ok. So, in order for the
primer to bind what you have to do; you have
to heat it. The heating is done till the temperature
reaches 95 degree centigrade. So, you make
sure because 95 degree centigrade means that,
all the DNA’s that are present in the universe
are except the extreme conditions all the
DNA’s will melt at this temperature ok.
So, these DNA will melt now. This is my original
DNA that will form single strands ok. Now
you have the primers also and primers are
given in excess large excess. So, what will
happen now, as you cool this the most likely
because the primers are present in excess,
if you do not add the primers then they are
going to self anneal with each other, but
as the primers are present so, what will happen,
your primers are present in excess so, now
the primers will hybridize with this single
strands that are separated by heating at 95.
So, after heating at 95 if you cool down to
say about 40 degree then what will happen,
the primers will join at the two ends and
then you heat it again to between keep the
temperature between 70 to 80 degree. So, if
you do that so, what will happen now? So,
you will get so, DNA polymerase will because
that is the polymerase which works the Taq
polymerase that works at very well at 70 to
80 degrees. So, this will happen so, you get
now two copies of the double stranded DNA,
you started with one copy now you get two
copies.
So, after one this is called one cycle. What
is the cycle? Cycle means you add you take
everything heat it to around 95 so, that it
becomes single stranded, then cool it to around
40. So, that things the primers bind to the
single strands and then you heat it back to
about 72 degree centigrade and then what will
happen, the polymerase will work and the polymerase
will complete the extension of the chain,
make the complementary strand. So, this is
the cycle.
Then what you do? Then again you heat it to
95 degree centigrade. If you heat it to 95
degree centigrade, now what you have; you
have this and you have this you have another
strand here another strand and this will be
your so, you have these 4 strands all separate
and down ok. And now you cool to about 40
degree the temperature may not be very accurate,
but the science is very clear. You heat it,
make it a single strand, you cool it so, that
it hybridizes with the primer and then you
heat it to about 70 degree so, that polymerase
can one can complete the elongation of the
chain.
So, what will happen here now all the DNA’s
will be will have the primers? So, here will
be the primer, actually it better write also
this is your 5 prime end this is your 3 prime
end. So, when the DNA is synthesized, this
is your 3 prime, this is your 5 prime, this
is your original DNA 3 prime to 5 prime so,
that will be 5 prime to 3 prime. So, you maintain
that ok.
So, this will be so, the first one is 5 prime
to 3 prime, the second one is 3 prime to 5
prime the third one is 5 prime to 3 prime
and this last one is 3 prime to 5 prime. That
is your original piece of DNA. Now when it
again cool so, the primer will because it
is 5 prime to 3 prime. So, the primer will
bind to the 3 prime end here. And this is
3 prime to 5 prime so, now the primer will
bind here. And this is 5 prime to 3 prime
so, the primer will bind here and this is
3 prime to 5 prime so, the primer will bind
here ok.
Now, what you will do? You again heat it to
72. So, now, what will happen everything will
be so, now, this will be your another piece
of DNA. This will be the piece of DNA this
will be sorry this will be your piece new
piece of DNA and this will be your new piece
of DNA. So that means, now you started with
1; that means, 2 to the power 0, now you have
2 to the power 1, you have 2 strands of double
stranded DNA and after two cycles you have
2 to the power 2; that means, 4 strands of
double stranded DNA.
So, now if you do n number of times so, ultimately
how many you will get? 2 to the power n. So,
if you do it 10 times suppose so, 10 times;
so, that will be 2 to the power 10 that is
a huge number ok. And to do it is a couple
of hours and you get these copies of double
stranded DNA. Most interesting the breakthrough
came in the polymerase chain in developing
this polymerase chain reaction process is
the discovery of this Taq polymerase, because
what happens usually the if you heat something,
normal proteins or normal enzymes if the temperature
is kept at 95 the enzyme loses its activity
and it becomes denatured and you cannot really
get back the original activity, that was a
problem.
But as soon as this is Taq polymerase is beautiful
that after the discovery of this the whole
thing can be automated. Earlier before the
discovery of Taq polymerase what you have
to do, you have to heat this all the time
you have to after cooling you have to all
the time add the DNA polymerase, every cycle,
because the polymerase will lose its activity
and denatured at 95.
But once this Taq polymerase was discovered
then what will happen, because this is not
denatured on the other hand it survives at
95 degree centigrade. It survives definitely
at 45, 40 degree centigrade, but its optimum
reactivity is at between 70 to 80. So, you
can depending on the number of copies you
need, you just do this number of cycles ok.
So, that becomes 2 to the power n, n cycles.
So, I think this is PCR reaction is repeated
usually 20 to 40 times, 25 cycles usually
takes about 2 hours. So, 2 to the power 25,
so, 100000 fold you increase ok. Step 1 I
said 25 step 2 anneal usually 40 and then
72 polymerase, extend the chains as I told
you. These are the now this is done by a machine
these days this is called thermocycler ok.
So, you can change the temperature and can
fix it at particular this thermo cyclers are
extremely good in maintaining the temperature
and also very quickly. Say you have to go
95 then quickly you have to drop to 40 and
then you have to take it to 72 ok. So, that
can be done in a machine called thermo cycler.
Actually the Taq DNA polymerase was purified
from the hot spring bacterium thermos aquaticus
that is the bacteria in 1976 and that gave
the that actually led the foundation of this
automated DNA polymerase chain reaction.
I think this is whatever everything as I said
this is by the way called the reverse primer,
because the extension goes in the reverse
direction of the DNA. And this is called the
forward primer, because this goes in the forward
direction ok. I think I told about all this
extension and then the number of copies that
you get. Now, this is interesting suppose
you have a DNA and you are interested only
to copy from this to that region ok. So, my
problem is I have a piece of DNA make it a
little bit smaller so, that and I want to
I am interested only to copy from these to
that ok.
So, suppose this is your 5 prime end this
is your 3 prime end, now if I want to copy
from this and that only this region so, I
have to use a primer which recognizes this
part and I have to use a primer which recognizes
this part. Because then only copying can be
done of this zone. I do not need a primer
from this side and from this side then, I
will get the entire piece of DNA as the copy
ok. However, one thing is important when I
do the 1st cycle the 1st cycle what happens
that this is the 1st cycle let me just erase
that erase I think ok. So, in the 1st cycle
what will happen let us see.
The original piece of DNA is this one and
then you have the red portion which started
from this one and then that goes up to the
end here. So, this is the piece of DNA that
you will get and from the other strand is
here and that will because the primer the
forward primer is here so, your DNA that you
will get will be up to this. So, the 1st cycle
you really do not get the your copy that you
wanted. You have extra one so, this is your
region of interest, but you are getting extra
after the 1st cycle.
Now, come to the second cycle the second cycle.
So, what will happen here? The 2nd cycle;
that means, the original this one is there
and in the 2nd cycle remember again the sorry,
take this one this is the 5 prime to 3 prime
and this is the 3 prime to 5 prime ok. So,
now, if you look at this piece only, where
there is a small red part here now the your
the this poly primer will bind here ok. And
for the other part this one which are the
smaller ones, but containing extra then what
you wanted that will have again a that will
have a strand like this and a red part and
a red part which is the up to this point sorry
and a red part I have actually come to the
end this is the red part.
So, now the next what I say that this is the
2nd cycle so, this part sorry this part will
have a primer attached here. So, when that
extends that will extend up to this point
and similarly for this part, the primer will
be attached somewhere here and when this is
extended that will form up to this part ok.
So, the from the 2nd cycle onwards you are
getting one double strand. So, when this is
the other strand again contains only the original
strand. I am not showing that other strand.
The other strand will have if I show it the
other strand will be the sorry this is green
the other strand will be the normal strand
containing the entire piece.
So, again I repeat, the first cycle you have
this is the primers where that will be attached.
You have to design the primers according to
the sequence here and according to the sequence
there. So, but the 1st cycle we are seeing
that you do not get the DNA only containing
your zone of interest. You get extra, this
is extra on this side and on the other side
this is extra. And then 2nd cycle when you
again melt it when you melt it from this strand
you will get the actual the zone of interest
a copy of that and from the other strand which
has got over end that also you get the; you
get the piece of interest.
So, this is your piece of interest and that
is your piece of interest. So, now, you get
after the second cycle this is your starting
point, you get the actual maybe I can you
get the actual ones the red and the red here
and the blue portion here ok. I hope that
is clear. So, that is your starting point.
So, two cycles you have to; you have to complete
two cycles in order to get the first copy
of the double strand DNA that you with containing
the region of interest. So, from then onwards
you will get the only this part that will
be copied because all the primers will either
bind here or bind there and that will be extended
up to the zone of interest.
So, here the number of copies, if I ask what
will be the number of copies after 10 cycles
of the zone of interest that will be 2 to
the power n minus 2, because you have to first
complete 2 cycles in order to come to a first
strand of the double strand containing the
zone of interest. So, after 10 cycles you
will get 2 to the power 8 copies ok. That
you remember and the other sometimes the other
problem is given also that if you have only
one strand of the DNA suppose you do not have
the double strand, we have only one strand
of the DNA which you want to copy then how
many copies you will get after 10 cycles.
First of all, whether you can apply PCR to
this or not? Yes, you can apply you just make
a primer, that is the reverse primer and you
also can write in the piece of paper that
if the complementary strand was present what
could have been your forward primer. So, you
add both these primers and the single strand
and you heat it and then cool as 95, then
40 and then 72. So, what will happen this
part will be converted, this primer because
it did not have the complementary strand to
start with that will remain there.
So, now, you have a double strand. So, one
cycle is required to make the double strand
and from then onwards you will get the double
strand because now this primer has the complementary
strand so that can bind here ok. So, now,
you will have basically 2 to the power n minus
1. So, yes it is possible to also do PCR reaction
on a single strand, but first cycle is needed
to make the double strand and then from then
onwards you can copy that DNA ok.
So, this is PCR, what are the usefulness of
PCR? PCR is so important has become a powerful
tool in molecular biology. One can start with
a single strand here. This is the foreign
scenes a suppose there is a homicide somewhere
and you want to identify who is the culprit,
who is the murderer? Now usually a hair of
the murderer can be isolated at the crime
scene ok, but from the single piece of hair
is very difficult to do the DNA analysis.
So, for that you have to amplify the DNA.
So, you isolate the DNA, how to amplify the
DNA? This is by PCR reaction, so, you take
the you do the PCR and then from the PCR you
do the DNA sequence analysis. And then you
compare with your suspect DNA suspect the
you have a group of suspect and from there
you can take the blood and then you isolate
the DNA and then you can again do the analysis
of the suspect and from that you can make
out who is the likely murderer. That is usually
not much uncertainty, because there is always
a piece of uncertainty in every study or every
evaluation. However, it has been found that
it is 1 in a billion that there could be a
chance of misidentifying the murderer ok.
So, that is one in forensic where the very
small amount of DNA is available and then
you can, now, only DNA sequence only DNA sequence
may not be; may not be enough to identify
the murderer. What is found that there are
certain repeat certain repeat which is, repeat
of base sequences which is present in every
individual. Repeat of certain base sequences
which are present; which are present in a
individual and that repeat is usually different
from individual to individual.
So, if you can identify those repeats and
then compare, that gives a better way of identifying
the person doing the homicide ok. So, not
only the DNA sequence, you have to see that
short repeats that are present in every individual
which vary from individual to another individual.
So, that gives you more rigorous way of identifying
the culprit ok.
Now, let us see identifying certain diseases,
like there are many genetic diseases which
are which we know that the like Huntington’s
disease or cystic fibrosis or there are some
viral diseases which remains viral induced
diseases which remains dormant for a long
time and that is like the HIV the Human Immunodeficiency
Virus where the viral load means the amount
of viral DNA will be very tiny in the body
system in the biological fluid ok. So, some
of these genetic disorders can be detected
with the help of DNA, like this Huntington’s
disease.
What is this disease Huntington’s disease;
is a genetic disorder characterized by abnormal
body movements and reduced mental abilities
ok. That means, their mental function is defective
also abnormal body movements and we have seen
unfortunately babies born with Huntington’s
disease. What is the reason for HD? It is
caused by mutation in the gene called Huntington
gene, HD. What is the defect? In individuals
with Huntington’s disease the HD gene that
the Huntington Huntingtin gene sorry Huntingtin
gene that is expanded.
That means, what happens this gene has a repeat,
I told you about some repeats in individual,
but that is a different, Huntington gene is
having a repeat of CAG; that means, this CAG
is repeated at regular intervals in the Huntington
gene. Now what happens, the non-Huntington
Huntingtin or if you say non HD individuals
when these people who are not suffering from
this disease this abnormality, the HD gene
has a pattern called trinucleotide repeats
as I said CAG, that repetition occurring less
than 30 times. That means, it is repeated,
but that repetition number of repetitions
is less than 30.
On the other hand, in HD individuals thus
the CAG, the CAG trinucleotide repeat occurs
more than 36 times. So, it is the number of
times if the repeat is number of repeat is
more than 36, you get these Huntington’s
disease. If it is less than 30, you are perfectly
normal and in between I think it is you have
a compromise. It is not very serious Huntington’s
disease, but serious Huntington diseases when
the number of repeats are greater than 36.
So, it has now incorporated originally when
the baby is born. It is a genetically it is
a genetic defect ok. So, PCR now to know whether
the a individual is suffering from these disease
HD, so what you do? You isolate the DNA and
then amplify by a via PCR and sequence and
see the number of repeats ok. If the number
of repeats again I say that if it is greater
than 36, then that individual will have Huntington’s
disease.
Cystic Fibrosis again, that is another cystic
fibrosis is a genetic disease characterized
by severe breathing difficulties and a predisposition
to infections ok. So, they suffer from infections
very frequently. CF, the cystic fibrosis is
caused by mutation in the cystic fibrosis
that is a gene transmembrane conductance regulator
gene called CTFR. We do not need, but it is
a specific gene where there is a mutation.
In non CF individuals the CTFR gene codes
for a protein that is a chloride ion channel;
that means, which makes a channel through
which the chloride ions move into the cell
or out of the cell ok. That is very important
for is it said that transmembrane conductance
that is extremely important the migration
of the chloride, because that is a charged
anion.
So, normal CF what it says that now in non
CF individuals this is perfectly ok, the CTFR
gene that is called the cystic fibrosis transmembrane
regulated gene conductance regulator gene
that is normally if it is normal then, it
expresses a protein which becomes a chloride
ion which creates a chloride ion channel ok.
In CF individuals person suffering from cystic
fibrosis mutation in CTFR lead to thick mucus
secretions in the lung and subsequent persistent
bacterial infection ok. Again, you can check
whether there is any mutation by comparing
with the healthy individual versus a CF individual
and then see whether there is any mutation
in the CTFR gene.
And the last one I told you I told you about
these viral infections the HIV human immunodeficiency
virus what happens, here the HIV does complete
destruction or very reduced amount of immune
response is present. So, it basically destroys
the immune response of a of a person ok and
but it keeps in a very dormant it stays in
a very dormant state for a long period of
time. So, HIV says it is a retrovirus we will
talk about that attacks that immune system.
We will talk about in a medicinal chemistry
aspect, but if you want to know because initial
the viral load is very little. So, the tiny
amount of viral DNA will be found in the infected
individuals body fluid ok.
So, in that case, because PCR is very important
that if you have whatever tiny amount do you
have if you can add the right primer then
it will be multiplied ok. You can amplify
that. So, therefore, if you do a PCR of the
body of the DNA isolated from the body fluid
and if you see a PCR product, because you
know what is the HIV gene DNA ok. So, you
know what the primer are. So, what you take
the body fluid add those primers do the PCR.
If you say a PCR product coming from the from
the bacterial fluid PCR product is generating
by the primer that you have added; that means,
there must be the viral DNA that is present.
Now where from this viral DNA is coming; that
means, the person is likely to be HIV positive.
If there is no PCR product the person is likely
to be HIV negative ok.
So, these are the PCR forensic I have already
told you about the forensic I told you about
this that there is this short repeated sequences
known as variable number of tandem repeats,
VNTR. This VNTR the number of repeats can
vary from 44 to 40 in different individuals.
Primers are chosen that will amplify these
repeated areas and the genomic fragment generated
give us a unique genetic fingerprint that
can be used to identify an individual. So,
you have to look not only for the sequence,
you have to use this VNTR; the variable number
of tandem repeats sequences and that will
give a fingerprint genetic fingerprint.
This is not the fingerprint that we know,
but this is a fingerprint of the gene. So,
that is why this is called a genetic fingerprint.
That is fingerprint means a method of identification
of an individual identifying individual. So,
through the genetic fingerprint we can now
match the genetic fingerprint of the blood
or the piece of hair that we obtained at the
crime scene and compare with the genetic fingerprint
of the suspected persons and by that you can
tell who the culprit is, ok?
So, that is I think we have now discussed
the r DNA technology and we have also discussed
this polymerase chain reaction. Polymerase
chain reaction is an in vitro process where
it is a very rapid one by which you can multiply
DNAs and then it has got many utilities as
I have shown and the r DNA technology on the
other hand that gives rise to the protein,
is very important ultimately if your target
is protein then you apply the r DNA technology
and then you amplify the DNA and get the protein
out of it ok.
So, that completes our nucleic acid for the
time being nucleic acid chemistry we started
with DNA RNA their structure and then the
processes, that melting the melting temperature
and then we have then followed or studied
the all these processes the in vivo and the
in vitro process of the multiplication, the
flow of information ok.
Thank you.
