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In the last session, we have discussed the gene cloning technique utilizing recombinant

DNA technology i.e. the r DNA technology. Now, we will discuss a completely in vitro

method of amplifying a piece of DNA by PCR. Amplify means making numerous copies

or clones.

PCR can make billions of copies of a target sequence of DNA in a few hours. So, it is

very useful. It was invented in 1984 by Dr. Mullis. He received the Nobel Prize within

after 5 years of this discovery. Every molecular biology lab will have one or many more

PCR machines because this is fully automated now.



(Refer Slide Time: 02:12)



How do you do this amplification? As I told you it is a laboratory version. It is an in vitro

version of DNA replication in cells. In last lecture, I have discussed how recombinant

DNA technology can be utilized in the cells to make copies of the DNA and to make

enough of the substantial amount of proteins that you require.

In PCR, you make copies of the DNA but you do not get the proteins out of it. Here the

DNA is in the test tube. It is not inside the living system. So, you do not get the proteins.

It is just a way to make the DNA. Remember in r DNA technology, you can make the

copies of the DNA as well as proteins also because it is being amplified inside the

bacterial cells.



(Refer Slide Time: 03:20)



In PCR, you need the DNA that you want to amplify. Then it requires a heat stable DNA

polymerase like Taq polymerase. DNA polymerase makes poly oligonucleotide in 5’-3’

direction. This enzyme works at a higher temperature.

Usually our proteins work in the biological system at 37 degree. It is the optimum

temperature. If the temperature goes above 500 C the enzyme slowly loses the activity.

Here 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 those bacteria must be having

and producing enzymes 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 you get the

cryo enzymes which work under cryo conditions. So, this is we are talking about a heat

stable DNA polymerase.

All the four oligo nucleotide triphosphates are mandatory requirements for synthesis of

DNA. As we are doing it in vitro all the four nucleotide triphosphates are required. You

have to add buffer and magnesium. When polymerase works the 3 prime OH attacks the

5 prime triphosphate and then the pyrophosphate is released. Magnesium takes care of



the negative charges through chelation. These two short single stranded DNA molecules

serves as primers.

These short double strands 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 strand but DNA polymerase requires

a small segment of double strand in order to make the complementary strand.

I need to take this in a Eppendorf and then add primers.

Now, what are these primers? The primers bind to the 3 prime end. This is binding at the

right most corner and this is binding at the leftmost corner. It has to bind from the 5

prime to 3 prime and then the DNA synthesis takes place by attack of the 3 prime OH

because here 3 prime OH is free.

Here also 3 prime OH is free. If you add nucleotide triphosphates they will be taken up

one by one depending on the sequence here. The reaction will proceed and you get the

DNA piece. So, you add the primer and then you also add a heat stable DNA polymerase

whose optimum activity is usually between 70 to 80 degree centigrade.

Taq polymerase works very well at this temperature. But there is a particular temperature

usually 72 degrees maintained by the machine. You have this piece of test tube or

Eppendorf and then you take the buffer, the magnesium, the DNA, 4 oligonucleotide

triphosphates, a heat stable DNA polymerase and the primers. So, initially the primers

will not join because it was basically the DNA was present as the double strand.

So, in order for the primer to bind you have to heat it. The heating is done till the

temperature reaches 95 degree centigrade. 95 degree centigrade means all the DNA that

are present in the universe will melt at this temperature.

So, these DNA will melt now. This is my original DNA that will form single strands.

Now you have the primers also and primers are given in excess large excess. If you do

not add the primers then they are going to self-anneal with each other. The primers will

hybridize with these single strands that are separated by heating at 95.

If you cool down to about 40 degree the primers will join at the two ends and then you

heat it again to keep the temperature between 70 to 80 degree. The Taq polymerase t



works very well at 70 to 80 degrees. Now you get two copies of the double stranded

DNA. This is called one cycle. What is the cycle? Cycle means you heat everything to

around 95. It becomes single stranded, then cool it to around 40. So, the primers bind to

the single strands and then you heat it back to about 72 degree centigrade. Then the

polymerase will complete the extension of the chain. 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 these 4 strands will separate. 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 complete the elongation of the chain.

All the DNAs will have the primers. This is your 5 prime end and this is your 3 prime

end. 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. When it is cooled the primer will bind to the 3 prime end. 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.

Now, what you will do? You again heat it to 72. Now this will be your piece new piece

of DNA and this will be your new piece of DNA. After two cycles you have 22 = 4

strands of double stranded DNA.

So, now if you do n number of times you will get 2n



number of strands. If you do it 10

times it will be 210 number of strands. You copies of double stranded DNA within a

couple of hours. Most interesting the breakthrough came in the polymerase chain

reaction process is the discovery of this Taq polymerase. If the temperature is kept at 95

the enzyme loses its activity and it becomes denatured. You cannot really get back the

original activity that was a problem.

After the discovery of Taq polymerase this whole thing can be automated. Earlier before

the discovery of Taq polymerase you have to heat, then after cooling you have to add the

DNA polymerase in every cycle because the polymerase will lose its activity and

denatured at 95.



But once this Taq polymerase was discovered it survives at 95 degree centigrade. It

survives definitely at 45, 40 degree centigrade but its optimum reactivity is at between 70

to 80. Depending on the number of copies, you just do this number of cycles. So, that

becomes 2n.

(Refer Slide Time: 17:40)



So 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. Now these are done by a

machine called thermocycler.

(Refer Slide Time: 18:38)



So, you can change the temperature and can fix it at particular temperature. These

thermo cyclers are extremely good in maintaining the temperature. You have to go 95

then quickly you have to drop to 40 and then you have to take it to 72. 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 in 1976 and that actually led the

foundation of this automated DNA polymerase chain reaction.

(Refer Slide Time: 19:04)



(Refer Slide Time: 19:24)



(Refer Slide Time: 19:26)



(Refer Slide Time: 19:29)



By the way, this is called the reverse primer because the extension goes in the reverse

direction of the DNA. This is called the forward primer because this goes in the forward

direction. Now, suppose you have a DNA and you are interested only to copy from this

region. So, suppose this is your 5 prime end this is your 3 prime end. Now if I want to

copy only this region I have to use a primer which recognizes this part. Because then

only copying can be done at 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. 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 you will get copy. You really do not get your copy that you wanted. You have

extra one. This is your region of interest but you are getting extra after the 1st cycle.

Now, come to the second cycle. So, what will happen here? When 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. So, then from the 2nd cycle onwards

you are getting one double strand. So, this is the other strand again contains only the

original strand. 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. In 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. In 2nd cycle, when you melt it from this strand you will get the actual

the zone of interest. From the other strand, you get the piece of interest.

So, this is your piece of interest and that is your piece of interest. After the second cycle,

this is your starting point. So, that is your starting point. You have to complete two

cycles in order to get the first copy of the double strand DNA that containing the region

of interest. So, from then onwards you will get only this part. That will be copied

because all the primers will either bind here or bind there. That will be extended up to

the zone of interest.

The number of copies of the zone of interest after 10 cycles will be 2n-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. If

you 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, can we apply PCR to this or not? Yes, you can apply you just make a primer

that is the reverse primer. You also can write forward primer for the complementary

strand. So, you add both these primers, the single strand and you heat it and then cool it.

This primer will remain free because it did not have the complementary strand to start.



Now, you have a double strand. 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 it can bind here. Now, you will have basically 2n-1. It is

possible to do PCR reaction on a single strand. Here first cycle is needed to make the

double strand and from then onwards you can copy that DNA.

(Refer Slide Time: 27:33)



What are the usefulness of PCR? PCR has become a powerful tool in molecular biology.

One can start with a single strand. Suppose there is a homicide somewhere and you want

to identify who is the murderer. Now usually a hair of the murderer can be isolated at the

crime scene, 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 and do the PCR and then from the PCR you do the DNA

sequence analysis. Then you compare with your suspect DNA. You have a group of

suspect and from there you can take the blood and then you isolate the DNA. 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. However, it has been found that it is 1 in

a billion that there could be a chance of misidentifying the murderer.



(Refer Slide Time: 28:41)



(Refer Slide Time: 29:21)



So, In forensic where the very small amount of DNA is available and only the sequence

of DNA may not be enough to identify the murderer. There are certain repeat of base

sequences which is present in every individual. So, if you can identify those repeats and

then compare, that gives a better way of identifying the person doing the homicide. So,

not only the DNA sequence, you have to see the 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.



(Refer Slide Time: 30:39)



There are many genetic diseases like Huntington’s disease or cystic fibrosis and there are

some viral diseases which remains dormant for a long time. It is like the HIV, the Human

Immunodeficiency Virus where the viral load means the amount of viral DNA will be

very tiny in the biological fluid of the body system. So, some of these genetic disorders

can be detected with the help of DNA like this Huntington’s disease.

(Refer Slide Time: 31:27)



Huntington’s disease is a genetic disorder characterized by abnormal body movements

and reduced mental abilities. In this case, mental function is defective and also there are



abnormal body movements. We have seen babies unfortunately born with Huntington’s

disease, HD. Huntington gene is expanded.

Huntington gene is having a repeat of CAG at regular intervals. The people who are not

suffering from this disease or this abnormality are called non HD individuals. For non

HD individuals repetition number is less than 30.

In HD individuals the CAG trinucleotide repeat occurs more than 36 times. If the repeat

of number is more than 36 you get these Huntington’s disease. If it is less than 30 you

are perfectly normal.

So, it has now incorporated originally when the baby is born. It is a genetic defect. You

isolate the DNA and then amplify by a via PCR and see the number of repeats of

sequence. If the number of repeats is greater than 36 then that individual will have

Huntington’s disease.

(Refer Slide Time: 34:15)



Another cystic fibrosis is a genetic disease characterized by severe breathing difficulties

and a predisposition to infections. So, they suffer from infections very frequently. CF,

the cystic fibrosis is caused by mutation in transmembrane conductance regulator gene

called CTFR. 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 it makes a

channel through which the chloride ions move into the cell or out of the cell. That is very



important for transmembrane conductance. That is extremely important migration of the

chloride because that is a charged anion.

The CTFR gene that is called the cystic fibrosis transmembrane regulated gene

conductance regulator gene. If it is normal then it expresses a protein which creates a

chloride ion channel. Mutation in CTFR leads to thick mucus secretions in the lung and

subsequent persistent bacterial infection. 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.

(Refer Slide Time: 36:24)



The HIV human immunodeficiency virus does complete destruction or reduction of

immune response. So, it basically destroys the immune response of a person but it stays

in a very dormant state for a long period of time. HIV is a retrovirus. We will talk about

that how it attacks the immune system in medicinal Chemistry part. If the viral load is

very little then tiny amount of viral DNA will be found in the infected individual’s body

fluid.

In spite of having tiny amount of DNA, it can be multiplied using PCR with addition of

the right primer. You know what the HIV gene is. So, you know what the primer is. So,

add those primers to body fluid to do the PCR.



PCR product is generating by the primer that you have added. There must be the viral

DNA. If this viral DNA is coming the person is likely to be HIV positive. If there is no

PCR product the person is likely to be HIV negative.

(Refer Slide Time: 38:25)



These are the application of PCR in forensic science. There are short repeated sequences

known as variable number of tandem repeats, VNTR. This VNTR can vary from 44 to 40

in different individuals. Primers will amplify these repeated areas and the genomic

fragment generated gives us an unique genetic fingerprint that can be used to identify an

individual. You have to use this VNTR. The variable number of tandem repeats

sequences will give a genetic fingerprint.

This is a fingerprint of the gene. So, that is why it is called a genetic fingerprint.

Fingerprint means a method of identification of an 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.



(Refer Slide Time: 39:48)



So, that is I think we have now discussed the r DNA technology and polymerase chain

reaction. Polymerase chain reaction is an in vitro process. It is a very rapid one by which

you can multiply DNAs and then it has got many utilities. On the other hand, r DNA

technology gives rise to the protein. It is very important. If your target is protein you

apply the r DNA technology and then you amplify the DNA and get the protein out of it.

So, that completes our nucleic acid. We covered structure of DNA RNA, the processes,

the melting temperature, the flow of information. Then we have studied all these in vivo

and the in vitro process of the multiplication.