Welcome back. In the last session, we have
revisited the processes of involved in the
central dogma of biology that means, the flow
of information from DNA to proteins and the
processes, mechanism of various processes,
mechanism of various enzymes have been discussed,
ok.
Now, we will discuss the amplification of
a piece of DNA that if we have a small amount
of DNA, then how can we get thousands and
thousands of copies of this DNA. Now, remember
copying of DNA in the biological system is
called replication that is the copying of
DNA.
But here we are talking about something which
is carried outside the living system in a
test tube or Eppendorf but utilizing the biological
the utilizing the different components of
the biological system that are involved in
replication processes. We will show you how
to do that but this is important to distinguish
between the two processes. Replication is
the process which takes place inside the organism,
living organism and that is what is called
in vivo process and the amplification that
we are talking about is carried out, outside
the living system and that will be called
or that is called an in vitro studies.
So, there are two processes one is called
in vivo, usually it is written in italics
or the other is in vitro; this is done inside
the living system, this is outside the living
system ok. So, our topic of discussion today
is that if we have a copy of a double strand
DNA, then how can we make without going into
the intricate machinery of the biological
system, how can we make copies of this; identical
copies of this piece of DNA. Now, this is
what is also called this process is called
cloning.
What is cloning; cloning is basically making
identical copy of anything that if you have
a writing in a piece of paper and if you make
a if you scan it or if you Xerox it, you will
get the clone of your original system ok.
So, cloning is nothing but making or creation
of identical copy or copies.
Now, this is this can be applied to a cell
also that if you copy the cell, if you can
be if you have one cell particular cell and
you make copies of that cell that will be
that is also cloning, but that is cloning
of the cells. If you say that DNA cloning,
if I say DNA cloning, then basically you actually
you are talking about that multiplying or
amplification of a piece of DNA that is what
is called DNA cloning. So, we are talking
about DNA cloning in this case.
Now, there are usually two methods that are
followed ok; one is called Polymerase Chain
Reaction or PCR based method, polymerase chain
reaction this is also called PCR and the other,
other is kind of semi it can called semi in
vitro process, because at some point of time
the second method this is suppose 1 and this
is 2. The second method utilizes at some point
of time the cells in a living organism.
So, it is kind of a semi in vitro analysis
and our technique. So, this is basically utilizing
the living system. So, one is utilizing one
is called PCR and the other is utilizing a
living system, we will discuss both and what
are the steps of this amplification, so what
you do first.
Suppose you have this piece of DNA, and what
I want is to copy suppose I want to copy this
region only, I do not require the full copy
of the DNA, but I want to copy the DNA of
this region ok. So, what you have to do first
you have to the steps are cutting the DNA
at precise locations.
So, you have to cut the DNA at precise locations,
but the cutting should not involve the segment
that you are interested to amplify that is
number 1. This cutting is done by sequence-specific
endonuclease because it is done inside the
DNA strand; so it is called endonuclease,
also they are called the restriction endonuclease
on the restriction enzymes.
May be in earlier discussion somewhere, I
mentioned about this restriction enzymes.
Restriction enzymes are endonucleases, which
recognizes which recognize specific sequence
and the beauty of this sequence is that they
are all palindromic sequence that means if
you read from 5 prime to 3 end whatever sequence
you get, if you read the other strand from
the same 5 prime to 3 prime end you will get
the same or identical sequence ok, so that
is called that means it has got a local C2
symmetry that if you turn it upside down,
you will get the same thing.
But remember this is local, you cannot turn
the whole DNA this way and then get the same
thing; it is just only a particular location,
this type of palindromic sequences are present
and they are recognized by these restriction
endonucleases, also called restriction enzymes.
So, they are also basically necessarily they
are molecular scissors. Now, what you do after
you chop this off what you need is a what
is called a vector means, you have to something
which carries this gene or carries this DNA
and then carries this DNA and then ultimately
that carrier plus this DNA of interest that
can be copied ok. So, this is not polymerase
we are talking about in general what is done,
we are talking about the second one.
In the living that means, amplification and
living system we are talking about that in
the beginning. So, you have the DNA piece
of DNA, this is your region of interest you
cut the DNA from the two sides using restriction
endonuclease or the restriction enzymes and
then incorporate into a carrier DNA, it is
a carrier in this case I will talk about this
carrier molecule, this carrier and then the
DNA is in becomes an integrated part of this
carrier and then this carrier has a property,
which can be copied when that is put inside
a bacterial cell.
Then we see if you have a bacterial cell and
if you put this suppose this is your carrier,
so if you can put this carrier molecule inside
the bacterial cell. So, when the bacterial
cell grows, so you will have bacterial cell
containing each bacterial cell containing
this carrier DNA hybrid, ok. This hybrid or
this is actually also called recombinant DNA
when you, because the carrier molecule is
a DNA is made up of DNA and this is also DNA.
So, this is what is called recombinant DNA.
Again I just quickly go through it that first
you cut the your DNA from the two sides without
cutting the zone of interest, and then you
attach it to the carrier DNA which is by the
way called a cloning vector, ok. A vector
is nothing but a delivery agent like we have
vectors for malaria, we know that what are
the vectors of transmission of one organism
microorganism into the human body via the
mosquito. So, the mosquito is a vector for
malaria, encephalitis, all these.
So, vector is nothing but it is a delivery
agent, it takes up the thing that I need to
deliver and then brings it to the bacteria,
and they are they are capable of self-replication,
but not by itself. When it is put in the bacterial
in a cell its say suppose it is a bacterial
cell, usually that is bacterial cell; and
then when the bacterial cell is it grows,
then each cell then they plas this is this
cloning. This is now a recombinant DNA, this
recombinant DNA also makes its own copy in
different cells and now the next thing if
you want to isolate this piece of DNA, we
have to chop the bacteria the outside layer
of the bacteria take this out, this out this
recombinant DNA and then utilize the same
endonuclease to cut it off from the from the
carrier molecule.
So, I can now say that first you have the
recombinant DNA, containing your DNA of interest
put it inside 
a bacterial cell, grow, isolate the recombinant
R DNA; Recombinant DNA, and then endonuclease
will give you the DNA of interest; copies
of because you will get multiple copies, because
from each cell you will get this you will
get this R DNA and from the R DNA, you will
get the copies of the DNA of interest, so
that is the system which is usually utilized.
Now, this has been largely replaced nowadays
by or a very quick technique, this is little
bit slow technique; however, this has got
one advantage is that if you can put it in
the bacterial cell, now this cell you can
preserve and whenever you need this recombinant
DNA, then you can ferment the bacterial bacteria
which will make copies and then you can isolate
the DNA.
So, this is; this can run in theory it can
run forever, because so long as you have the
bacterial strain containing this recombinant
DNA, then you can always get hold of the copies
of the DNA that you want ok.
And the other important factor is that if
these copies of if this DNA piece is ultimately
after transcription and then translation,
it makes a particular protein which you are
interested. Then you will although all the
time you will get, you do not have to break
the cells, because you are not interested
in the DNA any longer.
If you are interested only of the protein
that this DNA makes, then you just grow the
cells and then isolate the protein from the
cells or from the sometimes it is actually
inside the; outside the cytosol that can also
happen; or in many cases you can lyse the
cell, separate the DNA isolate the protein
and from the protein, you purify and isolate
your target protein.
I will give some example here, maybe right
now I can give you that suppose we know that
in diabetes there are two kinds; one is insulin
dependent, another is insulin independent.
So, some people who are suffering from diabetes,
they have to take insulin that is the insulin
dependent once ok; and insulin wherefrom the
insulin is obtained, earlier it was obtained
from the cow the bovine. So, it was bovine
insulin that was the one which the diabetes
people, diabetes patients were using ok.
Now, later on it was found that it is not
a very good practice to slaughter a cow, and
then isolate the isolate your insulin. First
of all it is not human insulin, there are
some differences in the bovine insulin and
human insulin; although there are good this
is there is good homology.
Homology means that the sequence of the amino
acids in the protein are very similar ok,
the highest homology the bovine has that is
why the you isolated you are the practice
was that you isolate the insulin and that
bovine insulin was used by the diabetes patient,
suffering from diabetes which is insulin dependent.
Later on it was found that if we can if we
can inscribe, suppose this is your insulin
gene and if that be the case if you can take
the insulin human insulin gene and put it
into as a recombinant DNA put it inside a
bacterial cell via this cloning technology,
then what will happen these cells because
it has got the insulin gene, so when it grows
it will make the insulin because it has got
the insulin gene and now whatever insulin
that are available in the market are all dependent
on the are all recombinant insulin ok, and
which is which is the also it makes the human
perfect human variation human variation of
the insulin, ok.
So, you see that the this technique is so
important, because if you want a protein particular
protein if you are interested, usually the
proteins are made in the cells very in very
small amount ok. If you want large amount
of the proteins, then what the your target
will be that you try to identify what is the
gene corresponding gene of that protein and
then take the gene and then put it as a recombinant
DNA that means, you attach it to the carrier
DNA or the vector DNA, and then put it inside
a bacteria.
If you are successful in that then you can
all the time you can get the protein, because
as the bacteria grows you will get the it
makes the protein and that is the modern day
technology for any laboratory; whether who
are doing proteomics or enzyme logy protein
related biology.
Now, the questions to be asked here that how
do you make this recombinant DNA that is number
1. The second is basically I think that is
the most important, because this recombinant
DNA is basically what we are talking about
that you have a vector DNA and you attach
your DNA of interest, ok. How will you ensure
that it has been attached, because when you
have this vector DNA some of the vector DNA
will remain as without any attachment from
outside, ok?
And some of the vector DNA will be will be
attached to this external DNA ok, it is like
very similar when I talked about the catalytic
antibodies, there was this type of problem
that some cells some immortals but they only
are made up of cancer cells and some cells
who are there, which are actually hybrid of
the cancer cell and the and other foreign
cells ok.
So, named that is the spleen cells which are
making the antibodies. So, they are also you
have to select that which are the cells which
are hybridoma and which are non-hybridoma
cells ok. Here which cells are which DNAs
are recombinant DNA and which DNA are only
the vector DNA.
So, you have to identify that so that is very
important and we will discuss that that how
it is done before that, let us talk about
a little bit of this of this restriction enzyme.
Restriction enzyme I told you that it recognizes
palindromic sequence, it recognizes palindromic
sequence and this is the palindrome A A G
C T T and if you write read from this side
A A G C T T same, so that is a palindrome
a six based palindrome and the enzyme that
recognizes it it is called HindIII.
So, HindIII recognizes this sequence and it
cuts at this point and that point, so between
the two As the phosphodiester bond is cut
is cleaved and that produces this type of
fragments and this is what is called this
that means, there is 5 prime protruding ends
or overhang you can say that means, the 5
prime overhang on this side and the 5 prime
overhang on this. Overhang means where there
is no complementary strand present, there
is no complementary strand present so that
is now this is what is called the sticky cuts.
Sticky cuts means again if you add these two,
then because this A matches with T, this G
matches with C. So, they will come and immediately
sorry, they will so just if you think this
is a block of wood kind of thing. So, then
what you have is basically sorry, so this
part goes inside and then because there is
perfect matching here. So, it goes inside
and then, you can again do the ligation ok.
So, you need a ligase to ligate these two
pieces but this is what is called the sticky
cuts, because there is a recognition point,
there is a overhang which recognizes each
other.
So, similarly there is this Pst1 another enzyme
which recognizes C T G C A G, again this is
a palindrome C T G C A G, ok; so this also
this gives a 3 prime overhang, 3 prime end
is extended ok.; and there is another type
of cleavage that is called blunt cut and that
is they done by EcoRV, EcoRV recognizes G
A T A T C again this is a palindromic sequence,
but it cuts; it is a blunt cut, it cuts straight
away at this at the same point and that gives
what are called blunt ends, ok.
Now, it is it is easy to ligate these two,
because they have a recognition recognition
arm, but here there is no recognition arm
because it is a blunt cut, but there are ways
we are not going into that details but there
are ways to also stick to this to again join
this together ok, but that is a much more
complicated once if you have blunt ends.
On the other hand people try to have sticky
cuts, because sticky cuts are easy to join
just at the DNA ligase, they will join each
other. So, this is what is the restriction
enzymes, there are many restriction enzymes
that have been isolated and this restriction
enzymes is present in bacteria and it is believed
that the bacteria, bacteria has evolved this
restriction enzymes in order to protect themselves
from the from virus from viral infection or
other bacterial in actually viral infection,
so that these restriction enzyme cuts this
external DNA and chops the DNA apart, so that
it cannot infect the bacteria ok.
I think this is what restriction enzymes perform
highly specific DNA cleavage reactions. Bacteria
evolved mechanism to protect themselves from
viral infections ok. So, if this is the viral
one the viral DNA and if this is the host
DNA, now you can always ask this question
that the sequence that is present in the virus
that has to be a palindromic sequence and
that has to be recognized by a restriction
enzyme, which is made by the host DNA but
the host DNA itself can have the same restriction,
same site which is recognized by the same
restriction enzyme.
So, how does it really protect itself from
the self cleavage? So, what it does one interesting
point is mentioned here that the bacteria
labels the A, the DNA self DNA with A added
in with a methylation.
So, it does a methylation of the adenine and
that actually indicates or gives an instruction
to the endonuclease that this is my self DNA
don’t cut it, so better cut the other ones
that means, the viral DNA which is not methylated
where the As are normal As. So that is the
bacterial way of identifying self and the
external ok. Just by putting the methyl group
to the host DNA that means, the bacterial
DNA.
I think now, we are talking about this the
question is how do we identify the recombinant
the cells which are making recombinant DNA.
Now, we did not talk about the vector yet
that we said that it is a piece of DNA which
has which has replicability, we can replicate
when it is put in a bacterial cell ok. So,
this vector is usually what is called what
is found to be what are called plasmid DNA.
Plasmid DNA is a circular DNA, this is called
plasmid DNA; plasmid DNA are circular, DNAs
are circular piece of DNA and these are usually
present, they are not usually always present
outside the it is not present in the chromosomal
DNA, it is not a part of the chromosomal DNA
but it is actually present within the cytosol,
suppose this is the bacterial cell and suppose
these are the chromosomal DNA that means,
containing all the information to make the
proteins, ok.
So, in addition to the chromosomal DNA the
bacteria has developed this what are called
plasmid DNA. The plasmid DNA are the circular
they are circular DNAs and they are they are
not their extra chromosomal DNA and bacteria
involved this plasmid DNA to confer resistance
to different antibiotics. So, this is basically
has evolved it offers resistance or gives
resistance capability.
Resistance to what, resistance to antibiotics
resistance capability I can say, antibiotic
I can add this word antibiotic resistance
capability is provided in the plasmid that
means, the plasmid has a gene if you go a
little bit extrapolate this part that the
plasmids are developed because of to give
to confer antibiotic resistance to the bacteria
that means, the plasmids are making some enzymes
which are destroying our antibiotic before
the antibiotic action is shown, ok.
The beautiful character of this means this
is also very dangerous, because this is the
mechanism by which bacteria developed resistance,
but what was the problem if bacteria had the
resistance gene, it is there in the chromosome.
The time taken from chromosome to express
to the protein is much more than the plasmids,
the plasmids can easily replicate, they have
very good replicability and I said big that
is why they are vector and they can also very
quickly infect other cells, which do not have
this plasmids. So, there could be cross infection
of this plasmid to the bacterial cell which
does not have this plasmid, ok.
And that is how the resistance can be spread
into the entire bacterial population very
quickly, because they are they do not you
do not have to unwind the entire chromosomal
DNA in order to get the; that resistance gene,
it is actually present in the plasmid. So,
plasmids are having these characteristics
that they have anti bacterial resistance genes
present there and also and they and that could
be multi, there could be many genes, one is
acting against tetracycline, another is acting
as penicillin ok, there are many antibiotics
we have class.
So, there may be different antibiotic genes
resistance genes present in the plasmid and
they can cross infect, so they can also easily
multiplied multiply ok. So, in this technique
what is I can show it here that basically
what is what happens here is that you take
a plasmid which has got, now plasmids can
be synthetic also; means now people there
are different plasmids that are available
which are made synthetically synthetic plasmids,
containing different genes expressing for
different proteins, ok.
Like let us take this one, this is called
what is called pBR322. This pBR322 plasmid
has two genes, one gene confers resistance
to the ampicillin, ampicillin is a penicillin;
and there is another gene which confers resistance
to tetracycline, so this is my starting vector
ok. So, this vector is pBR322 if you do not
remember the name, follow the principle.
The principle is that that it should have
the plasmid should have two identifying two
different kinds of genes, expressing for two
different proteins and that gives some the
resistance to two different antibiotics, but
there could be plasmids where there could
be one antibiotic resistance gene, another
could be if a gene for a protein which gives
color, so that is also possible means there
could be different variations of plasmids.
So, this one let us talk about this one, this
has got two types of resistance tetracycline
and ampicillin.
Now, what has been found that this ampicillin
resistance gene inside, there is a site which
is a restriction site for this enzyme Pst1
restriction endonuclease, so that means there
is a restriction site, these sites where the
restriction enzyme works are called restriction
sites. So, this gene has a restriction site
as a restriction site inside the ampicillin
resistance gene ok.
So, if you now treat this vector with Pst1
restriction enzyme, so what will happen this
will cut and this gives a sticky cut, I told
you last time that Pst1 gives a sticky cut
HindIII gives a sticky cut eco EcoRV that
gives a blunt cut, but this is a sticky cut,
so that cuts it into this type of pieces open,
this is the ring is not closed ok. Now, if
you have the foreign DNA, suppose I have the
foreign DNA like this and I am interested
only in this part. Now, if there is a restriction
site here suppose there is a restriction site
here and a restriction site here, these are
recognized suppose by Pst1, ok.
So, then what will happen then you will have
pieces where the ends the restriction this
after treating with the restriction site of
the foreign DNA, remember this part should
not be disturbed at all. So, you are cutting
at the little bit far on this side and little
bit far on this side and then this will have
similar kind of sticky ends.
So, this will be the picture and then when
you ligate, that means, this part now can
ligate with the protruding part or the over
end part of this end, and the other end can
ligate with the overhang part of the vector
DNA which is already treated with the same
restriction enzyme.
So, basically the principle is that you should
use the same restriction enzyme to cut your
external DNA, and also to cut the plasmid
and the plasmid is cut in such a way that
your ampicillin or any of the antibiotic resistance
- the marker, these are called marker genes
and these genes are cut one of the gene is
cut inside. So, that this if you now express
it in the bacteria, so because these two are
not joined. So, this is a disjointed gene.
So, that will not express the enzyme that
destroys the penicillin ampicillin ok. So,
that is the reason that why you got inside
the ampicillin resistance gene.
Now you add this part this is your foreign
DNA with the same sticky ends and this is
your vector DNA with the same sticky ends.
Now, when you add DNA ligase, what will happen,
some of the vector will just again bind to
each other because they can they can recognize
each other. So, some of the; this is the recombinant.
Now, some of the vectors are they not taking
any external DNA, but this external DNA can
recognize that part.
So, now some of the vector I can show it here.
Now, some of the vector will remain like that
remain as earlier. So, they just join with
each other again, but some of the vector will
can take this external piece of DNA. Suppose,
this is the external piece of DNA, and then
you complete the other part, so that means,
you have a now you have a this is recombinant
DNA, r DNA sorry DNA and this is your just
a vector DNA. So, you have now two types of
DNA one is the normal earlier one that means,
this one, and the other one is a vector is
the recombinant DNA.
The difference between the two is that see
remember this was your ampicillin resistance
gene and you have cut inside and then you
have put a foreign DNA in the r DNA recombinant
DNA. So, this recombinant DNA lacks the resistance
against ampicillin, ampicillin ok, because
your gene is disjointed because this is the
gene in between there is something foreign
DNA.
So, now what you do you take this all these
recombinant DNA plus the original vector DNA,
and put it inside that is not difficult. You
can transform E. coli cells that are a particular
type of bacterial cells gram negative bacteria
and you put this; put this DNA some are some
cells will have the vector DNA some cells
will have the recombinant DNA usually one
vector one plasmid in one cell ok.
Now, you grow this in a agar plate where the
bacteria grows can be cultured in a petri
dish, and this is continuity tetracycline.
So, when the bacterial cell, when this population
is transferred here and they are allowed to
grow, but the medium you have already added
tetracycline. If you have added tetracycline
remember tetracycline gene was not touched
at all. So, it was they are in the vector
DNA, and it was there in the recombinant DNA
both so that there will be bacterial colonies
everywhere ok. All the two types of bacterial
cells one containing the vector DNA and the
other containing the recombinant DNA, both
will grow here.
Now, what you do, you take another plate and
two plates, two agar plates, one containing
ampicillin and see now you take two plates
one plate containing tetracycline, and the
other plate containing ampicillin and tetracycline.
So, this is ampicillin and tetracycline – both,
and the other is only tetracycline, these
are both are antibiotics.
So, now, what will happen two matching positions,
that means, if you are taking a bacterial
colony from here, you are taking it at the
same position, that means, you make a grid
kind of thing you make a grid and then according
to the xy coordinate like this bacterial colony
is you place that here ok; and also you have
the same you make the grid and then matching
positions, that means, the colonies are transferred
from here to the same x y coordinate the matching
positions you have to transfer. Now, what
will happen here, because you have here tetracycline
only tetracycline.
So, what will happen that when you transfer
it, so tetracycline resistance means everything
will grow again just to cross check, everything
will grow because both the vector DNA and
the recombinant DNA has the tetracycline gene
but when you develop it into ampicillin plus
tetracycline, then only the colonies only
the bacterial cells which have got only the
vector DNA, because it is the vector DNA which
confers resistance to both ampicillin and
tetracycline.
So, the colonies that are growing here is
containing only vector DNA. So, now, you can
realize that this colony is not present here,
that means, it is not growing, so that means,
this is a recombinant this colony must be
having a r DNA. So, by this technique you
can compare at different points so, and then
like this one it is shown here this is not
there. So, if it is not there, that means,
this colony must be a recombinant DNA.
So, that is how now you take the colony and
then grow in another media, grow in a suitable
medium and then you get the copies of the
when the bacteria grow. So, you get copies
of the recombinant DNA.
As I told you now it is if you are interested
only in the DNA, then isolate the DNA. But
if you are interested in the protein that
this r DNA is making, then you isolate the
protein, but in both the cases you have to
lyse the bacterial cells, and then separate
the DNA nucleic acids and then isolate the
protein of interest. So, this is the this
is I told you this is kind of a semi in vitro,
because initially the reactions with an restriction
enzymes are done in in vitro, and then that
is transformed into the bacterial machinery
and then after that it is in vitro in vivo
technique ok.
So, this is one way is a beautiful way. Now,
many of the problems have been solved that
recombinant DNA technology can generate or
give copies of DNA as well as the proteins
that are the most interesting aspect that
carries out all the reactions that you want
ok. So, a very useful technique which was
developed in the I think the early 1980s,
this recombinant DNA technology was made available
and now it has become a routine technique
in any molecular biology lab ok.
The next session, we will discuss the entirely
in vitro method and that is called the polymerase
chain reaction. So, the second technique,
we first discussed now we will go back to
the first technique, which is the polymerase
chain reaction.
Thank you.
