Vectors: Restriction Sites - Biotechnology Principles, Biology, Class 12 Video Lecture

hello friends this video on
biotechnology principles part 13 is
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fear from exams so now let us talk about
the first important feature in a cloning
vector and that is origin of replication
so what do we mean by origin of
replication first of all now whenever we
talk about the process of replication
there has to be a specific site or the
DNA from where the process of
replication will start and that specific
point is known as the origin of
replication so the process of
replication starts from a specific
sequence on DNA and this specific
sequence is known as ma Legion of
replication which is often written as o
our I in short form now it is very
important to know that the process of
replication doesn't just start from
anywhere there are some specific
locations or specific sequences on DNA
from where it starts now why is it
important to know the origin of
replication that's because when the
following gene or the foreign DNA is
being introduced into the host cell our
main purpose is that the foreign gene or
the following DNA should be able to
replicate now for the foreign DNA to be
able to replicate it should get
connected to the origin of replication
that means it should be near that area
where the origin of replication is
located so that means it is important to
know the origin of replication of the
cloning vector so let us suppose this is
the vector so this is the cloning vector
which is for example and suppose this is
a plasmid DNA which is a circular DNA
which is capable of self-replicating but
still in this DNA also there will be
some specific point from where the
origin of replication starts so it is
important that the foreign gene or the
foreign DNA connects to this plasmid DNA
somewhere near the origin of replication
so that it can be a part of the process
replication so now if you look at this
gene even more clues it in a closer way
for example here we have taken the
example of pbr3 22 so what does PDI 322
it is the first widely used vector in
genetic engineering so this was the
first ever used vector so what does this
P be and why is the name so much
complicated so e V at 3:22 so here V
stands for plasmid because this is a
plasmid circular DNA so P stands for
plasmid or what does V and R stands for
so B and starves R stands for the name
of the scientists Bollywood and draw
trees so these are the scientists who
actually created this vector or who at
first who were they were the first ones
to describe this vector in the concept
of genetic engineering and that is why
it was named after them so that is what
P B are so these where the sign is
constructed this pbr3 22 so if you would
look at the origin of replication in
pbr3 22 you see this is where the origin
of replication is located so this is
that part of the DNA or did here there's
no sequence of DNA are located from
where the process of replication will
start now we need to remember that if we
want many copies or multiple copies of
the target DNA then the foreign piece of
DNA has to be linked to this sequence
right so because this is the place from
where the replication will start now if
the falling DNA that is the foreign
countries of DNA if it is linked at this
in this region then that will also
multiply so if this say the plasmid DNA
if it produces some 10 copies then the
foreign DNA will also cute
some penguins so it should be cloned to
a vector whose origin support high copy
number now not for all vectors it is not
necessary that their origin of
replication will support very high copy
number so that also needs to be taken
care that the origin of replication
supports high copy number that means if
the if the following VN is linked to the
origin of replication of that particular
vector it will definitely replicate
because it is linked to the origin of
replication
now since high copy number is associated
with that origin therefore a large
number of copies of DNA will be produced
so these are this is very important that
origin of replication should be located
and it should be associated with high
copy number only then the vector would
be a good cloning vector so here if you
look at this PBR 3:22 the origin of
replication in BB are 322 is derived
from eco line class net so the plasmid
is of e.coli so this is essentially
required for replication once inserted
inside a host and that in fact that is
the purpose of the vector why do we mean
vector so that the foreign DNA is able
to replicate once it is introduced
inside the host body so this there is
one important feature so this also
controls the copy number of linked DNA
as I said just now
so not all vectors origin will support
high copy number so we need to take care
that if you select the vector whose
origin supports a very high copy number
so if the copy number is either more
number of copies of the foreign DNA will
be produced the next important feature
is the restriction sites so what are
these restriction sites frog as the name
suggests restriction site is something
which is related to the restriction
enzymes now by now we know that all the
restriction endonuclease enzymes they
need specific recognition sequence only
when this
that sequence they do their job and what
is the job of the restriction
endonuclease entrance
their job is to cut DNA at specific
locations now what are those specific
locations are determined by the
recognition sequences so those sequences
wherever those sequences are present
they are known as the restriction sites
so restriction sites are extremely
important because wherever restriction
sites are present there you will have
the recognition sequence and dose
recognition sequence will actually
invite the restriction enzymes and then
the DNA will get cut so specific
recognition sites need to be present on
the vector for each restriction enzyme
and also another important thing is that
each restriction enzyme has a specific
recognition sequence or a specific
sequence which it recognizes right so
that means if you talk about different
enzymes like BAM
H 1 cell 1 you talk about eco r1 you
talk about bv u 2 you talk about VST 1
you talk about PB u 1 so there are so
many restriction enzymes but each of
these enzymes have got a specific
restriction site or a specific
recognition sequence so all those
recognition sequence need to be present
on the vector even if not for all of
them but at least for many of them the
restriction site should be present
because if those restriction sites are
not present then the restriction enzymes
will not be able to operate because
restriction enzymes operate only when
the recognition sequence or the
restriction sites are present now let us
look at PVR 322 so here when if as long
as we are discussing the cloning vector
we will take the example of PBR
322 so in PVR 322 as you saw here this
was the origin of replication this
portion is for origin of replication now
in the entire DNA some specific sequence
of DNA is divided or that they are
resolved towards a set of restriction
enzyme so here this region
which you see here this is for the
enzyme pvu
to a games here if you see this region
is for the enzyme cell one if you look
at this region this is for bam h1 you
look at this portion so this is for
another restriction enzyme but in the
tree you look at this region so here you
just portion and squat e o r1 here again
you have restriction sites for enzymes
PB u one and here you have PS t1 so
these are all different restriction
enzymes and all of them need a specific
recognition sequence to do their job and
that recognition sequence is present in
these specific regions now if you see
here this look this is just one vector
by piece one vector has multiple
recognition sites or multiple
restriction sites which contain the
recognition sequence for different
restriction enzymes so in this case what
will happen if you want this vector to
be cut by BAM h1 so now each one will be
able to cut it because the recognition
sequence for BAM h1 is present here so
by h1 will cut it somewhere in this
region so even if you want this to be
cut by eco r1 so that will also be able
to cut it because the recognition
sequence for eco r1 is also present here
but now if you think of a vector or a
plasmid DNA which doesn't have any
restriction site what will happen if
there are no restriction sites then the
restriction enzymes will not be able to
open it if they are not able to operate
then then this vector will not be able
to cut if you cannot cut the vector then
obviously your vector will not be able
to combine with the foreign piece of DNA
so recombinant DNA will not be able to
form so for the formation of
in DNA it is very important that your
vector can be cut at specific location
and for that it is important that
restriction sites are present on your
vector DNA
so rather DNA is either standing active
and they can do their job so here you
can see now once you have the
restriction sites when the restriction
enzyme will be able to cut it now once
it has been cut so a specific region or
a space is being created so that the cut
is foreign DNA so this one is the
foreign piece of DNA which contains our
gene of interest so this foreign piece
of DNA can now come and it can combine
to this plasmid DNA order vector DNA as
a result recombinant DNA is formed so
presence of restriction site is the
second feature which should be present
in a plural vector now the third feature
that needs to be present is the
selectable marker so let us see what are
selectable markers now selectable
markers are basically antibiotic
resistant genes so there are certain set
of genes which are present on the DNA
which are resistant to antibiotics now
here we will take the example of the
same cloning vector that is BB are 3 22
so here we have 2 selectable markers
present one is this a and P and the
other one is T et so what is a and P and
what is T et okay now when we say
antibiotic resistance so there are many
antibiotics which are present so the
antibiotics can be tetracycline
ampicillin chloramphenicol can
so these are all various antibiotics now
each cloning vector should have certain
antibiotic resistant genes which are
resistant at least against some of these
antibiotics so for example here in Billy
I think Eddie - you see that it has a MD
which is for ampicillin and PEP which is
for tetracycline so for these two
antibiotics
there are antibiotic resistant genes
which are present in PVR 322 so these
regions can actually provide resistance
against a tetracycline and ampicillin
right so what does this do why do we
need resistance against anti virus so
that resistance can be provided to the
host against because antibiotics so in
case of pili are 322 it will be now once
this recombinant DNA which is formed
with the help of PBR 322 and the foreign
gene so that once that recombinant DNA
enters inside the host body so it should
be able to provide resistance to the
host against ampicillin and tetracycline
and how does it help it also helps to
identify and distinguish between
transformants and lawn transformants so
here you have got two new terms
what are transform ins and non transform
ants so length for that we will have to
understand the process of transformation
now the presence of selectable marker is
very important but its importance is
understood or you are you are able to
understand its significance once it
enters the host cell once the
recombinant DNA enters into the host
body only then you realize the
importance of the presence of selectable
markers so now before we talk about
transformants and non transformants let
us first try to understand what is
transformation thank you please visit
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