200
BIOLOGY
gene gets ‘inactivated due to insertion’ of alien DNA, and helps in
selection of recombinants.
Selection of recombinants due to inactivation of antibiotics is a
cumbersome procedure because it requires simultaneous plating
on two plates having different antibiotics. Therefore, alternative
selectable markers have been developed which differentiate
recombinants from non-recombinants on the basis of their ability
to produce colour in the presence of a chromogenic substrate. In
this, a recombinant DNA is inserted within the coding sequence of
an enzyme, b-galactosidase. This results into inactivation of the
enzyme, which is referred to as insertional inactivation. The
presence of a chromogenic substrate gives blue coloured colonies if
the plasmid in the bacteria does not have an insert. Presence of
insert results into insertional inactivation of the â-galactosidase and
the colonies do not produce any colour, these are identified as
recombinant colonies.
(iv) Vectors for cloning genes in plants and animals : You may be
surprised to know that we have learnt the lesson of transferring genes
into plants and animals from bacteria and viruses which have known
this for ages how to deliver genes to transform eukaryotic cells and
force them to do what the bacteria or viruses want. For example,
Agrobacterium tumifaciens, a pathogen of several dicot plants is able
to deliver a piece of DNA known as T-DNA’ to transform normal
plant cells into a tumor and direct these tumor cells to produce the
chemicals required by the pathogen. Similarly, retroviruses in animals
have the ability to transform normal cells into cancerous cells. A
better understanding of the art of delivering genes by pathogens in
their eukaryotic hosts has generated knowledge to transform these
tools of pathogens into useful vectors for delivering genes of interest
to humans. The tumor inducing (Ti) plasmid of Agrobacterium
tumifaciens has now been modified into a cloning vector which is no
more pathogenic to the plants but is still able to use the mechanisms
to deliver genes of our interest into a variety of plants. Similarly,
retroviruses have also been disarmed and are now used to deliver
desirable genes into animal cells. So, once a gene or a DNA fragment
has been ligated into a suitable vector it is transferred into a bacterial,
plant or animal host (where it multiplies).
11.2.3 Competent Host (For Transformation with
Recombinant DNA)
Since DNA is a hydrophilic molecule, it cannot pass through cell
membranes. Why? In order to force bacteria to take up the plasmid, the
bacterial cells must first be made ‘competent’ to take up DNA. This is
done by treating them with a specific concentration of a divalent cation,
such as calcium, which increases the efficiency with which DNA enters
2015-16
22
BIOLOGY
gene gets ‘inactivated due to insertion’ of alien DNA, and helps i
n
selection of recombinants.
Selection of recombinants due to inactivation of antibiotics is a
g
on two plates having different
antibiotics. Therefor
e,
nt
alternativ
e
selectable markers have been developed which differentia
te
recombinants from non-recombinants on the basis of their ability
to
p
roduce colour in the
p
resence of a chrom
og
enic substrate.
In
this, a recombinant DNA is inserted within the coding sequence of
n of t
he
on
. Th
e
lonies if
sence of
dase a
nd
fied a
s
may b
e
ng
g
en
es
kno
wn
ells a
nd
am
pl
e,
is ab
le
no
r
ma
l
duce t
he
anima
ls
cells. A
gens i
n
m thes
e
nterest
acterium
ch is
no
hanism
s
imilarly,
deliver
agment
acterial,
Since DNA is a hydrophilic molecule, it cannot pass through ce
ll
membranes.
Wh
y
Wh
? In order to force bacteria to take up the plasmid, t
he
bacterial cells must first be made ‘competent’ to take up DNA. This
is
done by treating them with a specific concentration of a divalent cation,
such as calcium, which increases the efficienc
y
with which DNA enter
s
2015-1
6
220000
this, a recombinant DNA is inserted within the codin
g
se
qu
en
an enzyme,
b
-galactosidase. This results into inactivatio
n
en
zy
me, which is referred to as
insertional inactivati
on
presence of a chromogenic substrate gives blue coloured co
lo
the plasmid in the bacteria does not have an insert. Pre
se
insert results into insertional inactivation of the
â
-g
alactosi
da
se
the colonies do not pr
oduce any colo
ur
, these ar
e identi
fi
recombinant colonies.
(iv)
V
ectors for
VV
c
loni
ng
g
enes i
n
p
lants an
d
a
nimals :
Y
:
ou
m
Y Y
su
rp
rised to know that we have learnt the lesson of transferri
ng
into plants and animals from bacteria and viruses which have
k
this for ages how to deliver genes to transform eukaryotic c
el
force them to do what the bacteria or viruses want. For ex
am
Agrobacterium tumifaciens
, a pathogen of several dicot plants
to deliver a piece of DNA known as
T
-DNA’ to transfor
m
no
plant cells into
a
tumor
and direct these tumor cells to pro
du
r
chemicals required by the pathogen. Similarly, retroviruses in
a
ni
have the ability to transform normal cells into
cancerous
c
better understanding of the art of delivering genes by patho
ge
their eukaryotic hosts has generated knowledge to transfor
m
tools of pathogens into useful vectors for delivering genes of i
nt
to humans. The tumor inducing (T
i) plasmid of
Agr
ob
ac
r
r
tumifaciens
has now been modified into a cloning vector whi
ch
s
more patho
ge
nic to the plants but is still able to use the mec
ha
to deliver genes of our interest into a variety of plants. S
im
retroviruses have also been disarmed and are now used to
d
desirable genes into animal cells. So, once a gene or a DNA fr
ag
has been l
ig
ated into a suitable vector it is transferred into a b
ac
pl
ant or animal host (where it multi
pl
ies).
11.2.3
Competent Host (For Transformation with
Recombinant DNA)
Si DNA is hyd philic ol ul it ot th
BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
201
the bacterium through pores in its cell wall. Recombinant DNA can then
be forced into such cells by incubating the cells with recombinant DNA
on ice, followed by placing them briefly at 42
0
C (heat shock), and then
putting them back on ice. This enables the bacteria to take up the
recombinant DNA.
This is not the only way to introduce alien DNA into host cells. In a
method known as micro-injection, recombinant DNA is directly injected
into the nucleus of an animal cell. In another method, suitable for plants,
cells are bombarded with high velocity micro-particles of gold or tungsten
coated with DNA in a method known as biolistics or gene gun. And the
last method uses ‘disarmed pathogen’ vectors, which when allowed to
infect the cell, transfer the recombinant DNA into the host.
Now that we have learnt about the tools for constructing recombinant
DNA, let us discuss the processes facilitating recombinant DNA technology.
11.3 PROCESSES OF RECOMBINANT DNA TECHNOLOGY
Recombinant DNA technology involves several steps in specific
sequence such as isolation of DNA, fragmentation of DNA by
restriction endonucleases, isolation of a desired DNA fragment,
ligation of the DNA fragment into a vector, transferring the
recombinant DNA into the host, culturing the host cells in a
medium at large scale and extraction of the desired product.
Let us examine each of these steps in some details.
11.3.1 Isolation of the Genetic Material (DNA)
Recall that nucleic acid is the genetic material of all organisms
without exception. In majority of organisms this is
deoxyribonucleic acid or DNA. In order to cut the DNA with
restriction enzymes, it needs to be in pure form, free from other
macro-molecules. Since the DNA is enclosed within the
membranes, we have to break the cell open to release DNA along
with other macromolecules such as RNA, proteins,
polysaccharides and also lipids. This can be achieved by treating
the bacterial cells/plant or animal tissue with enzymes such as
lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).
You know that genes are located on long molecules of DNA
interwined with proteins such as histones. The RNA can be removed by
treatment with ribonuclease whereas proteins can be removed by
treatment with protease. Other molecules can be removed by appropriate
treatments and purified DNA ultimately precipitates out after the addition
of chilled ethanol. This can be seen as collection of fine threads in the
suspension (Figure 11.5).
Figure 11.5 DNA that
separates out can be
removed by spooling
2015-16
BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
0011
the bacterium through pores in its cell wall. Recombinant DNA can then
be forced into such cells by incubating the cells with recombinant DNA
on ice, followed b
y
placin
g
them briefly at 42
0
C (heat shock), and then
putting them back on ice. This enables the bacteria to take up the
recombinant DNA.
This is not the only way to introduce alien DNA into host cells. In a
method known as
micro-injectio
n
, recombinant DNA is directly injected
into the nucleus of an animal cell. In another method, suitable for plants,
cells are bombarded with high velocity micro-particles of gold or tungsten
coat
last
infe
DNA,
11
Reco
sequ
rest
liga
reco
medi
Let
11.3
Reca
with
deox
rest
macr
memb
with
poly
the
ly
so
Y
ou
YY
inte
treatment with ribonuclease whereas proteins can be removed by
treatment with protease. Other molecules can be removed by appropriate
treatments and purified DNA ultimately precipitates out after the addition
of chilled ethanol. This can be seen as collection of fine threads in the
suspension (Figure 11.5).
2015-1
6
22
00
lls are bombarded with h
ig
h veloci
ty
micro-particles of gold or tungsten
ated with DNA in a method known as
biolistics
or
gene gun
. And the
st method uses ‘disarmed pathogen’ vectors, which when allowed to
fect the cel
l,
transfer the recombinant DNA into the host.
Now that we have learnt about the tools for constructing recombinant
A, let us discuss the processes facilitating recombinant DNA technology.
1.3 P
ROCESSES
P
P
OF
R
OF
ECOMBINANT
RR
DNA T
NT
ECHNOLOGY
T
T
combinant DNA technology involves several steps in specific
quence such as isolation of DNA, fragmentation of DNA by
striction endonucleases, isolation of a desired DNA fragment,
gation of the DNA fragment into a vector
, transferring the
combinant DNA into the host, culturing the host cells in a
dium at large scale and extraction of the desired product.
us examine each of these steps in some details.
.3.1 Isolation of the Geneti
c
c
Ma
Ma
te
te
rial
(
(
DNDN
A)A)
call that nucleic acid is the genetic material of all organisms
thout exception. In majority of organisms this is
oxyribonucleic acid or DNA. In order to cut the DNA with
striction enzymes, it needs to be in pure form, free from other
macro-molecules. Since the DNA is enclosed within the
mbranes, we have to break the cell open to release DNA along
th other macromolecules such as RNA, proteins,
lysaccharides and also lipids. This can be achieved by treating
bacterial cells/
pl
ant or animal tissue with enzymes such as
so
zy
me
(bacteria),
ce
ll
ul
as
e
(p
lant cells),
ch
it
in
as
e
(fu
ng
us).
ou know that genes a
r
e located on long molecules of DNA
terwined with
pr
oteins such as histones. The RNA can be removed
by
eatment with ribonuclease whereas proteins can be removed by
Figure 11.5
D
NA
t
ha
t
separates out can be
removed by spooling
202
BIOLOGY
11.3.2 Cutting of DNA at Specific Locations
Restriction enzyme digestions are performed by incubating purified DNA
molecules with the restriction enzyme, at the optimal conditions for that
specific enzyme. Agarose gel electrophoresis is employed to check the
progression of a restriction enzyme digestion. DNA is a negatively charged
molecule, hence it moves towards th