upnacar

ne des
s
Jriveri CHAPTER11
e sh

rship ct
ned shu
with BIOTECHNOLOGY: PRINCIPLES
hove AND PROCESSES
hlett whtta
ped endi no Father oF Genchc Engineeung: Pau Bergh
Ording cone ecombinaut DNA Foww.d 84 Stanluy Cohun Hethe
y
Cohen Ca11.1 Principles of Biotechnolo9 Boyen (1972)
smids and we
ol celk ond 00s ofRecombnant DNA Biotechnology deals with techniques
o using
ng
ve

oranisms or enzymes from oran

Onen hosa
and
el and tenu processes useful to humans, In this sense, making
curd, bread or all mierobe-mediated
11.3 Processes of Recombant wine, which are
thatof Di DNA Technologu processes, could also be used
thought as a form of
nts of DNAT blotechnology However. t s
in a
restricted sense
today. to refer to such of those proCses i c h usc
els, which ca genetically modified organisms tonchig uictme on a
This breoita Plasmid of larger scale. Purther, mary other prgesfcelnkues are
ogy wos so c e u t Doeo cxple,tn vtro
Salmontlla Phimu and usin e A ie u n g
defectiyegedcareull pert 9f blotechnology.
TueEaropeap/Federiuon of Biotechnology (EFB) has
dedarftion of biotechnology that encompasses otn
thuditonalview and modem molecular blotechnology.
T g The The definition given by EFB 1s as follows:
and
integration of natural science organisms,
ells, parts thereof, and molecular analogues for products

services
11.1 PRINCIPLES OF BiOTECHNOLoGY
Among may. the two core techniques that enabled birth
of modernm blotechnology are:
Genetic
(0 engineering: Techniques to alter the
chemistry ol genetic material (DNA and RNA).
 Genuhc engineening
is hybidisoion ofDNA

these into host organismns and thus cha

OAOY
change the
ninciple of Bioechnologym p h e n o t y p e of the host organisn.

1) Bioprocess engunecrng teharice of sterile micr

contaminaton-free) ambience in chemical engineerin l
to enable of the
Genetic growth oniy desie crobe/eukaryotic cetl
Mantenonce large quantities for the manulacture of blotechnological pro

likeantiblotics, vaccines, enymes, ete.

of Seile enginung
t us now understand the conceptual developmient of the
principles
Covdution ol genetic engineeringg
of sexual
the
You probably
appreciate advantages
reproductlon o
sexual reproduction. The former provides oPportunities tor
varlations
and formulation of unique combinations ol genetic setup. some of
-ONA Gue beneticial to organisn as population. Asexua
which
may
be the as well the
technology oning reproduction preserves the genetic inlormaton, while sexual reproducton
permits variation Traditional hybridisation procedures used in plant and
Gene animal breeding.very often lead to inclusion and multiplication of
ransFer undesirable genes alongwith the desired genes. Te tecigues of genetic
engineering which include creation of
reconb NA, Use of
gene cloning and gene transfer,
to Isolate and introduce overcome this nitation and allow
only one opsetof desirable genes without
ntroducing undesirable genes jnto tje tget organism.
E
Tools Doyou know the likelafe ofapiece of DNA. which is
T-DNA transferred
intean gHep organisi? Most likely. this plece of DNA somehiow is We
T DNA not be ableto niuldipt ijsefin the progeny
would
mbl
echnploy *unDi1 when itged cells of the organism. But rlool
intecateefnto genome
the of the
Cang inberted along with the host DNA. This recipient,
it
is because themayalen
mulupYd the
of DNA hde
become of a part chromosome. which has
plece
the ability
replenteIh a
chromosome there is a DNA specific to1.2.1
ofigin 6f replication., which is sequence called
the

multuplication ofresponsible for initiating replicatio

Therefore. for the be yen
it needs to be a
alen any plece of DNA in an organis ucte
part of a
chromosomefs)
which
known
as ongin ol replication'. Thus, an alien has specilie sequen hylg
a

origin of DNA is linked with

replication,
so that,
muluply itsell in the host
this allen
piece DNA can replicate ana
of
ne
making organism. This can also be
called as cloning
mulupie
Let us now identcalcopies of any
o

focus on the first template DNA. cte

ecombinant DNA molecule. The
instance of the
construction of an
DNA emerged irom the construction of the first arunc ule
194
reslstance with a native possibility of linking
gene encoding a a recono
plasmid (autonomously
uchromoont DNol Salmonella typhimuurum. replicating creand
ept
resistance gene acconplished this in 1972 by
Stanley Cole
isolating the antiblote
ponsible for conferring anto NA Irom a plasmid which was
specitic locations became
became possible istance. The cutting of DNA a
with the
discovery of the 5 alled
sms Plasmi Con don ony
anA o
sml fragmawt of Dey
anceNesl as thoe sonciOLOG

DN
PNCPLES AND PROCESSES
sorsr restriction
aen t olecuiar
plasmid enaymes.
enaymes The cut

blnerotoechobel
i n k e d witth the d DNA. plece of DNA
hen
sfer the
These plasmld DN was y-To0
9-TooS Rqured:

C. ect
pleceONAattached to
vector to transfer protbably kknow that
You probabhv
vectors to

mosquito
sas h
s i d can be used as vector to o human body. Enz
elopmer e the DNA Into ne norganistn. The linking of antiblotie
th became
resistance gene (2) Vecfors
possible with the enyme DNA
e ctson cu
sexual repr bination of ctrcular
molecules and jolns their ends. This maklig
autonomously replicating DNA
a new (a) Passengen DNA

poreticSetutuniltie x created.n rou) Host

DA When this DNA is transfe
eherichia col, abaeteu ciosely related to Saimonellait could
HoSt eus

the po plicate ustng he hew

whle ptes. The ability to hosts DNA polymerase enyme and make multipie
multiply coples of antibiotic
Cedure
essed
Ecoll wras called eloning of
oC resistanceg
resistance gene in
E co
hence infen
a n hence
YouYou ccan Bene in E.in o
infer that there are three basic
n d mu odilying an organism steps geneticail
ne
techro dentißcation of DNA with desirable genes:
onbinant De r introduction of the identified DNA into the host: h (Roman
s No-)
limitationd a maintenar
maintenance of introduced DNA in the host and tranerfPthie
e DNK
cies

esirable g to t s p r o e n y
Name
Ssm.
o
S+aun
NA which h 11.2 TooLS OF
REcOMBINANTD oLOGY
so
this plece of DNiKow we know from the farecofngdcuslo tlateenetic
engineering or Orden in which
lls of the orgese nunant Dtelnde Cag beaccomplished only if we have the iso loled pm
ecipient.i my d os.etricto ennes. polymeraseenymesofligases.
thichos orgarsniet try to understandus
vectors
these in detail. some
tho stoain of Back
hich has the a11.2.1 Restriction Enzymes
NA
NA SCqua year(1963the two enzymes responsible for restricting the grow
the
pld
r inioas bacterlophagein Escherichla coll were isolated. One of these added
eof DNAI etyl groups to DNA, while the other cut DNA. The later was called
has spe a rietion endonuclease.
nNA Is liskr h e first restriction endonuclease-Hind l., whose functioning

can r Pended on a specific DNA nucleotide sequence w a s isolated and

led
e cal as tracterised fve years later. It was found that Hlnd Taways cut DNA
e c u l e s at a particular point by recognising a specie eguetee of
as the
Strcton ac o g nxbase
datan
pairs. This specifne base sequence is known
i t i o n sequence for Hind 1. Besides Hui toaye O
thetirste 195
e erncod
n 900 restriction enymes that have been isolated from overstrains
cteria each of which recognise diterentre
The convention for etter of the
name
y (b naming tne ome from the
and the second two letterrs come from species of
the spectes of
h e genus EooRI comes from
o r y o t i c cell from which they were isolated. eg.
alaem
nchla coll RY 13. In EcoRI, the letter R 1s derived from the name
of
Dacaia p o e themselves pm virwses by ragmening
o fthe

Viral DNA witk Endonvclea

 W+GAATTC recogni Hon sik of restithon endo nucea

EcoRT WOLOGY
Nuclea ses strain. Roman numbers following
tne
niames indicate the orde
whleh
isolated from that strain of bacteria:
7 he enzymes were Class of enzymes
Endo- Restrictiorn enzymes Delon calles
Exonucleo ses nucleases.
ol twO Kinds,
These are eronucieases and

-nucleoExonucleases remove nucleotides rom ine ci odie DNA whereas

endonucle

Cndonucieases make ctuts specinc posiuon wthiri Ohe DNA.

at

Kemove Make
restriction endonuciease tunc
a DNA sequence. Once t inds
pecting the lengh ordhe F
ognition sequence, it d
nucleohids c u t s at will bind to the DNA and q e wo strands of the doubl h a

from ends .hellx at speciflc polnts, it thon sugar Phosphate backbonesese

specuc (Figure 11.1(Eachrestrigtioi endonuclease recognises a specife p l e
F DNA et
posihion Pandrorne ncleotide séquences in the DNA
with inthON Action of Restriction enuyme
com
Thee cut both DNA EcoRl cuts the DNA between bases feretn
Whe
Enzymes: Gand Aonly when the sequence
GAATTC is present in the DNA ve th
tDNA orelgn DNA nd-to
0Lysing ug: Lysozymevector
&Cleaving tng V
Reshichion Endonuclease
EcoRI
CSynthesizing engDNA polymerope,
evsehonstiptau
(4) Joining Eng aaAR
DNA Ligase DNA fragnenta jon ticky ends)
e)Alkaline Phosphatose

ecombinant DNA

Pigure 11.1
Steps tn Ecob
tormation ol
recombinant DNA
enyme by action of restrietion endonuc
Erds df DNA
196 ogmuwh a shiky duu b Bases
Do you what
palindromes are?
that form the same These are groups of leten
c.g. "MALAYALAM". As a c a d both forward and backward
word is read in both directio ord-palindrome where the sian
Dse pairs that reads
same opalindrome in DNA is aentation
sequeno
Eco RI cleawes the DNA shrand
t produa
Shicky-ends
 NOLOGY N E S AND PROCESSES

cading i1s
s kept the same. For
n the two strands in 5 example, the followingequencess
directi sequences reads
hesame 33 direction. This is also true if
a d in
the 3 >
5 direction
Jon.
5
3
-GAATTC
-CTTAAG 5
3
Vectos/Ve hicle
DNA muSt
Restriction enugymes cut the strand of DNA a little away from the centre
se e Dalindrome sites, but
between the sarme two
ands. This leaves single stranded portions at bases on the opposite
the ends. There a
howe
bangng natmed
stretehes called sticky ends on each strand (Figure 11.1).(1 Yresena OT
Deseare so because
they lorm hydrogen bonds
splementary cut counterparts. This stickiness of the egdswitt ther (a) Selectobla
faptitate Manker
be action of the enyme DNA igase.
Restriction endonucleases are used in genetie pgneerng to
combinant' molecules of DNA. which are otplecduf DNA form from s Amp gne
ferent sources/ gehomnes.
When cut by the same restriction efyhe. Uhs resaltiant DNA fragments tet g
Kesto
e the siame Kind of steyhds and, these be jotned together Clonin9/
nd-to-end) using DNAigaies gare 11.2). sile
Vector Plasm ids 1ike
oo s t t b n enuyme cutting both foreigm
DNA

CpBR 322),
DNAthd vector DNA at specific
poin
Tr-plasmi4
- Bacheuopha ge
A oo
(-phage)
Lugases join forelgn
DNA to plasmid
Cos mid
6AC
Recombinant DNA

Molecule YAC

Transformation

Ecoll
oning o s Cells divide

recombinant
DNA technolo
of
are 1 1.2 representation
rammatic
 Y u SPlicing: echníqve oF insenion o f adasied
etar.
ino DNA of plasmid
gns OLOGY
ay have realised that normaly, unless one cuts the vector
tor and
the source DNA with the same restriction enzyme, the
recombinant vector
Gel-elechrophoresis molecule cannot be created.
Separation and isolation of DNA tragments: The cutting of DNA S
Mobiliky f DNA restriction endonucleases results n the iragnents of DNA. These frage
can be separated by a technique knoOw as gel electrophoresis. Stn
Cap onds ugon DNA fragments are negatively charged molecules they can be separatd

change sige o by forcing them to move towards the anode under an electric ficld throge
a
medium/matric Nowadays the most commonly used matrix is agaro
which is anatural polymer
oF DNA extrictea ito e c a ne DNAiragments
DNA separate (resolve) according to their size through sieving effect provy
by the agarose gel. Hence. the smaller the fragment
S e . the farther
Look at
moves.
the Fgure 113 and
guss hic
E end of the gel the
sample uDas loaded

Thepeparated DNA fragments can be

Wells
ised onlyafter staining the DNA
-DNA-
2etg wth compound
a known as
ethdium
Larget ds bromide followed by exposure to UV
radiation (you cannot see pure
fragments in thevsible light
DNA ller
without staining. You can see and
brlght ver
orange coloured bands of DNA in
ethidium bromide stained arecm
gelusees
eposed toUV ight (Figure 11.3).The
separated bands of DNA are cut out
Figure 11.3
A typlcal aguro0e gel from the
agarose gel and
cliectroporesis stowing from the gel plece. This stepextracted
is
fra
lane 1 an
ested
as elutiona The DNA knoangen
A ignernts dane 2 to 4) purified in this way fragments
are
Plosmid used
DNA
Tes
tjotningn gwith
recombinant b
them eloning vectoo.
CPBRTi-Plasmid
322, -11.2.2 Cloning Vectors
You know that
within
plasmids and bacteriophages have the
bacterlal cells independent of the control of ability
Bocenophag Bacteriopthages because of their high chromosomal
to
repilc
ta
A-phagt MA3 copy
nutmbers ot thetr number per cell, have
may have only one orgenome within the bacterial cells. Some s
198 plas
15-100 copies per cell. two copies per cell whereas
Theirnumbers can go even others n
OACYACc toink an alen plece of DNA with
muluply its numbers higher. 1t we a
bacteriophage or plasmid D
bacteriophage. Vectors equal to the
used at copy number of the P
nat uncy Deip
easy linking of present, are engineerea waY
from non-recombinants. foreign DNA and selection of inrecon inants
 he oFCHNOLOGYkNs AND PROCESSES

The sttn he following are te eatures that are

required to facilitate cloning
Selechve Makars
ert origin of replication (ori): This is
sequence a
where from Used to
eplication
starts and any
hey en n be made to replicate plece ol DNA when
linked to this
within the host cells his sequence IDENTIFY
Aneette esponsio OSOonng tne copy number of thhe linked DNA.
wants to
seguenceSa
So.
y coples of the target DNA IE shoud be
ste ctot woe origin support high copy number. Non-hrans Fomo
selectable marker In addition to ors, the
:
vector requires a
selectabie marker. whicth helps 1n kdentilying and eliminating non- Transfomad

ch end4 transtormants and

selectively permitting the growth of the
transtormants. Transtormation s a
procedure through which a
plece ot DNA IS introduced in a host bacteriunm (you will study the OP epre ssr tprime
dDNAfne
y after sa process n subsequent section). Normaly. the genes
erncodipe.
und knoe resistance toantibioties such as ampicillin.
etricyclltne or kanamycin. etc.. are
ehlarampheais
considered usejal
Hind , ExoRI:
wed by eqe stlectable
markers lor E. collhe normal E. coli cells dgapt carryetance eshichion ensys
caninot sep against any of these antiblotics
1 the via cloning sites: In order to lin u EcoR 1 C l a i n d
alien
ing. c DNA. the vectos Pru
d bands da very few, Peferabl ie BamH
romide sta recognition sttes tondhe bortfimonly
usedurstretorenanes: Presence of
ght Pa more thariouk redognition sites within
pBR322 Sal1
nds ol DNe the veeto9 kenerate several
rose gel a fragmeñts, iwtiich will complicate the CANtet
The
dece. 1his sp gene cloning (Figure 11.4).
ligation of alien DNA is carried out at CAhbiohc n u l a
The DAA
a restriction site present in one ot the
his
recombinas
two antibiotic resistance genes. ror
example, you can ligate a forelgn DNA
ori:ongin
Figure 11.4 E o l cloning vector pBR322
of ch
winc a r
at the BamH I site of tetraeycune
resistance gene in thhe vector pBRS22
showing restrictlon site
The recombinant plasmids w l ose
Pe IPst 1. Cla n. ori an
tetracycline resistance due to nserto antiblotic resistance genes
foreign DNA but can still be selccted ADandtcro nes 1o
the protetns Involved in the
chrams

out from non-recombinant ones oy epuicatot or the plastid.

plating the transformants o

growing on
S a r

medium. The transtormants

ctracycline containing a
medium a r e
then rlisietreu on cun
e a so t h e
picillin containing r e c o m b i n a n t s will groW In amplcilin
e The
nhe ntaning tetracycline. not on that containing tetracycline. But,
but
containing medium botlh the
o n the m e d i u m containing
recombinants will grow
on gene heips in
ublotics, In this case, o n e
antibiotic
resistanc
antoiote reslstance
erd

wafm transformants, whereas the o t h e r

ecting the
o Aed w Seleckab
Hntibiohics (Awmpicillin, Ttra yelive)
Maakers in Biotechnology
 OOG
of alien
gene gets inactivated due to
DNA.
DNA, and
inserion helps in
selection o
Selection of recomblrnants due to inactvation of antibioties
isa
cumbersome procedure becaisetires simultaneous
on two plates having different antibiotics. Therefore, alt g
selectable markers have been developed whlch different
ecombiniants rom non-recombinants on the basts of theirabilty

to produce colour in he preacnce oLachromogenic substrate)

this, a recombinant DNA Is inserted within the coding neose
a nenzyme B-galactosidase. 1his results into inactvationcqueniceof tof
heed

gene for symthesis of this enzyme. which relerred to as tnsertlonal

is
inactivation. The presence of a chromogenic substrate gves bluet t
Vector coloured colonles if the plasmid in the bacteria does not have
an No
insert. Presence of insert results into
insertionalnacjvation the
of let
-galactosidase gene and
colonies do the
ot praucdny colour
these are ldentifed as
Plans recombinant colonle
Anim (IV) Vectors for cloning genes in plants and aninmals : You may be 1.3
surprised to know that we have lem tie lejon of transiering genes
HgApbocivm
into plants and animals fromabcthid and vinises which hive knowm
tum ipcien Ketro this for ages-
bow to delogenGtginsorm eukaryotic cells and ct
Vinses force them to do
whaPthe.bicteria or viruses want. For example.o
Aarobactettimtkmiackris, a
pathogen of several dicoLplantajs able mbi
Natwna Genhe to deivea piecerot DNA
known as TDNA to translorm normlun
plnt el inó atumor and direct these tumor cells to produce the
Enginun of Plau u ie pathosED. Similarly, retrovinuses in anma us e
Drvebe ability to transform normal cells into cancerous cells.
A
better
understandingof the art of delivering .1
genes by
their eukaryotie hosts has
generated knowledge to
palhogis llth
ot
transtorn
tools pathogens into useful vectors for deliveringgenes to
of lintere
to
humans. The tumor inducing (T) plasmid of
Gene hans Fer tumyaciers has now been moditled into a Agrobacteriumyrib
cloning vector whlch is t
npaaoenic to the plants but is still
able to use the ctic
Biolishe/ to deiver
8ehes of our interest into a variety of mechanistns
Mico
Heot
Shock inJethim n also been disarmed and pliants. mbrar
desirable genes into animal cels. So, once a
are now used to
dei
Disamite gene or a DNA fragnent
has been gted into a sultable vector it is transferred
into
into a
hem Plant or animal host (where it a
bactersal
bacite
muluplies). act

200
11.2.3 Competent Host
Recombinant DNA) (For Transformation with
knos
Since DNA
is a
bydrophilic.
molecule. it cannot win
membrancs. Why? In order to force
bacterial cells must
done bw treating
em tirst e
bea speet 'competent
with made
pass
bacteria to take up theth
plasmid,
e t e n t to take up DNA. This 1s
atme
tn
e

such as calcium, which increases

peciic ooncentration of adivalent cat
he the efficlency
efficiency with which
which DNA ente D
 cNOLOGY x AND PROCESSES

ctevaton ede
akres slz
Acs. Th
cterium through pores
in its ell wall.
edinto such cels by tncubating the Recombinant DNA can then
loped nd
followed
them
by placing them
bacK on iee. This
brielly at
cells with recombinant DNA
42C heat shock). and then Isolaon en
Gethic Makna

hrehe base
enables the bacteria to
nbinant DNA. take up the

in he This is not the only way to introduce aliern DNA CaLu Teoled wih
hod knowrm as micro-injection. recomblirnant DNAinto host cells. In
1s directly
a

s into ina
s Telerre the nucleus of an animal cell. In another method, sultable lorinjected Lyso zyme
ogenle
e bombarded with high
veloeity micro-particles of gold or plants.
ed with DNA in a method known as biolistics or tungsten Cellolase
hactertSubsinte
rüonal t t
method uses
disamed
And
vectors, which when allowed to
pathogen
the cel. trnsier the recombinant DNA into the host.
gene gun. the

Chiinor
do not Now that we have leamt about the tools for
constructing recombinant RNase
onles ut let us discuss the
processes faclitating
reoombinant DNA
Prokoses
technology
nimals:11.3 PROCESSES OF REcOMBINANT DNA TECHNOLOGY DNA
lesson dw
d vinises whes bant DNA technology involves several steps in specifle
nsform e Suenice such as 1solation of DNA. fragmentation of DNA
a
rictionn endonucleases, Isolation of a desired
Les WanL Fr ton of the DNA fragment into a vector, DNA Irent.
Oaenlai mbinant DNA into the host, eulturine tietape bost in a
DM O ransim ndium at large scale and extracto ghtng duprocuct.
c tunor eis s examine cachef theve ficp g m è debaik.
llarty, retrowinunt
Material (DNA)
ells into canema 3.1olatien pr the enetic
ering genes byP all that aucoaclk is the genetic material of all organisms
nowledge to tn thout exet ruoh. In majority of organisms this is
acid o r DNA. In order to cut the DNA with
rdcin w ibonuclele
plasnu etion enzymes. It needs to be in puC c . lreCIun.ohc the
Since the DNA Is enelosed witnin
a Clon.de Omolecules.
cell opet to release DNA along
able to s ranes, we have to break the RNA,
other macromolecules such as protelns,
variety d
and are . o accharides and also liplds. This can be achieved by treating
sere seredmiva
i bacteríal cells/plant or animal tissue with enymes s u c h a s
gure 11.5 DNA hat

Iungus. eONEd by poolind

O etbacteria). cellulase (plant cellsl. chitinase of DNA
know th genes are located long nolecules on

The RNA c a n be removed by

wined with proteins such as histones.
O r m a t i o n w i l l

can be removed by
nent with proteins
ribonue s can
can be removed
be r et by approprae
n t with protease. Othet h o c s the a d d i t i o
purified DNA ultimatelyprecipitates outalier
and thireads n ie
cnno collection ot nne
This can be seen a s
hanol:
n rt ota
take

hslon (Flgure 11.5)

wofa
 OOGY
Specitic Locations
11.3.2 Cutting of DNA at ape

Reshrchon Restriction enyme digestions are periomed by' incubating p

me digesuo me.
the r e s t r i c t i o n eniyme,
at the
at
optimal conditions for M
the optimal condition o
molecules with sis f thatoN
locaes sDecifc enzyme. Agarose uhe
restriction enzyme cestion. DNAis nes e c k
digestion. DNAisane a
Poesslon
of a
towards the positive cleeto argd ed a

it moves
SeqUma olecule, hence
Palind
Alind ome
ome
(Figure 11.3). The prox eral processes,
of ONA The joining ol DNA vo
After havine e
ain
DNA as well a s the vector DNA with a specific rest n enyme,
source
the cut out from
gene of interest' the source DNA and the cut w
vector dto
Cut to Fom space are mixed and ligase is added. 1his results in the preparatior

recombinant DNA

11.3.3 Amplification of Gene of Interest using PCR

Blunt SHcky PCR stands for Polymerase Chain Reaction. In this reactlon, muluipe ec
End end coples of the gene (or DNA of tnterest is synthesised tn vitro ustng twoAPres

Regiorn to be amplited resistar

s the he

spread t

nsforma
uide
unpicilin
Denaturatlon
ence of
electabl
1.3.5 C
Primers Annealing
ben you
o a bact
ost a
DNA pomerse-
deo etid
auhve high kuenv irah

expres-
inical du
Extenslon Alter h
-s ditions-
30 ycles der F
there
202 es ex
eln.
Anplifed sma
cting
Figure 11.6
ratior
Polyherase chan teacton (PCHO
) Primer annealng :
Each cycle has three Thecem
used B/
and u) Extenslon of
primers
steps: 0)
Dena tio
euseds
ded fro
Tag pomrase annealiun 2 Exlensinw