BioTECHNDLOGY : kINCIPLEs 4 PRoceSsEc
" Biotechn ology Biolechxo logy us she technique ing
bve angontcmà 7o panducis ard
Vn humans.
The Cwopian fodvahon ) Buotrcbnolegy (ErP) ode hnes
biotrchnolog 9
The jntegralion ) natwoal science and ogonismi,
(ellspah Jhese 2, and mslecula anal ogl o
producs and dervice
" Bistechno logy deals wite
’ iorobe- mediated processes(nakiny ewrd, bread, wint eh)
’fh vito feriisattom (test tube baby Programne ).
’ Syathesi and wslngA ?gee
DNA vac clne
Cotsechig degecive
Pincsles 9 Biokechno loqy
1- Cone technuques moden biotechnolog4
jeheing- The technique jn wahich geneke
mateial /oNA l RNA) s hemcally
ltese ard inhodueed lo hott
phenotype.
Maintenance ) sterile
ambiance Jnchenicali
desined puc2obe
Putayot+ cell 4he nanuacture
nanu d otbiatie,
vaccints, enyre) e
2- Baslc Atehs n 9enehcally meoli fying an gonism
2
Idenilicahion 9 DNA wiih besira ble
Thacihonal hybidi saHon deads to in clusion and
mulhblicatin unolesi rabe aenes al
disirabte genes. n genete du desirable
are hdueed.
genes
Avecbor
b) Dboduchion' îolenified bNA Jnto hast : SNA Auch
as plasmid is sed to dlelives an alien piece
9 DNA Jrto host
(© Maintenance ) Dahoaluced DNA Jn kost and tanfe
aien DN
the DNA to ib Prgeny: doApiece
not
aenAeguenee
hare
Called ohigin ol epttah n'Chi) needed fos
astug epicätin . So ih cannat pt~y ikeye
çn prageny cells oganis. Hence ali en
Jntegrated it eaipievt ensne
alog
with host bNA,
-The pocesA jaining and insenig o 7aegn
a
bNA ndott oad opoduee
piece7enehc
meus nehe
Conbi notlo
Combinatton iN called ReCombinat
DNAechnolgg
was podueed by
fisst ecom binaut (2DNA)
Stade Cehen Hesbet Boyer (1972).
They ibolated an .Mbtotc
eniatanee qene (DNA þiece)
artsbloic
monella typhlwlu. t
a platnid ) Sal
nked ut a bla.mid ve chor t transleed in E. Coll, as
a helt, he gere wsa eepressed muttip ued in E. cali.
Töes S) Recombingnt NA Tehnoloy .
Restsichon Enz4mes (mole culas Aisso)3
1-
DNA at specifc Aitea Jnta
The en241neA hat cut
rucleases.
thgmenk. enymes called
lo aclass
They belo restichng
" În 1963, uo en24nes responuble were iholated
ne
in E. cali.
t.cali. we
grooth Q Bacteiophage The othes
netyl groups to bNA
en2ne added
enyme)[Reticion endonuele
ane] cut SNA
(AesbicHon been
estiHon enyme have
Moie than qoa ) bactesia.
ibelate *\om oreh 2.30 Aaund
) he Reticion Enymes y
Namisg
Fist Jetesjncicates gerus. The Aecond
lettes irclcates
cell som ulich they were isolated
No. = the odes in lich t
TRonan en2ymeA were isolated.
fr that stoin 9 bochevo
Types a Restucton Enzymes
ucleotides io He ends
"Erorwcdeases TheH 2emore
) "DNA.
" Endorucle ase), heu cut peciic pei#i witt the bNA
Ey- Eca R1.
’ They bind do pe ca tic 2ecngnihm Aequencel ) the bNA
ardcut Jho tuso hands 'at the speciie point.
’ The fist estoihian endo ntclease i Hind I.TH et
DNA mole cules by 2ecoqricing a spe cifie Bequenee
6 base pairs . This i
Ae cogniion equenoe koi Hindl TL.
Kesticion eudonuclea\es ecogses n a xpecifiq
te DNA,
pandomic nucleotide equenoe
reada
eods
base pais that
1 t s a Sequence t i v o A r a n o l a i n 5 ' 3 d i s e c i o n
e ame
cion
nd in 3 5 ' dire For EcoRI u
Palindhomic nucdeotide Aequence
Eg>
5-GAA TTC-3
3-cTTAAG- 5
bases
Éco R uts t e DNA b
The en244 me cuts both GtA amly when the sequence
DNA Ahands atsame ie GAATTe
ÒNA.
JS present jn
Vectr NA Foreign bNA.
AA
SHcky end
SHcky en.
DNA fragments jain at shcy end
Ke cornhbinant DNA
ecombinant DNA
Stebs un fosmah on
by Eco Ri.
’Kesthicion en2yne
enzne cut he Atraud
hom the Cenb.e A the balindomic ite., but
beween the Aame fuso b e m
oposte Atrauda. Thi deavesendAingle shranded
AhretcheA at the They
ovelag,Aickyends.
Called
They
usith thelr coplnentay cect counte pa
This AHckiness kaciitad acio 9 the en2yrne
DNA
Ahe
ushen cutt by same esticon enzyrne
relullant
by
DNA fragnents hore he sa me
kind
Ahc-ends
and tese re jained bgathe
by DNA -igae
farein DNA
DNA
Sarne esictin enyme cutig
Cplasid
both foreign DNA 4 vecor DNA at specificpainh
Ligases jan oregn
fo
DNA to plamid.
DNAbinauTechuolesy Recombìnant NA maleeule
4 Trans formaton
(rast O) ¬.aa. Celening hott)
Divide
2. Clonng Vecr 1
oforeign
DNA
segrneut and eplicatel Jnid cell jboit
Eg Pladics, Boctenichhage ete.
" PlasrricÙs ’ Plamds ale autenom0Usy
rculor erha- chmosool
eplicalug
DNA
baceria. Some plabmids have only 1-2 copy percel
Rad
Qnd sne have Is-16D copies
pe cell
nurmben peocell)
their genome Ahave
witin
very igh copy umbes
tue baceial ella.
e when the cloni ng vectos are meltpued n host,
Ahe iaked ctaning
DNA i also,muliplied to
the numbes.equalto he copy numbes
Necto.
featanes regwred for cloning ito a vector
oigin greplication (eni) -
" This i seguenee fiom wshere reptication stak.
" Aþiece - sNA iNked to oe Can resicate within
tae st cel. Thi ib abso conholiug he copy
rumbe á inted bNA. So, for gettinà many
Copie & he taget DNA, t ahautd be cloned
n a ve cher ushge
gin utpos igh copynumbo.
(o) Selectable nasker (m gene)
helbs to elect tansformnts
Tti a
and eleminate
ene that the Non-tnsfomnants,
DNA ænodreed æna hot
a biece
"1fbacteium, i ia called tron fur rmation. Such
bacteia Caled Jrarsfor mart. 1f Jranfes mcie
docs no take place , iti transfosmant.
Selectable maskess £. coli includes the genoenceding
resistance Jo anibiots ike omþicilin, Jehraycin
Ompicilin,
kanamycin e c. No5mal Ecoi cetle
chlesophe nl cel
these antbioica,
have no Nesistance against
Cling ies
These tthe eco gmii¡n ie jo. 2etichan en24me.
he alien DNA, he ve ctr need a
To link
Singe o vey Peio recoqrition sites,.
Mere han ome Aecogmtin ites, geneates serera
fraamens.t compli cates Jhe bNA gene clering
eLiaati on alien bNA s caied o t at a hestickon
te pÁsent Jn one & the wo anibioiG TeUstance
gene.
E - I n vecher pBR322,oreign bNA i igated at
Bam HI ite tetacyctine esishance gene.
ks a nelt, necombinant plamid i fomed. 14
does net occuw, it is called
igoten
no- heCombiran plasmid.
EcoRI ClaI Hind I |Resticion ite = Hind I1
ScoRI. bam H1, Sal I,
PvuI Bam H 2 Pru I, st I, cla I.
Pst I amp tesk
BR322.
Sal T "Ant bioHe Aelskqnce gene
ampk, et R.
- Codes bos the proten
.Rof Jnvalred n the
vech
pBR322. ro plald
eplication
when a orcign
that 9ene N Jnocivated, called
1
bactesio qene
çnseional ina ctivahon. Hee, the 9e combl nant
plamid ose tetacyline e|tance due
cinhehtim toseign DNA.
btlA.
" ahen he. pladuds e 3nhoduced into
obtained~
C. coli cels. 3types cceell. a))e
>Nm-Transfor mans : hey hare no lasrmid. So
They
theyae not reulant
sto
eithe dehra uyeline or ampicillin.
’ Tranomant ih nsn-ecombinant plasid .
batha etracuyeline t ompi ollin
hey are hesistant to
Transtomank ith Aeeombiriant blalmud: They ae
rEsiStant on
to ampiclin.
Can be
Recanbinant plasnmids ca selected sut tom
non-ecesmbinant one by placing
anufor mants
on ampicin medium "Then te
on Jetra cyctine medium,.
ae dians}esed
heu etnenki ran gro jn ampiillin mediun but
nst on Jettoay eine, But non- econbinants
both Ahe qvti biota
Thus, Gne antibig tic -helbs to Aetect the
anibiotie Aesis taut
haro nant, The tndcthrated
ecombinant,
gene'
" But hiss tpe selecion ecom bingts
prcedune beco .e it neods
a difleut
Ainultane oes ang
plaing on 2 platea da ving
ing differeut
anibicttca. So, altanatve elechable makea
have dereloped bascd achromo?enie
on abibby to produce
substance
Celowr Jn pesence achramo
20712
(substrate).
Di Hu, a ec combinant DN A is Jnieted jnto
theceding qeneAequence (genc) an enymne , Pqalecosidase
an(isertienal
is jnactvated jnacivation).
So,
Such coleues do not produce amy olous, These are
jdeiied as ecombirant colony.Lawa
have
ies
E he plamid on bacteia faila to unset, it
blue Coloured eolonies in preence
Coloured chromagenic
ubsfrate.
Vectsfor cloning genes on plants animal :
be
Genehc tot Aome paogens
vechis jeh deliveinj genes
tranleimed Jnts welul vects
to blat k animals.
harobachesium tume ka dens (a þakcgen a may du cat jiont)
can eli ve a piece 96) DNA (T-DNA) Jo tanfom
nonal plant celQ jnto a tumo. These tumoi
pathogen,
cells preduee te chemicaa required by the
The tumo jnducing (Ti) plamid Aapobokoium
modifed Jnto a cloring vechor
tametaciens ça madified
net bathogeie but can
but can ube mtcharuSA
which i
to delire gene q intrest jno plant.
nemal cells
" Retaoviruses jn anumals can traham
re wsed to delie
Jnto cancerou celll, So, they
desirable genes Jnto animal cetl.
3. Compelent Hest (fos transfotmaltion wik Reembing t ani
Us a kydeplillic vote cae, it connst pas
Singl bNA memmbrane . So ba ches lal cells a
"ough
cell
modl ompelent" o toke up allen bNAn plasmid
follas concentat on
* Theat boctesial cella witt, a Ahrciic
Qdivalent caion (ealeium)
jn cell sall
DNA entes tho ba ceum
resmbinant DNA on jce,
Dncubale the cells wih
at y2'e (heat ahock)
Place hem breiclby
Put them back on Jce
Bacteia utb secombinant DNA.
take up
hoshcella.
" Other method to Jnhduce alien DNA in
injechons: Dr this; hecombinat DNA s
* Micio-
directy unjeeted Jnto nucles
an ànmal cell.
gun): In hi, cells ame bombarded
* Bioictica(gene uofth iyh elocitey
micropatHele
methed
4 goldor unglen eoated uitaabNA, Tkin
o plans.
*bisas med pah nqen vecho ; hey ine ct the
the
9ecombirant DNA into he host.
Eq> Agrobaclesium tume}aciens
Paoceses ) Recombina nt DNA Technoloy
I.Lsolahn 6en chc Mateial (oNA):
stth
" Treat he bacteial cele/plant
enzymel ike soz4me ( ba bo chehia), celldase (plants),
chihase (ungu) et The cell is broken Aeleaäi vg
DNA ther macrornelecules (RNA, protein, upid
and pslySacchatides).
with 2iboruclease.
RNA i hemoved by reatng
Protein ae hemoved by Jreating wit protease
Ghes melecules re Aermove by abbro pri ahe
theatments.
bA u
wshen chiled eHhans is added, pui Cred
collectio¡n tine thread
precibi tated out as
in
locatorns:
2. Cutting 4 DNA at specifie
PuiteA ÞNA 0s Jncubated wh the esticHm
enyme As a result DNA s oligested. Tese
technique
DNA
fagmens are seþatated by a
called Gel Elecbiopheresi,.
Wels - DNA Bands
Smallest
phoresd
el- unbiyeste
Lanel irahon bNA
fragments
Lane 2toy: igraim
digested oNA
t wsed tofrgmens.
check
the progresston resticcHon
enzyme digeatiom,bNA
moves tobads anoole
sepaated according to thair
DNA AMagrment, ae 4 the ogarose el (a
fhom e a weedN. The Xmaler
þolymer ethated
more tather.
Aized
tagmut Aebeated usith ve ctor DNA also.
precesi cs bright oang
Can be
be een
SNA Atoine d
band heu theu raiation.
Celeured
and ecbose d to uV
utth Ethidium b m i d e .
intrest
s called Eluion. The cutout
and cut vecto! a e .mined ard igase
recombinan DNA
addedo t creates
3. Amblifcahen a Gene terest wsig PcR B
4
.Pslymesase chain Reacton (PcR) i the Auntheii
mulhþle. cobies the gene jntrest in vitho
2 sets - prumes,and,th enzyne DNA plmece
. Pimes are Anall chemieally synthelised
oisonucleetides that are, csmplementary
Jntrest)at Aigh temb. (ac to sepaate the
ond. Each hand act aA JemMdeto
joininy to briers (at s2c)
at the 3'end DNA Jenplates.
Cxtenuin :: ii the addition o) nudesthdes Io
fn
Athe btimer wiga Jhe thermosakle
DNA polymerase Ta
Called Toq, polymease .
1 Usolaled omn hocteiUm, Thms aquaies
1t 2emainl acire
in high mperature duins
igh toandod
Jhe denatusahen dokle DNA.
Through Continu ou 3ehlicaian, tho bNA 2egmmtut
is ampiied wpth 1'bilion capies.
qments
The amsfie d omei con be to Ssgae
with a vecei ko ftber doring
Region o be ampiied
-3'
I
Heat Denatain
3
3 Paime 5
Paimer Strands separared. Anealig
Ls'
DNApolymelase
CTa polymerase)
deoyucleottdes
Extensin
s'
30 ycle
(~ bion timu)
4. nsesHen Recnbinert DNA Jnto Host lele ?
Aeclentaost) cel Jo)gonicm, hey dake up DNA t om
a 2eCom bhont
binGnt DNA beaing ampiein heslsontgene
hanslesied into C. coll es, tho h, Lost cell
be tomes ampion- hrea/s tont celi.
the honfoime d colls aie13e Apread on
coxtahing awipicilin, ny banyeimonk w'u Ugroa
Uinhanslrmed secipiint col wldie,
5, sulaining dhe 7oieigr qene paodudt :
he aim Reconbinon ANA Technsloy (DT) i
to phoduee desiaable biotein.
exbrevsed
a piotein encodi ng fereign gene
heterotogous hode
eembina ut psotein.
The ceLLs usic thavt feeign aene e ron
wsed to
in
JaboratoAy. The ulures ale
exiract the desiaed biotein and
Bepaiaion techniques.
also be mullibied de
Cultue ystem . Hese, he wied edium
while meium
ut Aide
the eido . maintains tha
a cH ve aud podces a
Ce msre
moe
desired proteind.
Jange
BioRCACTORS
the vesael ushich Aaus mateial)
*These ae
Conveated to
re
biolegcally specjic ernymes,
ioobial, slant, aimal or kuman
producks etc,
Bioreacons are Used
to procuce Jarge quantiie
productk. They Can Proces t60 -.laote. culture,
A bisseactor þoovidet opirmal qrot Condli Hos ike
pH, Tempe\atwe, ubshat, Aalt, vitamis, okygen.to
qet desired proddut
mest eommonly wed bioreocts ae
Atiig type (stiased-Jank biohea
dor),
ae a
AidlBase Molo
pH Conbol
foam
Breake
Flat Ertrainmeut
Steaa fot Bladed
Bubbles
Srilisatn impeller: Jnciea
lultre baothy te Oz
’ Steile a'r
trous7e
area
Simle sisred- Tonk
Bioreacor
Spoged Sired- Tank
Bioreccor tayough uhech
stesile açr bubbles
are
Aporged.
wita cUvec base to
uually oylirbhical he eacto content. The
7acilitale
Atirer lacitates ud ox4en a vailibi liy.
the
AUeanaíveby, aú Can be bukbled Jhug
eact.
. Tke BioveacBs has
>foan ehal Aytem
pH conhol
Sampling oit.(n peniodie wikdraual j uulbur),
6. Douon sheam Paesslnq :
It s a Selie proceses uch as eboraHon
andl paiAcahon prduchs after biosyuthetie stage.
"The product foimlated with Aitable prcsemmet
preseoratres.
Such lgosimulahon wtergoes thonsugh eliwcal taials
Ataict
qualitcoel Jetins
