bio ppt by soorya.pptx

8/9/2019 BIO PPT BY SOORYA.pptx

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BIOTECHNOLOGY:PRINCIPLES, PROCESSESANDAPPLICATIONS

BY

B.H.SOORYA RAO

CLASS XII


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DEFINITION

Biotechnolo ! "e#l$ %ith techni&'e$ o( '$in li)e o* #ni$+$o* thei* co+ onent$ o* en-!+e$ (*o+ o* #ni$+$ to *o"'ce

*o"'ct$ #n" *oce$$e$ '$e('l to h'+#n$.

The E'*o e#n Fe"e*#tion o( Biotechnolo ! EFB/ ('*ni$he$it$ i+ lic#tion #$:

The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products andservices.


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PRINCIPLES OF MODERN BIOTECHNOLOGY 1. GENETIC ENGINEERING The techni&'e o( #lte*in che+i$t*! o( enetic +#te*i#l, i.e., tIt (#cilit#te$ the *o%th #n" +'lti lic#tion o( onl! the "e$i*e"2. CULTURE TECHNIQUES The techni&'e o( #lte*in che+i$t*! o( enetic +#te*i#l, i.e., tIt (#cilit#te$ the *o%th #n" +'lti lic#tion o( onl! the "e$i*e"


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TECHNIQUES OF GENETIC ENGINEERING

The technique of genetic engineering includes :(i Cre!tion of reco"#in!nt $N%

(ii Use of gene cloning& !nd(iii Gene tr!nsfer' ith this technique& )e c!n

isol!te !nd introduce onl* the desired genes)ithout introducing undesir!#le genes into

t!rget org!nis"s'


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WHAT IS CLONING?

Clonin o* +#2in +'lti le co ie$ o(#n! te+ l#te DNA nee"$ th#t #n #lien

DNA i$ lin2e" %ith the o*i in o(*e lic#tion # "e$i*e" DNA $e&'ence*e$ on$i1le (o* initi#tin *e lic#tion/ $oth#t #lien (o*ei n0"e$i*e"/ iece$ o(

DNA c#n *e lic#te #n" +'lti l! it$el( inthe ho$t o* #ni$+.


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CRE%TION OF RECO+,IN%NT $N% The first recombinant DNA was constructed by Stanley Cohen and

Herbert Boyer in 1972 by;(i) Isolatin the antibiotic resistance ene by cuttin out a !iece of

DNAfrom a !lasmid which was res!onsible for !ro"idin antibioticresistance# The cuttin of DNA at s!ecific location was done bymolecular scissors $ restriction enzymes #

(ii) The cut !ieces of DNA were then lin%ed with the !lasmid DNA#(iii) The !lasmid DNA acts as "ector to transfer the !ieces of DNA attached

to it& to the reci!ient host bacterium#(i") 'in%in of antibiotic resistant ene (alien DNA) with !lasmid DNA

("ector) was !ossible with en yme DNA ligase, which oins the cutends of DNA molecules#

(") *ence& a new circular& autonomously re!licatin DNA created in vitro isformed called as recombinant DNA.

("i) It re!licates usin the new host+s DNA !olymerase en yme and ma%esmulti!le co!ies# The ability to multiply copies of antibiotic resistantgene in E. coli is called cloning.


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TOO-S OF RECO+,IN%NT $N% TECHNO-OG.

(i) ,estriction en ymes(ii) -olymerase en ymes

(iii)'i ases(i") .ectors(") *ost or anisms


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RESTRICTION ENZYMES

Restriction enzymes belong t o a cl ass of enzymescalled nucleases. They are of two t ypes:

(a) Exonucleases : Remove n ucleotides from theends/terminals of DNA.

(b) Endonucleases : Cut the DNA at specic

position of N-bases anywhere i n the l ength exceptends.


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RESTRICTION ENDONUCLEASES- AN INTRODUCTION

Linn and Arber (1963) isolated two en zymes from E. coliresponsible f or rest ricting the g rowth of bacteriophage, oneof them added m ethyl group (CH 3 -) to DNA while t he ot hercut the DNA into segments and is called restrictionendonucleases.

Smith, Willox and Kelley (1968) isolated andcharacterized the rst restriction endonuclease f rom

Haemophilus inuenzae bacterium called it Hind-II. Hind- II always c ut DNA at a particular site/point by recognizing

a specic s equence o f six base p airs c alled recognition

sequence for Hind-II. Today more t han 900 restrictionenzymes have b een isolated from over 200 strains ofbacteria, each of which recognizes d ifferent recognitionsequences.


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RESTRICTION ENDONUCLEASES (CONTD)

The rec ognition sequence i s pa lindromic w here t he sequ ence of base pa irsreads the same on b oth the DNA strands when the or ientation of reading iskept the same, i.e., 53 or 35 direction e.g. 5- GAATTC- 3 3- CTTAAG-5.

Each RE function by inspecting the length of a DNA and then binds t o theDNA at recognition sequences.

RE cut the t wo strands of DNA double h elix a t specic points i n their sugar-phosphate b ackbone, a little aw ay from the cen tre of palindromic s ite, butbetween the s ame t wo ba ses on both the s trands. As a res ult single s trandedportions are l eft at the en d of DNA in each strand called sticky ends.

Unwanted sel f ligation of vector DNA molecules by re moving phosphategroup from the 5 end of a DNA molecule, leaving a free 5 hydroxyl group,using alkaline p hosphate f rom bacteria (BAP) or c alf intestine ( CAP).

These s ingle s tranded D NAs in each strand form hydrogen bonds with theircomplementary cou nterpart (single st rands of alien DNA). This s tickiness ofthe en ds facilitates the act ion of enzyme D NA ligase. Hence R E are u sed ingenetic engineering t o form Recombinant molecules of DNA


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RESTRICTION EN$ONUC-E%SES (contd

/hen cut by the same ,0& the resultant DNA fra ments ha"e the same%ind of stic%y ends and these can be oined to ether usin DNA li ase#


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NO3ENCLAT4RE OF RESTRICTION

EN5Y3ES The 6*$t lette* o( the n#+e co+e$ (*o+ en'$ #n" the ne7t t%o

lette*$ (*o+ the n#+e o( the $ ecie$ o( the $ ecie$ o( the 1#cte*i'+*o2#*!otic cell/ (*o+ %hich the! #*e i$ol#te".

The ne7t lette* co+e$ (*o+ the $t*#in o( the 1#cte*i'+.

The Ro+#n n'+1e* (ollo%in the$e (o'* lette*$ in"ic#te the o*"e* in%hich en-!+e$ %e*e i$ol#te" (*o+ th#t $t*#in o( the 1#cte*i'+, e. .

#/ Eco R8I i$ i$ol#te" (*o+ Escherichia coli RY 9 1/ Hin"8 II i$ (*o+ Haemophilus in uenzaec/ B#+ H8I i$ (*o+ Bacillus amyloliquefaciens

"/ S#l8II i$ (*o+ Streptomyces albus.


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CLONING VECTOR

Clonin ;ecto* incl'"e$ l#$+i" #n" 1#cte*io h# e$, %hich h#)ethe #1ilit! to *e lic#te %ithin 1#cte*i#l cell$ #n" in"e en"ent o(the cont*ol o( ch*o+o$o+#l DNA. A )ecto* $ho'l" h#)e )e*! hi hco ! n'+1e* o( thei* eno+e %ithin thei* 1#cte*i#l cell $o th#t #lin2e" #lien iece o( DNA, c#n +'lti l! it$ n'+1e* e&'#l to theco ! n'+1e* 1! )ecto* e+ lo!e".

;ecto*$ '$e" #t *e$ent #*e en inee*e" in $'ch # %#! th#t the!hel e#$! lin2in o( (o*ei n0#lien DNA #n" $election o(*eco+1in#nt (*o+ non8*eco+1in#nt.


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CLONING ;ECTOR


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FEAT4RES OF ;ECTOR

Follo%in #*e the (e#t'*e$ th#t #*e *e&'i*e" to (#cilit#te clonin in# )ecto*.. S+#ll $i-e o( )ecto*

O*i in o( *e lic#tion o*i/: Thi$ i$ the $e&'ence (*o+ %he*e*e lic#tion $t#*t$ #n" # iece o( DNA lin2e" to thi$ $e&'ence c#n

1e +#"e to *e lic#te %ithin ho$t cell$. Thi$ $e&'ence #l$o cont*ol$the co ! n'+1e* o( )ecto* DNA o* lin2e" #lien DNA.

Selectable mar er : It hel $ in i"enti(!in #n eli+in#tin non8t*#n$(o*+#nt$ #n" hel in $electin tho$e ho$t cell$ %hich cont#inthe )ecto*, i.e., t*#n$(o*+#nt$.

Cl!"#"$ %#te% : The )ecto* $ho'l" h#)e # (e% o* #tle#$t one


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VECTORS FOR CLONING GENES INPLANTS AND ANIMALS

In plants, the T i-plasmid (Tumour inducing p lasmid)of bacterium Agrobacterium tumefaciens has beenmodied (does not cause tumour ) is used as acloning vector .Similarly, retroviruses also make t he n ormal animalcells i nto cancerou s cel ls. The retroviruses h ave alsobeen disarmed and are n ow used to deliver desirablegenes i nto a nimal cells.

Once a ge ne or a DNA fragment has been ligated intoa vector, it is t ransferred into a bacterial, plant oranimal host.


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/ROCESSES OF RECO+,IN%NT $N%TECHNO-OG.

IT IN;OL;ES THE FOLLO?ING STEPS :

(i) Isolation of DNA

(ii) ra mentation of DNA by ,0s(iii) Isolation of desired DNA fra ment(i") Am!lification of the ene of interest(") 'i ation of DNA fra ment into "ector ("i) Transferrin the recombinant DNA into host("ii) ulturin the host cells in a culture medium at lar e scale("iii) 03traction of desired !roducts#


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/ROCESSES OFRECO+,IN%NT $N%TECHNO-OG.

IN $ET%I-


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DNA that separates out can be remove b! spoo"#n$


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%. C&TTING DNA AT S'(CIFIC LOCATION (! Fr!g"ent!tion of $N% is c!rried out #* incu#!ting 0urified $N%

"olecules )ith restriction en3*"e !t o0ti"!l conditions of te"0er!ture !nd 0Hfor th!t s0ecific en3*"e'

(# %g!rose gel electro0horesis technique is e"0lo*ed to chec4 the 0rogression of restriction en3*"e digestion'

(c The si"il!r 0rocess is re0e!ted )ith 1ector $N%'


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). S('A*ATION ANDISOLATION OF DNA

F*AG+(NTS

(! $N% fr!g"ents #eing neg!ti1el* ch!rged&c!n #e se0!r!ted #* forcing the" to "o1eto)!rds !node under !n electric field through! "ediu"2"!tri5 (!g!rose ' The s"!ller thefr!g"ent si3e& the f!rther it "o1es'

(# $N% fr!g"ents c!n #e 1isu!li3ed #*st!ining #* st!ining $N% )ith ethidiu"

#ro"ide follo)ed #* e50osure to U6r!di!tions' ,right or!nge colour #!nds in$N% #ec!"e 0ro"inent in the gel' These0!r!ted #!nds of $N% !re cut out fro" gel!nd e5tr!cted fro" the gel 0iece' This ste0 is4no)n !s e"ut#on.

,c- /urified $N% fr!g"ents !re used for

reconstructing reco"#in!nt $N% #* 7oiningthe" )ith cloning 1ectors'


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. A+'LIFICATION OF G(N(S OF INT(*(ST &SING 'C*

! /CR st!nds for po"!merase cha#n react#on. In this re!ction& "ulti0leco0ies of gene 2$N% of interest !re s*nthesised in 1itro using t)o sets ofpr#mers.

# The en3*"e $N% 0ol*"er!se'c The en3*"e e5tends the 0ri"er using the nucleotides 0ro1ided in the

re!ction !nd the geno"ic $N% !s te"0l!te'd Continued $N% re0lic!tion of seg"ent of $N% c!n #e !"0lified to

!00ro5i"!tel* 8 #illion ti"es i'e'& 8 #illion co0ies !re "!de'e Re0e!ted re0lic!tion is 0ossi#le #* the use of ther"ost!#le $N%

0ol*"er!se (isol!ted fro" #!cteriu" Thermus aquaticus )hich re"!ins

!cti1e during high te"0er!ture induced den!tur!tion o dou#le str!nded$N%'

f %"0lified fr!g"ent c!n #e used to lig!te )ith 1ector for further cloning'


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/. 0OINING OF DNAs (! %fter cutting $N% )ith s0ecific RE& the cut out gene of interest fro" the

source $N% !nd the cut 1ector )ith s0!ce !re "i5ed !nd lig!se is !dded'

(# This results in the 0re0!r!tion of reco"#in!nt $N%'

. INS(*TION OF *(CO+2INANT DNA INTO HOSTC(LL3O*GANIS+

There !re se1er!l "ethods of introducing the lig!ted $N% into reci0ient

cells'Reci0ient cells !fter "!4ing the" 9co"0etent to recei1e& t!4e u0 $N% 0resent in its surrounding' So& if ! reco"#in!nt $N% #e!ring gene

for resist!nce to !n !nti#iotic (e'g'& !"0icillin is tr!nsferred into E. coli cells& the host cells #eco"e tr!nsfor"ed into !"0icillin;resist!nt cells' If

)e s0re!d the tr!nsfor"ed cells on !g!r 0l!tes cont!ining !"0icillin& onl* tr!nsfor"!nts )ill gro)& untr!nsfor"ed reci0ient cells )ill die' Since& due to !"0icillin resist!nce gene& one is !#le to select ! tr!nsfor"ed cell in the 0resence of !"0icillin' The !"0icillin resist!nce gene in this c!se is c!lled

! se"ectab"e mar4er.


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5. O2TAINING TH( FO*(IGN G(N( '*OD&CT

Ulti"!te !i" of !ll reco"#in!nt technologies is to 0roduce !desir!#le 0roteins #* e50ression of reco"#in!nt $N%' The foreigngene gets e50ressed under suit!#le conditions& #* culturing the hostcell on ! suit!#le "ediu"'


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*(CO+2INANT '*OT(IN

Reco"#in!nt 0rotein is 0roduced if !n* 0rotein encodinggene is e50ressed in heterologous host'

(i The tr!nsfor"ed cells cont!ining cloned gene of interest

!re gro)n in cultures


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BIORE&CTORS

Bio*e#cto*$ #*e '$e" (o* *oce$$in l#* e )ol'+e o(c'lt'*e (o* o1t#inin the *o"'ct o( inte*e$t in l#* e&'#ntitie$. In 1io*e#cto*$ the *#% +#te*i#l$ #*econ)e*te" into $ eci6c *o"'ct$, e. ., en-!+e$.

A 1io*e#cto* *o)i"e$ the o ti+#l con"ition$ (o*#chie)in the "e$i*e" *o"'ct 1! *o)i"in o ti+#l

*o%th con"ition $'ch #$:

i/ te+ e*#t'*e ii/ H iii/ $'1$t*#te i)/ $#lt )/ )it#+in$ )i/ o7! en


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2IO*(ACTO*S


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A--'I ATI4N5 46I4T0 *N4'4 8


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RE&SE&RCH &RE&S INBIOTECHNOLOGY

Th*ee c*itic#l *e$e#*ch #*e#$ o( 1iotechnolo ! #*e :

i/ P*o)i"in the 1e$t c#t#l!$t in the (o*+ o( i+ *o)e" o* #ni$+'$'#ll! # +ic*o1e o* '*e en-!+e.

ii/ C*e#tin o ti+#l con"ition$ th*o' h en inee*in (o* # c#t#l!$tto #ct #n"

iii/ Do%n$t*e#+ *oce$$in technolo ie$ to '*i(! the*otein0o* #nic co+ o'n".


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BIOTECHNOLOGICAL APPLICATIONS INAGRIC4LT4RE

T'ree !(t#!"% )!r #"crea%#"$ )!!* (r!*+ct#!" :

9. A *oche+ic#l 1#$e" # *ic'lt'*e=. O* #nic # *ic'lt'*e

. Genetic#ll! en inee*e" c*o 1#$e" # *ic'lt'*e.

Gree" Re,!l+t#!" h#$ $'ccee"e" in t*i lin the !iel" o( c*o $ "'eto :

i/ I+ *o)e" c*o )#*ietie$ii/ 4$e o( 1ette* +#n# e+ent *#ctice$iii/4$e o( # *oche+ic#l$, i.e. (e*tili$e*$, in$ectici"e$ #n" e$tici"e$

etc.


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0N0TI A''8 4DI I0D 4, ANI5 5 ( 4)

!lants are useful in many ways# 5ome of their uni:ue features are

1# ore tolerant to abiotic stresses such as cold& drou ht& etc#2# *a"e reduced de!endence on chemical !esticides (!est


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PEST 8 RESISTANT PLANTS Se,eral "emat!*e% (ara%#t#%e a -#*e ,ar#et !) (la"t% a"*

a"#mal% #"cl+*#"$ '+ma" be#"$%.

9. A ne+#to"e eloidegyne incognitia in(ect$ the *oot$ o( to1#cco l#nt$ #n"c#'$e$ # *e#t *e"'ction in !iel".=. A no)el $t*#te ! %#$ #"o te" to *e)ent thi$ in(e$t#tion %hich %#$ 1#$e"

on the *oce$$ o( RNA inte*(e*ence RNAi/. RNAi t#2e$ l#ce in #lle'2#*!otic o* #ni$+$ #$ # +etho" o( cell'l#* "e(en$e. Thi$ +etho"in)ol)e$ $ilencin o( # $ eci6c +RNA "'e to # co+ le+ent#*! "$RNA+olec'le th#t 1in"$ to #n" *e)ent$ t*#n$l#tion o( the +RNA $ilencin /.

. The $o'*ce o( thi$ co+ le+ent#*! RNA co'l" 1e (*o+ #n in(ection 1!)i*'$e$ h#)in RNA eno+e$ o* +o1ile enetic ele+ent$ t*#n$ o$on$/th#t *e lic#te )i# #n RNA inte*+e"i#te.

>. 4$in !grobacterium vectors, ne+#to"e8$ eci6c ene$ %e*e int*o"'ce"into the ho$t l#nt .

@. The int*o"'ction o( DNA %#$ $'ch th#t it *o"'ce" 1oth $en$e #n" #nti8

$en$e RNA in the ho$t cell$.. The$e t%o RNA $ 1ein co+ le+ent#*! to e#ch othe* (o*+e" # "o'1le

$t*#n"e" "$RNA/ th#t initi#te" RNAi #n" th'$, $ilence" the $ eci6c +RNAo( the ne+#to"e.

. The con$e&'ence %#$ th#t the #*#$ite co'l" not $'*)i)e in # t*#n$ enicho$t e7 *e$$in $ eci6c inte*(e*in RNA.

. The t*#n$ enic l#nt the*e(o*e ot it$el( *otecte" (*o+ the #*#$ite


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Gene the*# ! i$ # collection o( +etho"$ th#t #llo% co**ection o( # ene "e(ectth#t h#$ 1een "i# no$e" in # chil"0e+1*!o *e&'i*e" # +ech#ni$+ to $%itch o #

"e(ecti)e ene #n" to $'1$tit'te # he#lth! ene co !. The 6*$t clinic#l ene the*# ! %#$ i)en in 9 to # >8!e#* ol" i*l %ith#"eno$ine "e#+in#$e ADA/ "e6cienc!. Thi$ en-!+e i$ c*'ci#l (o*

the i++'ne $!$te+ to ('nction. The "i$o*"e* i$ c#'$e" "'e to the "eletion o( the ene (o* #"eno$ine "e#+in#$e.In $o+e chil"*en ADA "e6cienc! c#n 1e c'*e" 1! 1one +#**o% t*#n$ l#nt#tion

in othe*$ it c#n 1e t*e#te" 1! en-!+e *e l#ce+ent the*# !, in %hich ('nction#lADA i$ i)en to the #tient 1! in ection. B't the *o1le+ %ith 1oth o( the$e # *o#che$ th#t the! #*e not co+ letel!c'*#ti)e.A$ # 6*$t $te to%#*"$ ene the*# !, l!+ hoc!te$ (*o+ the 1loo" o( the #tient#*e *o%n in # c'lt'*e o't$i"e the 1o"!.

A ('nction#l ADA cDNA '$in # *et*o)i*#l )ecto*/ i$ then int*o"'ce" into the$el!+ hoc!te$, %hich #*e $'1$e&'entl! *et'*ne" to the #tient. Ho%e)e*, #$the$e cell$ #*e not i++o*t#l, the #tient *e&'i*e$

e*io"ic in('$ion o( $'ch enetic#ll! en inee*e" l!+ hoc!te$. Ho%e)e*, i( the ene i$ol#te (*o+ +#**o% cell$ *o"'cin ADA i$ int*o"'ce" into cell$#t e#*l! e+1*!onic $t# e$, it co'l" 1e # e*+#nent c'*e.

GENE THER&PY


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MOLECULAR DIAGNOSIS

Recombinant DNA Technology, polymerase chain reaction

(PCR), and Enzyme-lin ed !mm"nosorbent Assay (E#!$A) aresome o% techni&"es diagnosis beca"se '

1. Very low concentration of bacteria or virus (before appearance of visiblesymptoms of disease) can be detected by amplification of their nucleic acids.

2. PCR is now used to detect H V in suspected ! "# patients.$. ! probe is a piece of sin%le stranded "&! that is ta%%ed with a radioactive

molecule and it is used to find it complementary by hybridi'ation.. Presence of normal or mutated %ene can be detected by this method .


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TR%NSGENIC %NI+%-S

Trans$en#c an#ma"s are those that have ha the#r DNA man#pu"ate toposses an e6press an e6tra or 7ore#$n $ene8 e6amp"e trans$en#c rats8 rabb#ts8p#$s8 sheep8 co98 etc.

Rea%!"% )!r $e"et#cal m!*#/cat#!" !) a"#mal%8' Normal physiology and development


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E T H

I C A L I S S U E S

Modication of living organisms by human withoutregulation and some ethical standards can not beallowed.

The modications and uses of living organisms forpublic servi ce h ave r esulted in problems with thegranting of patents for them.

Hence, Govt. of India has se t up organization suchas GEAC (Genetic E ngineering Approval

Committee) which will make decisions regardingthe va lidity of GM research and the sa fety ofintroducing GM organisms for public ser vice.


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BIOPIRACY

Bio iracy is the term used to refer to the use of bioresourcesby N s and other or ani ations without !ro!er authori ationfrom the country and !eo!le concerned without com!ensatory!ayment#

(i) inancially rich industriali ed nations but !oor in biodi"ersitye3!loit the bioresources and traditional %nowled e of de"elo!in andunderde"elo!ed !oor countries rich in biodi"ersity#

(ii) 5ome nations are de"elo!in laws to !re"ent such unauthori ed

e3!loitation of their bioresources and traditional %nowled e#

SO3E ?ONDERF4L FACTS


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SO3E ?ONDERF4L FACTS

m!+%e -#t' '+ma" ear

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