Genetic recombination

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1. Genetic Recombination
By: Bijaya Kumar Uprety

2. Contents
•Transformation,conjugation,transduction,
protoplastfusion.
•Genecloningandtheirapplications.
•Developmentofhybn'domaformonoclonal
antibodies.
•Studyofdrugsproducedbybiotechnology
suchasActivase,Humulin,Humatrope,and
HBetc.

3. Auxotroph:A mutated microorganism having nutritional
requirements that differ from those of unmutated microorganisms
from the same strain.
Cloning vector:genetic element into which genes can be
recombined and replicated
Conjugation:transfer of genes from one prokaryotic cell to another
by a mechanism involving cell-to-cell contact and a plasmid
Diploid: a eukaryotic cell or organism containing two sets of
chromosomes
Electroporation:the use of an electric pulse to induce cells to take
up free DNA
Gene disruption:use of genetic techniques to inactivate a gene by
inserting within it a DNA fragment containing an easily selectable
marker. The inserted fragment is called a cassette, and the process
of insertion, cassette mutagenesis
WORKING GLOSSARY

4. Genetic map:the arrangement of genes on a chromosome
Genome:the total complement of genes of a cell or a virus
Genotype:the precise genetic makeup of an organism
Hybridization:formation of a duplex nucleic acid molecule
with strands derived from different sources by
complementary base pairing
Molecular cloning:isolation and incorporation of a
fragment of DNA into a vector where it can be replicated
Haploid:a cell or organism that has only one set of
chromosomes
Mutagens:agents that cause mutation
Mutant:an organism whose genome carries a mutation
Mutation:an inheritable change in the base sequence of
the genome of an organism

5. Nucleic acid probe:a strand of nucleic acid that can be
labeled and used to hybridize to a complementary molecule
from mixture of other nucleic acids
Phenotype:the observable characteristics of an organism
Plasmid:an extra chromosomal genetic element that has no
extracellular form
Point mutation:a mutation that involves one or only a very
few base pairs
Polymerase chain reaction (PCR):a method used to amplify
a specific DNA sequence in vitro by repeated cycles of
synthesis using specific primers and DNA polymerase
Recombination:the process by which parts or all of the DNA
molecules from two separate sources are exchanged or
brought together into a single unit.

6. Restriction enzyme:an enzyme that recognizes and makes
double-stranded breaks at specific DNA sequences
Shotgun cloning:making a gene library by closing random
DNA fragments
Site-directed mutagenesis:a technique whereby a gene
with a specific mutation can be constructed in vitro
Synthetic DNA:a DNA molecule made by a chemical
process in a laboratory
Transduction:transfer of host genes from one cell to
another by a virus
Transformation:transfer of bacterial genes involving free
DNA

7. INTRODUCTION
•Geneticrecombinationistheprocessbywhichgeneticelements
fromtwoseparatesourcesarebroughttogetherinasingleunit.
•Atmolecularlevel,recombinationcanbethoughtofasthe
movementofgeneticinformationfromonemoleculeofnucleic
acidtoanother.
•ThegeneticexchangeoccurringbetweenhomologousDNA
sequencesfromtwodifferentsourcesistermedasgeneral
recombination.Forthistohappen,identicalsequencesonthetwo
recombiningmoleculesarerequired.
•Theprocessofgeneticexchangewhichoccursineukaryotesduring
sexualreproduction(meiosis)isanexampleofthistypeofgenetic
recombination.

8. •Figure 1.Duringmeiosis,
homologous
recombination can
produce new
combinations of genes as
shown here
betweensimilar but not
identical copies of human
chromosome 1

9. Molecular Events in General Recombination
•Atmolecularlevel,recombinationhasbeenstudied
onlyinprokaryotesandviruses.
•Theprocessistoocomplicatedtobeanalysedinthe
eukaryotes.
•Inbacteria,generalrecombinationinvolvesthe
participationofaspecificproteincalledtherecA
protein.
•TherecAproteinisspecifiedbytherecAgene.Therec
Aproteinishelicalinstructureanditwrapsitself
aroundtheDNAhelix,facilitatingrecombination.
Note:RecAproteinRecAgenemutationinRecA
decreasedlevelofgeneticrecombination.

10. •BacteriawhicharemutantinrecAshowmarkedly
decreasedlevelsofgeneralrecombination.
•Anoverallmolecularmechanismofgeneral
recombinationbeginswithanickinoneofDNA
moleculeswhichleadstotheformationofashort
single-strandedsegment.
•Thehelix-destabilizingproteinscombinesatthissite
andaidsinopeningupoftheDNAdoublehelix.
•TherecAproteinbindstothesingle-strandedfragment
andpositionsitinsuchawaythatannealingoccurs
withacomplementarysequenceintheadjacent
duplex,simultaneouslydisplacingtheresidentstrand.
Note:nickinoneofDNAstrandbindingofhelix
destabilizingproteinsrecAproteinsbindstosingle
strandedfragmentdisplacementofresidentstrand.

11. •Exchangeofgeneticmaterialoccursbetween
homologouschromosomesleadingtothe
formationofrecombinantDNAstructures.
•ThemeansbywhichDNAfragmentsare
introducedintotherecipientare:
transformation,transductionandconjugation.

12. Asimplifiedversionofonemolecularmechanismof
recombinationisshowninfigureinthenextslide)
HomologousDNAmoleculespairandexchangeDNA
segments.
Themechanisminvolvesbreakageandreunionof
pairedsegments.Twooftheproteinsinvolved,asingle-
strandedbinding(SSB)proteinandtheRecAprotein.
Notethattherearetwopossibleoutcomes,depending
onwhichstrandsarecutduringtheresolutionprocess.
Inoneoutcometherecombinantmoleculeshave
patches,whereasintheotherthetwoparental
moleculesappeartohavebeencutandthenspliced
together.

14. In order to detect physical exchange of DNA segments, the
cells resulting from recombination must be phenotypically
different from the parents.
Detection of Recombination
Strains that lack some selectable characteristic that the
recombinants will possess. For instance, the recipient may
not be able to grow on a particular medium on which the
genetic recombinants selected can.
Various kinds of selectable and nonselectable markers (such
as drug resistance, nutritional requirements, and so on) may
be used.

15. (1) Transformation, which involves donor DNA free in
the environment
(2) Transduction, in which the donor DNA transfer is
mediated by a virus
(3) Conjugation, in which the transfer involves cell-to-
cell contact and a conjugative plasmid in the donor cell
Three main processes of genetic recombination in prokaryotes
fragments of homologous DNA from a donor chromosome are
transferred to a recipient cell

16. Genetic Transformation
•Griffith’s Experiment:
Griffith'sexperiment,conductedin1928byFrederick
Griffith,wasoneofthefirstexperimentssuggestingthat
bacteriaarecapableoftransferringgeneticinformation
throughaprocessknownastransformation.
Griffithusedtwostrainsofpneumococcus(Streptococcus
pneumoniae)bacteriawhichinfectmice–atypeIII-S
(smooth)andtypeII-R(rough)strain.TheIII-Sstraincovers
itselfwithapolysaccharidecapsulethatprotectsitfrom
thehost'simmunesystem,resultinginthedeathofthe
host,whiletheII-Rstraindoesn'thavethatprotective
capsuleandisdefeatedbythehost'simmunesystem.Until
Griffith'sexperiment,bacteriologistsbelievedthatthe
typeswerefixedandunchangeable,fromonegeneration
toanother.

17. •Inthisexperiment,bacteriafromtheIII-Sstrainwerekilledbyheat,
andtheirremainswereaddedtoII-Rstrainbacteria.Whileneither
aloneharmedthemice,thecombinationwasabletokillitshost.
GriffithwasalsoabletoisolatebothliveII-RandliveIII-Sstrainsof
pneumococcusfromthebloodofthesedeadmice.Griffith
concludedthatthetypeII-Rhadbeen"transformed"intothelethal
III-Sstrainbya"transformingprinciple"thatwassomehowpartof
thedeadIII-Sstrainbacteria.
•Today,weknowthatthe"transformingprinciple"Griffithobserved
wastheDNAoftheIII-Sstrainbacteria.Whilethebacteriahad
beenkilled,theDNAhadsurvivedtheheatingprocessandwas
takenupbytheII-Rstrainbacteria.TheIII-SstrainDNAcontainsthe
genesthatformtheprotectivepolysaccharidecapsule.Equipped
withthisgene,theformerII-Rstrainbacteriawerenowprotected
fromthehost'simmunesystemandcouldkillthehost.Theexact
natureofthetransformingprinciple(DNA)wasverifiedinthe
experimentsdonebyAvery,McLeodandMcCartyandbyHershey
andChase.

19. •Transformationisaprocesswherecertain
‘competent’bacteriaareabletotakeupfreeDNA
releasedbyotherbacteria.
•TheDNAistakenuponlyinrelativelysmallamount
andcanbeacquiredonlyinasingleevent.
•Onlycertainstrainsarecompetentandthisability
seemstobeaninheritedpropertyoftheorganism.
•Competenceincertainbacteriaisgovernedby
certainproteinswhichincludemembrane-associated
DNA-bindingprotein,cellwallautolysin,andvarious
nucleases.

20. •Increasedtransformationefficiencyincertainspecies
ofbacteriamaybeduetothedeficiencyincertain
DNases,whichnormallydestroyincomingDNA.
•Thenatureofthecellsurfacealsoplaysanimportant
roleindeterminingwhetheracellcantakeupDNA.
•Duringtransformationcompetentbacteriafirstbind
DNAreversiblyandsoonthebindingbecome
irreversible.
•CompetentcellsbindmuchmoreDNAthanthenon-
competentcells(asmuchasthousandtimesmore).
•ThetransformingDNAisboundatthecellsurfaceby
aDNA-bindingprotein.

21. •TheDNAiseitherincorporatedcompletelyintothecelloris
degradedbythehost’snucleaseenzyme,whereonestrandis
degradedandtheotherstrandistakenupbythehostcell.
•TheincorporatedDNAgetsassociatedwithacompetence-
specificprotein,whichremainsattachedtotheDNAsegment
preventingiffromthenucleaseattackuntilitreachesthehost
chromosomewheretherecAproteintakesover[recAisa
productofrecgenes(recombinationgenes).]
•TheDNAisthenintegratedintothegenomeoftherecipient
byarecombinationalprocessandrecAproteinsarereleased.

22. Gene Recombination in Bacteria
•Microorganismscarryoutseveraltypesofgene
recombination,themostcommonofthembeing,
generalrecombination.
•Thisresultsinareciprocalexchangeofgenes
betweenapairofhomologouschromosomes.
•Itcanoccuratanyplaceinthechromosomeand
itresultsfromthebreakageandreunionof
chromosomesleadingtocrossingover.
•Theproductsofrecgenessuchasrec-Aprotein
playanimportantroleinrecombination.

23. •Inbacterialtransformation,anonreciprocaltypeof
recombinationtakesplace.
•Apieceofgeneticmaterialisinsertedintothe
chromosomethroughtheincorporationofasingle
strandtoformastretchofheteroduplexDNA.
•Anothertypeofrecombination,importantinthe
integrationofvirusgenomesintobacterial
chromosomes,issite-specificrecombination.
•Transfectionisaprocesswherebacteriacanbe
transformedwithDNAextractedfromabacterial
virusratherthanfromanotherbacterialcell.

24. •Transfectionhasbecomeausefultoolfor
studyingthemechanismoftransformation
andrecombinationbecause,thesmallsizeof
thephagegenomeallowstheisolationofa
nearlyhomogeneouspopulationofDNA
molecules.

26. (a)Binding of free DNA by a membrane-
bound DNA binding protein.
(b) Passage of one of the two strands
into the cell while nuclease activity
degrades the other strand.
(c) The single strand in the cell is bound
by specific proteins, and
recombination with homologous
regions of the bacterial chromosome
mediated by RecA protein occurs.
The introduction of DNA into cells
by mixing the DNA and the cell
Transformed cell

27. The mechanism of bacterial transformation

28. Agrobacterium tumefaciensMediated Gene Transfer
What isAgrobacterium tumefaciens?
ØBacterial plant pathogen found in the soil that results in tumorous growths and/or roots to
develop in infected plants (“Agrobacteriumtumefaciens” 2001)
ØThis infection is known asCrown Gall Disease(Deacon 2002)
ØThe bacteria transfers a tumor-inducing (Ti) plasmid located in a section of its DNA (known
as T-DNA) into the nucleus of an infected plant cell.
ØThe newly introduced Ti-plasmid is incorporated into the plant genome and is consequently
transcribed (Sforza2002)
ØThe T-DNA that is integrated into the plant genome contains cancer-causing oncogenic
genes and genes that synthesize opines which are excreted by infected Crown Gall cells and
are a food source forAgrobacteriumtumefaciens(González-Cabrera 1998)

29. Transduction
•TransductionisaprocesswhereDNAistransferredfromcell
tocellthroughtheagencyofviruses.
•Suchgenetictransfersfromthedonortotherecipient
throughthevirusescanoccurintwoways,onebeing
generalizedtransductionwheredefectivevirusparticles
randomlyincorporatefragmentsofthehostDNA(virtually
anygeneofthedonor)andtransferittotherecipientcell.
•ThesecondisspecializedtransductionwheretheDNAofa
temperatevirusexcisesincorrectlyandbringsadjacenthost
genesalongwithit,andonlygenesclosetotheintegration
pointofthevirusaretransduced.

30. •Theefficiencyofgeneralizedtransductionisverylow
comparedtothatofaspecializedone.
•Transductionhasbeenfoundtooccurinavarietyofbacteria.
Allphagesdon'tparticipateintransductionandallthe
bacteriaarenottransducible.
•Butthisphenomenonissufficientlywidespreadanditplays
animportantroleingenetictransferinnature.
•Transductionhasbeenfoundtooccurinavarietyof
prokaryotes,includingcertainspeciesoftheBacteria:
Desulfovibrio,Escherichia,Pseudomonas,Rhodococcus,
Rhodobacter,Salmonella,Staphylococcus,andXanthobacter,
aswellasthearchaeanMethanobacterium
thermoautotrophicum.

31. Generalised transduction
•DiscoveredbyZinderandLederberg.
•ThiswasfirstextensivelystudiedinthebacteriumSalmonella
typhimuriumwithphageP
22
•Whenthepopulationofsensitivebacteriaisinfectedwiththe
phage,eithertemperateorvirulent,theeventsofthephage
lyticcyclemaybeinitiated.
•Inalyticinfection,thehostDNAoftenbreaksdownintovirus-
sizedpiecesandsomeofthesepiecesbecomeincorporated
insidevirusparticles.
•Uponlysisofthecell,theseparticlesarereleasedwiththe
normalvirusparticles,sothatthelysatecontainsamixtureof
normalandtransducingvirusparticles.

32. •Whenthislysateisusedtoinfectapopulationof
recipientcells,mostofthecellsbecomeinfected
withnormalvirusparticles.
•However,asmallproportionofthepopulation
receivestransducingparticleswhoseDNAcannow
undergogeneticrecombinationwiththehostDNA.
•Sinceonlyasmallproportionoftheparticlesinthe
lysateisofthedefectivetransducingtype,the
probabilityofadefectivephageparticletransferring
aparticulargeneisquitelowandusuallyonlyabout
onephageparticleinonemilliontransducesagiven
marker.

33. Generalized transduction

34. In above figure in text box 2 instead of reproduction
its replication…………….

35. Specializedtransduction:occursonlyinsometemperate
viruses;DNAfromaspecificregionofthehost
chromosomeisintegrateddirectlyintothevirusgenome-
usuallyreplacingsomeofthevirusgenes.
Generalizedtransduction:hostDNAderivedfromvirtually
anyportionofthehostgenomebecomesapartofthe
DNAofthematurevirusparticleinplaceofthevirus
genome.

36. Specialized transduction
•Incontrasttogeneralized(non-restricted)transduction,which
resultsintransferofanygenefromdonortorecipient
bacterialcell,specialized(restricted)transductionisthat
whichleadstothetransferofonlyspecific(restricted)genes
fromdonortorecipientcell.
•Specializedtransductionismediatedbythosetemperate
bacteriophages(e.g.,lambda(l)phage,mu(m)phageandf80
phage)thatusuallyincorporate(integrate)theirDNAintothe
bacterialchromosome.
•Thephage-DNAiscalledprophageinitsintegratedstatewith
thebacterialchromosome;thebacteriumhavingaprophage
issaidtobelysogenic,andthisphage-host-relationshipis
calledlysogeny.

37. •Lysogenictemperatephagesspontaneouslyswitchoverfrom
lysogenictolyticstateatalowrate(aboutonein195celldivisions)
innature,ortheymaybeinducedtodosobyirradiationwith
ultravioletlight.
•Duringthistransition,theprophageisusuallyexcisedpreciselyfrom
thespecificsiteofintegrationinitsexactlyoriginalform.But
occasionally,itmayexciseimpreciselysothatittakeswithitthat
specificportionofbacterialchromosomewhichliesclosetothesite
ofprophageinsertionandleavesaportionofitsownDNA
remainingintegratedwithinthebacterialchromosome.
•Suchprophageiscalledspecializedtransducingprincipleandis
packagedintoadevelopingphageparticleinsidethehostbacterial
cell.Phageparticlesodevelopediscalledspecializedtransducing
phageandisreleasedafterthehostbacterialcellundergoeslysis.

38. •Onlythosespecializedtransducingphagesareviablethat
containanamountofgreaterthan73%andlessthan110%of
thephage-DNA.Whenaviablespecialized-transducing-phage
infectsanewbacterialcell,itsspecialized-transducing
principlethatalreadycontainsspecificportionofbacterial
chromosomeinsertsintotherecipientbacterialchromosome
thusmakingthelatterdiploidforthatspecificbacterialgene
(partialdiploidorheterogenoteormerogenote).Sincethe
specializedtransducingphageis'defective'phageasithaslost
somegenesduringtheexcision,itfunctionsinrecipient
bacterialcellonlywhenthelatterisalreadyinfectedby
anotherphage(termedashelperphage)thatcontainsthe
missinggenes.

42. Direct contact between two conjugating bacteria is first made via
a pilus. The cells are then drawn together for the actual transfer
of DNA.
Bacterial conjugation (mating) is a process of genetic
transfer that involves cell-to-cell contact.
Conjugation

43. Conjugation involves a donor cell, which contains a
particular type of conjugative plasmid, and a recipientcell,
which does not.
Thegenes that control conjugation are contained in the tra
region of the plasmid. Many genes in the tra region have to
do with the synthesis of a surface structure, the sex pilus .
Only donor cells have these pili,
Thepilimakespecificcontactwithareceptoronthe
recipientandthenretract,pullingthetwocellstogether.
Thecontactsbetweenthedonorandrecipientcellsthen
becomestabilized,probablyfromfusionoftheouter
membranes,andtheDNAisthentransferredfromonecell
toanother.

44. •Conjugationinvolvingthetransferofanentireplasmidisthemost
commonform.TheF
+
plasmid(Fisfertilityfactor)undergoesrollingcircle
replication,meaningthatitisreplicatedasalinearsinglenucleoside
ratherthanacompletecircularstrandofDNAasoccursinreplicationof
thechromosome.AfterpassingthroughthepilusintotheF
-
recipientcell,
thecomplementarynucleosideissynthesizedandtheplasmidislinked
intocircularformbyligase.
•Hfr(highfrequencyrecombination)conjugationoccurswhenaplasmid
fromtheF
+
donor,previouslyrecombinedwiththechromosometo
produceanewHfr
+
cell,ispartiallycopiedalongwithchromosomalgenes
byrollingcirclereplicationandpassedtotherecipientcell.TheDNA
fragmentiscompletedandrecombinedwiththerecipientchromosome,
butsincealloftheplasmidDNAwasnottransferred,therecipientremains
anF
-
cellandcannotparticipateinfurtherconjugationwithothercells.

50. Protoplast fusion
•Protoplastsarethecellsofwhichcellwallsareremovedandcytoplasmic
membraneistheoutermostlayerinsuchcells.
•Protoplastcanbeobtainedbyspecificlyticenzymestoremovecellwall.
•Protoplastfusionisaphysicalphenomenon,
•Duringfusiontwoormoreprotoplastscomeincontactandadherewith
oneanothereitherspontaneouslyorinpresenceoffusioninducing
agents.Byprotoplastfusionitispossibletotransfersomeusefulgenes
suchasdiseaseresistance,nitrogenfixation,rapidgrowthrate,more
productformationrate,proteinquality,frosthardiness,drought
resistance,herbicideresistance,heatandcoldresistancefromonespecies
toanother.
•Protoplastfusionanimportanttoolsinstrainimprovementforbringing
geneticrecombinationsanddevelopinghybridstrainsinfilamentousfungi.
•Protoplastfusionhasbeenusedtocombinegenesfromdifferent
organismstocreatestrainswithdesiredproperties.Thesearethe
powerfultechniquesforengineeringofmicrobialstrainsfordesirable
industrialproperties.

51. •In bacteriology, a protoplast may be defined as —‘the sphere
remaining after Gram-positive bacteria have had their cell
contents lysed; and the bacterial cell wall constitutes are
absent’.
•Productionofhybridplantsthroughthefusionofprotoplasts
oftwodifferentplantspecies/varietiesiscalledsomatic
hybridizationandsuchhybridsareknownassomatichybrids.
•Thetechniqueofsomatichybridizationinvolvesthefollowing
foursteps:
1.Isolationofprotoplasts
2.Fusionoftheprotoplastsofdesiredspecies/varieties
3.Selectionofsomatichybridcells,and
4.Cultureofthehybridcellsandregenerationofhybridplants
fromthem.

52. •Significance of Protoplasts Fusion : The various cardinal
significance of protoplast fusion are, namely :
(1) For hybridization between genera’ or species that are
incapable to cross by the normal and conventional method of
sexual hybridization, and
(2) Significance fully realized in plant kingdom by virtue of the
fact that the hybrid cells are capable of being inducted to
regenerate into whole plants consequently.
•Two types of protoplast fusion:
Spontaneous fusion
Induced fusion

53. Somatic hybridization technique
1. isolation of protoplast
2. Fusion of the protoplasts of desired species/varieties
3. Identification and Selection of somatic hybrid cells
4. Culture of the hybrid cells
5. Regeneration of hybrid plants

54. Spontaneous fusion
•Protoplastduringisolationoften
fusespontaneouslyandthis
phenomenon is called
spontaneousfusion.
•Simplyphysicalcontactis
sufficienttobringaboutthe
spontaneousfusionamongthe
similarparentalprotoplasts.
•Duringtheenzymetreatmentfor
theisolationofprotoplast,itis
foundthatprotoplastsfrom
adjoiningcellsfusethroughtheir
plasmodesmatatoforma
multinucleateprotoplast.

55. •Spontaneousfusionisstrictlyintraspecificandgive
risetohomokaryon.
•However,oncetheprotoplastsarefreelyisolated,
theydon’tfusespontaneouslywitheachother.
•Anexceptionistheprotoplastfrommicrosporocytes
ofsomeplantsoflilyfamilywherefreelyisolated
protoplastfusespontaneously.Thistypeof
spontaneousfusionhasbeenusedtoproduce
intergenericfusione.g.thespontaneousfusionof
microsporocyteprotoplastofLoliumlongiflorumand
Trilliumkamtschalicum.

56. Induced fusion
•Fusionoffreelyisolatedprotoplastsfromdifferent
sourceswiththehelpoffusioninducingchemical
agentsisknownasinducedfusion.
•Normally,isolatedprotoplastsdon’tfusewitheach
otherastheiroutersurfacecarriesnegativecharges
aroundthemandtheyrepeleachother.
•Sothistypeoffusionneedsafusioninducingagent
orsystemwhichactuallyreduceselectronegativityof
theisolatedprotoplastsandallowthemtofusewith
eachother.

57. Various techniques for induced fusion
(a)Inanimals:Sendaivirus(inactivated)—is
requiredtoinitiatetheprocessofinduced
fusion,and
(b)Inplants:
•PEGtreatment;
•NaNO3treatment;
•Calciumiontreatment;
•andelectricalimpulse—areneededto
achievethephenomenonofinducedfusion.

58. PEG treatment
•Polyethyleneglycol(PEG)inducedprotoplastfusionisthe
mostcommonlyusedasitinducesreproduciblehigh
frequencyfusionaccompaniedwithlowtoxicitytomostcell
types.
•Theprotoplastmixtureistreatedwith28-50%PEGfor15-30
min,followedbygradualwashingoftheprotoplaststo
removePEG;protoplastfusionoccursduringthewashing.
•Thewashingmediummaybealkaline(pH9-10)andcontaina
highCa
2+
ionconcentration;thisapproachisacombinationof
PEGandhighpH-highCa
2+
treatments,andisusuallymore
effectivethaneithertreatmentalone.

59. •PEG is negatively charged and may bind to
cations like Ca2+, which , in turn, may bind to
the negatively charged molecules present in
plasmalemma (plasma membrane).
•During the washing process, PEG molecules
may pull out the plasma lemma components
bound to them.
•This would disturb plasma lemma organisation
and may lead to the fusion of protoplasts
located close to each other.

60. Calcium ion treatment
•BhojwaniandRazdan*(1983)devisedamethodinvolving
centrifugation(spinning)oftheprotoplaststakenupina
fusion-inducingsolution(0.05MCaCl2.2H2Oin0.4M
mannitolatpH10.5)for30minutesat50°C,
•Afterwhichthetubeswereincubatedatwater-bath
maintainedat37°Cforadurationrangingbetween40-50
minutes,whichcausedfusionofprotoplaststotheextentof
20-50%.
•However,themethodprovedtobesuperiorincomparisonto
othermethodsincertaincases,whereasthehighpH(10.5)
turnedouttobetootoxicinotherinstances.

61. Electrofusion Technique
•Itisamoreselectiveandlessdrasticapproach
whichutilizeslowvoltage(65-80Vcm
-1
)
electricpulsestoaligntheprotoplastsina
singlerowlikeapearl-chain.
•Thealignedprotoplastscanbemoved,witha
micromanipulator,andpairsofprotoplasts
may be isolatedinindividual
microelectrofusionchambers.
•Thepairsofprotoplastscanbefusedbyavery
briefpulseofhighvoltage(500-1000Vcm-1).

62. •Alternatively,theprotoplastsmaybesubjectedto
masselectrofusion.
•Insuchcasethepopulationofprotoplastsis
subjectedtohighvoltageaftertheyarebrought
closetoeachotherbythelowvoltagecurrent.
•Thehighvoltagecreatestransientdisturbancesin
theorganisationofplasmalemma,whichleadsto
thefusionofneighbouringprotoplasts.
•Theentireoperationiscarriedoutmanuallyin
speciallydesignedequipment,calledelectroporator.

63. Fusion induced by Sodium or Potassium Nitrate
•In this method, equal densities of protoplast from two
different sources are mixed and then centrifuged at 100 g
for 5 mins to get a dense pellet.
•This is followed by addition of 4 ml of 5.5% sodium
nitrate in 10.2% sucrose solution to resuspend the
protoplast pellet.
•The suspended protoplasts are kept in waterbath at 35
0
C
for 5 mins and again centrifuged at 200g for 5 mins.
[g = (1.118 ×10
-5
) R S
2
]; where g is relative centrifugal
force, R is radius of rotor and S is speed of centrifuge in
revolution per minute (RPM).

64. •Thepelletisonceagainkeptinwaterbathat30
0
Cfor30
mins.
•Fusionofprotoplasttakesplaceatthetimeofincubation.
•Thepelletisagainsuspendedby0.1%sodiumnitratefor5-10
mins.
•Theprotoplastsarewashedtwicewithliquidculturemedium
byrepeatedcentrifugation.
•Finally,theprotoplastsareplatedinsemisolidculture
medium.
•Thefrequencyoffusionisnothighinthismethodandsodium
nitrateistoxictocellathighconcentrationyetthisisoneof
theusefultechniqueforprotoplastsderivedfrom
meristematiccells.

66. What is cloning????
•Cloninginbiologyistheprocessofproducing
similarpopulationsofgeneticallyidentical
individualsthatoccursinnature.
•Cloninginbiotechnologyreferstoprocesses
usedtocreatecopiesofDNAfragments
(molecularcloning),cells(cellcloning),
ororganisms.

67. What is DNA cloning?
•When DNA is
extracted from an
organism, all its
genes are obtained
•In gene (DNA)
cloning a particular
gene is copied
(cloned)

68. •To"cloneagene"istomakemanycopiesofit.
•Actofmakingmanyidenticalcopiesofgene.
•Genecanbeanexactcopyofanaturalgene.
•Genecanbeanalteredversionofanatural
gene.
Theterm“genecloning”coversawiderangeof
techniquesthatmakeitpossibletomanipulate
DNAinatesttubeandalsotoreturnittoliving
organismswhereitfunctionsnormally.
What is Gene cloning?

69. Restriction Enzymes
•Bacteria have learned to "restrict" the possibility of attack
from foreign DNA by means of "restriction enzymes”.
•Cut up “foreign” DNA that invades the cell.
•Type II and III restriction enzymes cleave DNA chains at
selected sites.
•Enzymes may recognize 4, 6 or more bases in selecting
sites for cleavage.
•An enzyme that recognizes a 6-base sequence is called a
"six-base cutter”.

70. Type II restriction enzyme nomenclature
•EcoRI –Escherichia colistrain R, 1
st
enzyme
•BamHI –Bacillus amyloliquefaciensstrain H, 1
st
enzyme
•DpnI – Diplococcus pneumoniae, 1
st
enzyme
•HindIII –Haemophilus influenzae, strain D, 3
rd
enzyme
•BglII – Bacillus globigii, 2
nd
enzyme
•PstI – Providencia stuartii 164, 1
st
enzyme
•Sau3AI –Staphylococcus aureusstrain 3A, 1
st
enzyme
•KpnI – Klebsiella pneumoniae, 1
st
enzyme
Why the funny names?

71. Figure 1.1The basic steps in gene cloning.

72. •EversincetheBritishscientists,in1997,carriedoutthe
successfulcloningofsheep(namedDOLLY)bymeticulously
transferringthenucleusfromanudder-cellofanadultsheep
rightintothecytoplasmofanenucleatedfertilizedegg.
•Subsequently,theresulting‘egg’wasneatlytransplantedinto
theuterusofasurrogatemotherwhereiniteventually
developedjustlikeanormalzygote
•Ultimatelyintoa‘normallamb’thathasnowgrownintoa
normaladultsheep.
•Therefore,onemayrightlyconcludeandinfer,basedonthe
aboveactualrealisticexperimentalevidences,that—
•‘whencompleteanimalsaredulyaccomplishedfromthe
somaticcellsofananimal’—itisusuallytermedas‘animal
cloning’.

73. Cloning of Dolly (eg of animal cloning)

74. Cloning of dolly

75. Cloning process
(i) DNA—cloning,
(ii) Cloning larger DNA fragments in specified cloning vectors,
(iii) Cloning Eukaryotic DNAs in bacterial plasmids,
(iv) Cloning Eukaryotic DNAs in phage genomes,
(v) Cloning cDNAs
(vi) Expression cloning.
(vii) Amplifying DNA : The Polymerase Chain Reaction (PCR)

76. DNA-cloning
•TheDNAcloningisnothingbutabroadbasedtechniquewherebylarge
amountofaparticularlyDNA-segmentareproduced.
•Usually,theDNAsegmentwhichistobeclonedisfirstlinkedtoavector
DNA,thatservesasavehicleforcarryingforeignDNAintoasuitablehost
cell,suchasthebacteriumEscherichiacoli.
•Thevector(i.e.,E.coli)essentiallycontainssequenceswhichinturn
permitstobereplicatedwithinthehostcell.InordertocloneDNAswithin
bacterialhoststwotypesofvectorsarecommonlyemployed,namely:
(a)TheDNAsegmenttobeclonedinintroducedintothebacterialcellby
firstjoiningittoaplasmidandsecondly,causingthebacterialcellstotake
uptheplasmidfromthemedium,and
(b)TheDNAsegmentisjoinedtoaportionofthegenomeofthebacterial
viruslambda(λ)whichissubsequentlyallowedtoinfectacultureof
bacterialcells.Thus,ahugequantumofviralprogenyareproduced,each
ofwhichcontainstheforeignDNAsegment.

77. •Byfollowingeitherofthetwomethodsstatedabove
techniquestheDNAsegmentoncegetsinsideabacterium,it
willundergothereplicationprocesswiththebacterial(or
viral)DNAandpartitionedtothedaughtercells(orprogeny
viralparticles).
•Inthismanner,theactualnumberofbacterialcellswhichare
actuallyformed.
•Besides,cloningmayalsobeemployedasaversatilemethod
toisolateinapureformanyspecificDNAfragmentamongsta
relativelylargeheterogeneouspopulationofDNAmolecules.

79. Cloning larger DNA fragments in specified cloning vectors
•Ithasbeenobservedthatneitherplasmidorlambdaphage(λ)vectorsare
adequatelysuitableforcloningDNAswhoselengthismorethan20-25kb.
Thishasrevitalizedtheinterestofresearcherstolookintothe
developmentofseveralothervectorswhichmightfacilitatetoclonemuch
largersegmentsofDNA.However,themostimportanttothesevectors
aretermedasyeastartificialchromosomes(YACs).
•YACsarenothingbutartificialversionsofanormalyeastchromosome.
Theynormallycompriseofalltheelementsofayeastchromosomewhich
areabsolutelynecessaryforthespecificstructuretobereplicatedduring
S-phaseandsubsequentlysegregatedtodaughtercellsduringmitosis,
including:
•Oneofmoreoriginsofreplication,
•Havingtelomersattheendsofthechromosomes,and
•Acentromeretowhichthespindlefibersmaygetattachedduring
chromosomeseparation.

80. •Invariably,theYACsaredesignedinsuchafashionsoastoprovide
essentially:
(a)Agenewhoseencodedproductpermitsthoseparticularcellshavingthe
YACtobeselectedfromthosethatlacktheelement,and
(b)TheDNAfragmenttobeclonedlikeothercells,subsequentlytheyeastcells
shallpickupDNAfromtheirrespectivemediumthatcatersforthepath
wherebyYACsareintroduceddirectlyintothecells.
•IthasbeenobservedthatDNAfragmentsclonedinYACsrangetypically
from100kbto1,000kbinlength.Example:‘Therestrictionenzyme
usuallyrecognizestheeight-nucleotidesequenceGCGGCCGC,whichin
turnspecificallycleavesmammalianDNAintofragmentsapproximately
onemillionbasepairslong’.Fragmentsofthislengthcannowbe
introducedconvenientlyintoYACsandsubsequentlyclonedwithinhost
yeastcells.

81. Fig. Strategy for using YAC

82. Cloning eukaryotic DNA in bacterial plasmid
•AforeignDNAintendedtobeclonedisstrategicallyinserted
intotheplasmidtogivebirthtoarecombinantDNAmolecule.
•However,theplasmidusedforDNAcloningareexclusivelythe
modifiedversionsofthoseoccurringinthebacterialcells.
•Consequently,thebacterialcellsareabletotakeupDNA
fromtheirmedium.Thisparticularphenomenonistermedas
‘transformation’andformsthebasisforcloningplasmidin
bacterialcells.
•Fig.2.8.representstheDNAcloningusingbacterialplasmid.
Firstofalltherecombinantplasmidseachcontaininga
differentforeignDNAinsertareaddedtoabacterialculture
(E.coli)whichhasbeenpreviouslytreatedwithCa2+ions.

83. •ThesebacteriaaregainfullystimulatedtotakeupDNAfrom
theirrespectivesurroundingmediumuponexposuretoabrief
thermal-shocktreatmentyieldingplasmidDNA(purified).
•Secondly,humanDNAarealsoobtainedinthepurifiedform.
SubsequenttreatmentofhumanDNAandplasmidDNAwith
EcoR1resultintothecleavageofhumanandbacterialDNA
intovarioussizedfragments.
•Now,thesesmallfragmentsjointogethertoyield
recombinantDNAswithDNAligaseandthusgiverisetothe
plasmids.Thesepopulationofplasmidsinvariablycontain
varioussegmentsofhumanDNA.
•Incubation of these plasmids with E. coli cells under controlled
experimental parameters ultimately yields plasmid that are
free from E coli.

84. •Ithasbeenobservedthatonlyaverysmall
percentageofthecellsarecompetenttopickupand
retainoneofthecombinantreplicatemolecules.
•Onceitistakenuptheplasmidundergoesreplication
autonomouslywithintherecipientandis
subsequentlypassedontoitsprogenyduringcell
division.
•Theisolatedrecombinantplasmidscanthenbe
treatedwiththesamerestrictionenzymesusedin
theirformation,thatreleasestheclonedDNA
segmentsfromtheremainderoftheDNAwhich
servedasthevector.Thus,theclonedDNAcanbe
separatedfromtheplasmid.

86. Cloning eukaryotic DNAs Phage genome
•Abacteriophase,ormorecommonlyaphageisavirusparticlewhichinfectsa
bacterialcell.
•Infact,aphageparticlenormallycomprisesoftwoessentialcomponents;first,a
phageheadthatcontainsthegeneticmaterialandsecondly,atailthroughwhich
thegeneticmaterialisinjectedintothehostcell.
•Interestingly,oneofthemostbroadlyexploredofthethesephageparticles,
termedBacteriophageLambda[orbacteriophage(λ)],hasmoreorlessturnedout
tobeacommonlyemployedcloningvector.
•Thegenomeoflambdaisalinearanddouble-strandedDNAmoleculehaving50kb
length.
•Inusualpractice,themodifiedstrain(mutant)employedinmostcloning
experimentscontainstwocleavagesitesfortheenzymesEcoR1thatultimately
fragmentsthegenomeintothreelargesegments.
•However,thetwooutersegmentsessentiallycontainallinformationsrequiredfor
theinfectiousgrowth,whereasthemiddlefragmentcouldberejected
convenientlyandreplacedsuitablybyapieceofDNAupto25kbinlength.

87. •Thetwooutersegmentsofthebacteriophageundergosplicingwith
eukaryoticfragmenttoresultintotheformationofrecombinantDNA.
Consequently,therecombinantDNAmoleculescanbepackagedinto
phageheadsinvitroandinturnthesegeneticallyengineeredphage
particlemaybeemployedtoinfecthostbacteria.
•Oncegainingentryintothebacteria,theeukaryoticDNAsegmentis
adequatelyamplifiedalongwiththeviralDNAandsubsequentlypackaged
intoanaltogethernewgenerationofvirusparticlethatarereleasedwhen
thecellundergoeslysis.
•Thereleasedparticlethusobtainedinfectnewcells,andwithoutanyloss
oftimeeitheraplaque(Azoneoflysisorcellinhibitioncausedbyvirus
infectiononalawnofcells)oraclearspotinthe‘bacteriallawn’isvisible
distinctlyatthesiteofinfection.Eachplaque,whichisnothingbutazone
oflysis,possessesmillionsofphageparticle,eachcarryingasinglecopyof
thesameeukaryoticDNAsegment.)

88. Fig. Sequence for cloning DNA fragments in Lambda (I)
phage.

89. Cloning cDNA
•Thecentraldogmaofmolecularbiologyoutlinesthatinsynthesizing
proteins,DNAistranscribedintomRNA,whichistranslatedintoprotein.One
differencebetweeneukaryoticandprokaryoticgenesisthateukaryoticgenes
cancontainintrons(interveningsequences)whicharenotcodingsequences(in
contrastwithexonswhicharecodingsequences),andmustberemovedfromthe
RNAprimarytranscriptbeforeitbecomesmRNAandcanbetranslatedinto
protein.Prokaryoticgeneshavenointrons,sotheirRNAisnotsubjecttosplicing.
•Oftenitisdesirabletoexpresseukaryoticgenesinprokaryoticcells.Asimplified
methodofdoingsowouldincludetheadditionofeukaryoticDNAtoavector,
sometimesaprokaryotichost,whichwouldthentranscribetheDNAtomRNA
andthentranslateittoprotein.However,aseukaryoticDNAhasintrons,and
sinceprokaryoteslackthemachinerytosplicethem,thesplicingofeukaryotic
DNAmustbedonepriortoaddingtheeukaryoticDNAintothehost.ThisDNA,
whichwasmadeasacomplementarycopyoftheRNAandhasnointrons,is
calledcomplementaryDNA(cDNA).Toobtainexpressionoftheproteinencoded
bytheeukaryoticcDNA,prokaryoticregulatorysequenceswouldalsobe
required(e.g.apromoter).

90. •ThecloningthathasbeendescribedherewillworkforanyrandompieceofDNA.
ButsincethegoalofmanycloningexperimentsistoobtainasequenceofDNA
thatdirectstheproductionofaspecificprotein,anyprocedurethatoptimizes
cloningwillbebeneficial.OnesuchtechniqueiscDNAcloning.
•TheprinciplebehindthistechniqueisthatanmRNApopulationisolatedfroma
specificdevelopmentalstageshouldcontainmRNAsspecificforanyprotein
expressedduringthatstage.Thus,ifthemRNAcanbeisolated,thegenecanbe
studied.
•mRNAcannotbecloneddirectly,butaDNAacopyofthemRNAcanbecloned.(In
thisregard,thetermcDNAisshortfor"copyDNA".)Thisconversionis
accomplishedbytheactionofreversetranscriptaseandDNApolymerase.The
reversetranscriptasemakesasingle-strandedDNAcopyofthemRNA.
•ThesecondDNAstrandisgeneratedbyDNApolymeraseandthedouble-stranded
productisintroducedintoanappropriateplasmidorlambdavector.

91. •InordertoclonecDNAs,firstofallasizablepopulationof
mRNAisisolated;
•secondly,itisemployedasatemplatetoprovideasingle-
strandedDNAcomplement;
•thirdly,theresultingproduct(singlestranded)isduly
convertedtoadoublestrandedpopulationwiththehelpofa
DNApolymerase;
•andfourthly,theyarefinallycombinedwiththedesiredvector.
ItisquiteevidentthatessentiallymRNApopulationstypically
consiststhousandsofaltogetherdifferentspecies,andaswith
experimentsemployinggenomicDNAfragments,theclones
shouldbeinvariablyscreenedtoisolateonlyoneparticular
sequencefromaheterogeneouspopulationofrecombinant
molecule.

92. Fig: Sythesis whereby cDNA
get cloned in plasmid.
itmaybeobservedthatwhen
polypeptide(A)andmRNAare
annealed,itprovidesasmall
segmentofprimerattachedto
poly(A)tothetailofmRNA.Now,
withthehelpofreserve
transcriptasetheprimertopoly
(A)getsfullydeveloped.Alkali
helpsintheseparationofDNA
andRNAstrandstogiveriseto
fullydevelopedprimeralone,
whichontreatmentwithRNA
polymerase1yieldsthecombined
product.Theresultingproduct
whendigestedwithS1nuclease
twoseparatestrandsofthe
primerandpoly(A)areobtained.
Lastly,integratecDNAintothe
plasmidvectorthatwillproduce
abacteriumwhereinDNAcanbe
cloned.

93. Expression cloning
•ExpressioncloningisatechniqueinDNAcloningthatusesexpression
vectorstogeneratealibraryofclones,witheachcloneexpressingone
protein.
•Thisexpressionlibraryisthenscreenedforthepropertyofinterestand
clonesofinterestrecoveredforfurtheranalysis.
•AnexpressionvectorisarelativelysmallDNAmoleculethatisusedto
introduceandexpressaspecificgeneintoatargetcell.Oncethe
expressionvectorisinsidethecell,theproteinthatisencodedbythe
geneisproducedbythecellulartranscriptionandtranslationmachinery.
•Usuallytheultimateaimofexpressioncloningistoproducelarge
quantitiesofspecificproteins.
•Expressionvectorsareusedformolecularbiologytechniquessuchassite-
directedmutagenesis.Ingeneral,DNAvectorsthatareusedinmany
molecularbiologygenecloningexperimentsneednotresultinthe
expressionofaprotein.

94. •Expressionvectorsareoftenspecificallydesignedtocontainregulatory
sequencesthatactasenhancerandpromoterregions,andleadto
efficienttranscriptionofthegenethatiscarriedontheexpressionvector.
•Expressionvectorsarebasictoolsforbiotechnologyandtheproductionof
proteinssuchasinsulinthatareimportantformedicaltreatmentsof
specificdiseaseslikediabetes.
•Expressionvectorsareusedformolecularbiologytechniquessuchassite-
directedmutagenesis.Ingeneral,DNAvectorsthatareusedinmany
molecularbiologygenecloningexperimentsneednotresultinthe
expressionofaprotein.
•Expressionvectorsareoftenspecificallydesignedtocontainregulatory
sequencesthatactasenhancerandpromoterregions,andleadto
efficienttranscriptionofthegenethatiscarriedontheexpressionvector.
•Expressionvectorsarebasictoolsforbiotechnologyandtheproductionof
proteinssuchasinsulinthatareimportantformedicaltreatmentsof
specificdiseaseslikediabetes.

95. :::::Note:::::
•AllvectorsusedforpropagationofDNAinsertsinasuitablehostarecalled
cloningvectors.
•Butwhenavectorisdesignedfortheexpressionofi.e.productionoftheprotein
specifiedby,theDNAinsert,itistermedasexpressionvector.
•Asarule,suchvectorscontainatleasttheregulatorysequences,i.e.promoters,
operators,ribosomalbindingsites,etc,havingoptimumfunctioninthechosen
host.
•Therearemany typesofcloningvectors.Genetically
engineeredplasmidsandbacteriophages(suchasphageλ)areperhapsmost
commonlyusedforthispurpose.Othertypesofcloningvectorsincludebacterial
artificialchromosomes(BACs)andyeastartificialchromosomes(YACs).
•Inthecaseofexpressionvectors,themainpurposeofthesevehiclesisthe
controlledexpressionofaparticulargeneinsideaconvenienthostorganism
(e.g.E.coli).Controlofexpressioncanbeveryimportant;itisusuallydesirableto
insertthetargetDNAintoasitethatisunderthecontrolofa
particularpromoter.SomecommonlyusedpromotersareT7
promoters,lacpromoters(blapromoter)andcauliflowermosaicvirus's35s
promoter(forplantvectors).

96. Applications
ProteinProduction-Anexpressionvectorwith
proteinproducingDNAisimportedintoacell
throughtheuseofplasmidsintoabacterial
cell.Thiscausesthebacterialcelltothen
producesaidprotein.Thismethodcanbe
usedinmedicinetofarmproteinsonawide-
scalesuchasInsulin,Erythyroprotein,or
TissuePlasminogenActivator.

97. Ingenetics,apromoterisa
regionofDNAthatfacilitates
thetranscriptionofa
particulargene.Promotersare
locatednearthegenesthey
regulate,onthesamestrandand
typicallyupstream(towards
the5'regionofthesense
strand).
Anenhancerisashortregion
ofDNAthatcanbebound
withproteins(namely,thetrans-
actingfactors,muchlikeaset
oftranscriptionfactors)to
enhancetranscriptionlevels
ofgenes(hencethename)in
agenecluster.
Fig. The creation of an expression vector with which proteins
can be grown.

98. Amplifying DNA: The polymerase chain reaction (PCR)
•Gene Amplification through Polymerase chain reaction
•The polymerase chain reaction is one of the powerful gene
amplification technique developed by Kary Mullis in 1985.
•It generates microgram quantities (upto billion copies) of DNA
copies of the desired DNA (or RNA) segment, present even as
a single copy in the initial preparation, in a matter of few
hours.
•The PCR utilizes the following:
1. DNA preparation containing the desired segment to be
amplified
2. Two nucleotide primers (about 20 bases long) specific, i.e.
complementary, to 3’-borders (the sequences present at the
3’-ends of the two strands) of the desired segment,

99. •Thefourdeoxynucleosidetriphosphates[dTTP(deoxythymidine
triphosphate),dCTP(deoxycyctidinetriphosphate),dATP(deoxyadenosine
triphosphate)anddGTP(deoxyguanosinetriphosphate),andaheatstable
DNApolymerase,e.g.Taq(isolatedfromthebacteriumThermus
acquaticus),pfu(fromPyrococcusfuriousus)andVent(fromThermococcus
litoralis)polymerases.PfuandVentpolymerasesaremoreefficientthan
theTaqpolymerase.
Procedure of PCR:
•At the start of PCR, the DNA from which a segment is to be amplified, an
excess of the two primer molecules, the four deoxynucleoside
triphosphates and the DNA polymerase are mixed together in the reaction
mixture that has appropriate quantities of Mg
2+
•The following operations are now performed sequentially:
Denaturation:
•The reaction mixture is first heated to a temperature between 90-98
0
C
(commonly at 94
0
C) that ensures DNA denaturation. This is the
denaturation step. The duration of this step in the first cycle of PCR is
usually 2 min at 94
0
C.

100. Annealing:
•Themixtureisnowcooledtoatemperature(generally,between40-60
0
C)
thatpermitsannealingoftheprimertothecomplementarysequencesin
theDNA.Asarule,thesesequencesarelocatedatthe3’endsofthetwo
strandsofthesegmenttobeamplified.Thisstepiscalledannealing.
•Thedurationofannealingstepisusually1minduringthefirstaswellas
thesubsequentcyclesofPCR.Sincetheprimerconcentrationiskeptvery
highrelativetothatofthetemplateDNA,primer-templatehybrid
formationisgreatlyfavouredoverreannealingofthetemplatestrands.
PrimerExtension:
•ThetemperatureisnowsoadjustedthattheDNApolymerasesynthesizes
thecomplementarystrandsbyutilizingthe3’-OHoftheprimers,this
reactionisthesameasthatoccursinvivoduringreplicationoftheleading
strandofaDNAduplex.
•TheprimersareextendedtowardseachothersothattheDNAsegment
lyingbetweenthetwoprimersiscopied.
•Thedurationofprimerextensionisusually2minsat72
0
C.

101. •Thecompletionoftheextensionstepcompletesthe
firstcycleofamplification;eachcyclemaytakefew
minutes.
•Itshouldbenotedthattheextensionofprimer
continuestillthestrandsareseparatedduringthe
denaturationstepofthenextPCRcycle.
•Theproductoffirstcycleisthe‘longproduct’.
•Numeroussubsequentcycletakesplacesimilarto
firstcyclethusamplifyingthethegeneexponentially.

104. Development of hybridoma for monoclonal antibody
(Mas)
•Ahybridomaisahybridcellobtainedbyfusing
antibody–producingcellandamultiplemyeloma
cell.(Multiplemyelomaisacancerofplasmacell.)
•ORItmaybedefinedasahybridcellobtainedby
fusingB-lymphocytewithusuallyatumorcellofthe
antibodyformingsystemorofB-lymphocytes(these
arecalledmyelomas).
•Thehybridcellsthusproducedpossesstheabilityto
produceantibodiesduetotheB-lymphocyte
genomeandthecapacityforindefinitegrowthin
vitroduetothetumor(myeloma)cellinvolvedinthe
fusion.

105. •Therefore,hybridomacellsareeitherculturedin
vitroorpassagedthroughmouseperitonealcavityto
obtainmonoclonalantibodies.Thisiscalled
hybridomatechnology.
•B-lymphocytesareisolatedfromthespleenofan
animal,e.g,mouse,whichhadbeenimmunizedwith
theantigenagainstwhichmonoclonalantibodiesare
toberaised(produced).
•Myelomacellsareselectedformainlytwofeatures:
(1)thesecellsmustnotproduceantibodies
themselves,and

106. •(2) they must contain a genetic marker e.g.
HGPRT
-
trait (hypoxanthine –guanine
phospho-ribosyltransferase), which permits an
easy selection of the resulting hybrid cells.
•When HGPRT
-
cells are fused with B-
lymphocytes, the resulting cell population will
consist of (1) hybrid cells (hybridomes), (2)
myeloma cells (3) B-lymphocytes.
•This cell population is now cultured in HAT
medium containing the drug aminopterin.

107. •Similarly,theB-lymphocytesdonotgrowforlong
periodsoftimeintissuecultureandeventuallydie.
•Incontrast,onlythehybridomacellsproliferateon
theHATmediumsincetheB-lymphocytegenome
makesthemHGPRT
+
andtheyhavethecapabilityfor
indefinitegrowthfromthemyelomacell.
•Thushybridomas(myeloma+B-lymphocytehybrid
cells)areselectedbyusingasuitableselective
mediumlikeHAT,whichallowsonlythehybridomas
toproliferate.

108. •Thenextstepconsistsofidentificationandisolationofthe
hybridomacellsproducingantibodiesspecifictotheantigen
usedforimmunizationoftheanimals.
[Thehybridomacellsaresuspended,suitablydilutedand
distributedintomicro-wells,onecellineachmicro-well,and
allowedtogrow.Thehybridomacellsgrowandsecrete
antibodiesintothemedium.Thesupernatantfromeach
micro-wellissampledandassayedforthepresenceof
antibodiesspecifictotheantigeninquestionusingoneofthe
assaymethodse.g.ELISA.Wellscontainingtheantibodies
specifictotheantigenareidentifiedandthehybridomacells
fromthemisolatedandclonedtoensurethatahybridoma
cloneproducesantibodiesofasinglespecificity.]

109. •Oncethedesiredhybridomaclonehasbeen
obtained,itismultipliedeitherinvitroorin
vivotoobtainmonoclonalantibodies.
•Invivoproductionsysteminvolvesinjectionof
hybridomacellsintotheperitonealcavityof
isogenicanimals,collectionoftheasciticfluid
andseparationoftheantibodiesfromit.
•Invitroproductioninvolvesgrowing
hybridomacellsaregrowninvitroinasuitable
largescaleculturesystemandthemonoclonal
antibodiesarepurifiedfromthesecultures.

110. HAT medium
•HATmediumisoneoftheseveralselectivemediausedforthe
selectionofhybridcells.
•Thismediumissupplementedwithhypoxanthine,
aminopterinandthymidine,hencethenameHATmedium.
•Antimetaboliteaminopterinblocksthecellularbiosynthesisof
purinesandpyrimidinesfromsimplesugarsandaminoacids.
•However,normalhumanandmousecellscanstillmultiplyas
theycanutilizehypoxanthineandthymidinepresentinthe
mediumthroughasalvagepathway,whichordinarilyrecycles
thepurinesandPyrimidesproducedfromdegradationof
nucleicacids.

111. •Hypoxanthineisconvertedintoguaninebytheenzyme
HGPRT,whilethymidineisphosphorylatedbythymidine
kinase(TK);bothHGPRTandTKareenzymesofthesalvage
pathway.
•OnaHATmedium,onlythosecellsthathaveactiveHGPRT
(HGPRT
+
)andTK(TK
+
)enzymescanproliferate,whilethose
deficientintheseenzymes(HGPRT
-
and/orTK
-
)cannotdivide.
•Thus,onemayfuseHGPRTdeficienthumancells(designated
asTK
+
HGPRT
-
)withTKdeficientmousecells(denotedasTK
-
HGPRT
+
).Theirfusionproducts(hybridcells)willbeTK
+
(due
tohumangene)andHGPRT
+
(duetomousegene)andwill
multiplyontheHATmedium,whilethemanandmousecells
willfailtodoso.

112. Application of monoclonal antibodies (Mabs)
•Diagnosticapplication:
WhenMabsareusedtodetectthepresenceof
aspecificantigenorofantibodiesspecificto
anantigeninasampleorsamples,this
constitutesadiagnosticapplication.Some
examplesofdiagnosticapplicationsareas
follows:
1.Mabsareavailablefortheunequivocal
classificationofbloodgroupse.g.ABO,Rh,
etc.

113. 2. Mabs are applied for a clear and decisive detection
of pathogens involved in disease (disease diagnosis).
3. Mabs can be used for the accurate detection of
specific chromosomes of a given species.
Therapeutic application:
1. Antibodies specific to a cell type, say, tumor cells,
can be linked with a toxin polypeptide to yield a
conjugate molecule called immunotoxin. The
antibody component of immunotoxin will ensure its
binding specifically and only to the target cells and
the attached toxin will kill such cells.

114. 2. Mabs can be administered to provide passive
immunity against diseases.
3. Mabs are very useful in the purification of
antigens specific to pathogens; these purified
antigens are used as vaccines.
Immunopurification:
The highly specific interaction of an antibody
to the antigen is used to purify antigens
present in small quantities as a mixture with
several types of molecules; this is known as
immunopurification.

115. Drugs produced by biotechnology
•TheEuropeanFederationofBiotechnology(FEB)considers
‘biotechnology’as—‘theintegrationofnaturalsciencesand
organisms,cells,partsthereof,andmolecularanaloguesfor
productsandservices.’
•NewBiotechnologicalprocessesessentiallyembracealmost
allmethodsofgeneticmodificationbyrecombinantDNAand
cellfusiontechniques,togetherwiththe‘magictouch’ofthe
moderndevelopmentsoftheso-called‘traditional-
biotechnologicalprocesses’.
•Interestingly,theseprocesseswill,inmanyinstances,function
atrelativelylowtemperature,willconsumelittleenergy,and
willrelymainlyoninexpensivesubstratesforbiosynthesis.

116. •Althoughtherearealargenumberof‘drugs’that
havebeenevolvedviathebiotechnological
processes.Fewofwhicharelistedbelow:
(i)Altepase[Activase®],
(ii)HumanInsulin[Humulin(R)],
(iii)Humatrope:GrowthHormone,and
(iv)HepatitisB[RecombinantHB(Merck)—aHepatitis
Bvaccine]

117. Alteplase [ Activase®]
•Activase®(Alteplase)isFDA-approvedfortreatmentofmyocardial
infarction(heartattack),acuteischemicstroke(bloodclotinthe
brain)&acutemassivepulmonaryembolism(bloodclotsinthe
lungs).
•Activaseisoneoftherecombinanttissueplasminogenactivator,or
r-tPAandisproducedbyrecombinantDNAtechnology.
•Forcertainpatients,Activasemayimprovethechancesofrecovery
fromstrokewithlittleornodisability.PatientscanreceiveActivase
onlyiftheybegintreatmentwithin3hoursaftertheirstroke
symptomsstartandonlyaftertheyhavehadascantoruleout
bleedinginthebrain.
•Tissueplasminogenactivator(abbreviatedtPAorPLAT)is
aproteininvolvedinthebreakdownofbloodclots.Itisaserine
proteasefoundonendothelialcells,thecellsthatlinetheblood
vessels.

118. •Asanenzyme,itcatalyzestheconversionofplasminogentoplasmin,the
majorenzymeresponsibleforclotbreakdown.Becauseitworkson
theclottingsystem,tPAisusedinclinicalmedicinetotreatonlyembolicor
thromboticstroke.Useiscontraindicated(notadvisable)inhemorrhagic
strokeandheadtrauma.
•tPAmaybemanufacturedusingrecombinantbiotechnologytechniques.
tPAcreatedthiswaymaybereferredtoasrecombinanttissue
plasminogenactivator(rtPA).Recombinanttissueplasminogenactivators
(r-tPAs)includealteplase,reteplase,andtenecteplase(TNKase).
•Storage: Alteplase need to be stored preferably at –20°C or even below in
perfectly sealed containers.

119. •Units:Theactivityofalteplasemaybemeasuredintermsof
InternationalUnits(IU)byemployingthe2ndInternational
StandardfortheTissuePlasminogenActivatorestablishedin
1987,althoughitisanusualpracticetoexpressthedosesby
weight.TheSpecificActivityofalteplaseis580000IUs.mg
–1.
•Pharmacokinetics : It has been duly observed that alteplase
gets cleared from the plasma, chiefly via metabolism in the
liver.
Note: IU is the amount of an enzyme that catalysesthe transformation of 1 micromole of substrate per
minute (under defined conditions of pH, concentration, and temperature)

120. Uses and Mechanism of Action :
•Thevariousapplicationsandpossiblemechanismofactionof
‘alteplase’areasfollows:
(1)Itisathrombolyticagent,whichisapredominantrepresentative
ofasingle-chainformoftheendogenousenzymetissue
plasminogenactivatormeticulouslyproducedbytherecombinant
DNAtechnology.
Verymuchsimilartotheendogenoustissueplasminogenactivator,
itconvertsfibrin-boundplasminogentothecorrespondingactive
formofplasmin,therebycausinginmarkedandpronounced
fibrinolysisanddissolutionofclots.
(2)Alteplaseisemployedverymuchakin(similarinqualityand
character)tosteptokinasebothinthemanagementand
treatmentofthrombo-embolicdisorders,specificallythe
myocardialinfarctionandvenousthrombo-embolism.

121. (2)Alteplasehasalmostnegligibleeffectuponthecirculating,
unboundplasminogen;andhence,maybetermedasafibrin-
specificagent.
Itwasperhapsthoughtthatfibrinspecificitycouldbean
absolutenecessityforminimisingtheprevailingriskof
ensuinghaemorrhageintimatelyassociatedwiththe
applicationofthrombolytics;althoughthelatestfibrin
specificdrugsusuallygiverisetoappreciablebleedingin
comparisontothenon-specificthrombolytics.

122. Humulin : Humulin®
•Humulin:Humulin®isthebrandedproductofthefamouspharmaceutical
manufacturer,Lilly,containinghuman-insulinanditshostofvariants,being
producedbyitindifferentcountriesacrosstheglobe.
DescriptionofInsulin:
•Insulinisapancreatic-hormoneessentiallyinvolvedintheregulationofblood-
glucoseconcentrationsandalsohavingaspecificroleintheproteinandlipid
metabolism.
•Inusualpractice,thehuman,porcine,bovineormixedporcine-bovineinsulinis
administeredtosuchpatientshavinginsulin-dependentdiabetesmellitusinthe
managementandcontroloftheirblood-glucoseconcentrations.
Itmayalsobeusednecessarilyincertainnon-insulin-dependentdiabetics.Insulinis
alsoanessentialcomponentoftheemergencymanagementandcontrolofdiabetic
ketoacidosis.
Insulinis—‘ahormoneproducedbytheβ-cellsoftheisletsofLangerthansof
thepancreasandessentiallycompriseoftwoseparatechainsofaminoacids,
theAandBchains,joinedtogetherbytwodisulphidebridges’.

124. Human insulin
•Humaninsulinisadimercomprisingonechainof21amino
acid(Achain)andtheother30aminoacids(Bchain).
•BoththechainsAandBarederivedfromasinglepolypeptide
chain,andareheldtogetherbytwodisulphidebridges.
•Generally,theinsulingenecodesforpreproinsulin,a
polypeptidecontainingtheAandBpolypeptidesoftheactive
insulin,andaconnectingpolypeptidethatisabsentfrom
matureinsulin.
•Insulinisprocessedfrompreproinsulinviaproinsulinby
enzymaticcleavageoftheconnectingpolypeptidefromtheA
andBchains.

125. Production of human insulin
•Oneoftheprocessesdeveloped
byElilillyincollaborationwith
Genetechinvolvesthefollowin
steps:
•Thegenes(=DNAsequences)for
chainsAandBofinsulinwere
synthesizedseparately.Each
genecontainsmethioninecodon
at5’endandstopcodonat3’
end.
•Thesegenewereintegrated
separatelywiththelacZαgene
(encodingβ-galactosidase)of
twoseparatepBR322plasmids.

126. •TheseplasmidsweretransformedintoE.colistrains.
•Thetransformedbacteriathusproducesthefusionproteinof
AchainandBchainseparately.
•TheAandBchainswereseparatedbyamethionineresidue
fromtheβ-galactosidasesequenceencodedbylacZα.
•Therefore,theinsulinsequenceswereseparatedfromtheβ-
galactosidasesequencesbytreatingthefusionproteinswith
cyanogenbromide.
•ThepurifiedchainsAandBwerethenattachedtoeachother
bydisulphidebondstoproduceinsulin.

127. •Thismethodhoweverturnedouttobeaninefficient
reaction.
•Therefore,agenerepresentingB,CandAChainswas
synthesizedandexpressedinE.coli;inthiscase,the
interveningchainisremovedproteolytically
followingspontaneousfoldingoftheproinsulin
molecule.

128. Hepatitis B [Recombivax HM (Merck) —A Hepatitis B Vaccine]
•TheRecombivaxHB(Merck),ahepatitisBvaccine,isoneofthemost
recentandsignificantdevelopmentsinthefieldofrecombinantDNA
technology,thatessentiallycompriseofhighlyspecificantibodieswhich
actlikemagicbullets.
•Ithasbeendulyobservedthathepatitistendstocauseasevereacute
infectionandmayultimatelyleadtochronicinfectionandpermanent
liverdamage.ItisessentiallycausedbyhepatitisBvirus(HBV);and
recognizedasanenvelopedanddouble-strandedDNAvirus.
•Ithasbeenadequatelyrevealedthroughmeticulousstudiesthat
individualswhoareatthemostvulnerableandgreatestriskforinfection
include:IV-drugabusers(e.g.,morphine/heroinaddicts);homosexual
men;HBV-infectedmothers;andaboveallthehealthcareworkers.
•Various steps being followed in a sequential manner with regard to the
production of a genetically engineered vaccine e.g., Hepatitis B Vaccine.

130. •Step1:Geneticmaterial(DNA)isextractedfromtheensuinghepatitisvirus.At
thisstagethe‘surfaceproteins’essentiallyprovokeanimmuneresponse.
•Step2:The‘individualgenes’areadequatelyanalyzedandidentified.
•Step3:The‘specificgene’whichcategoricallydirectsproductionofsurface
proteinislocatedcarefully.
•Step4:InthismostcriticalstepsthegeneisremovedfromtheviralDNAand
insertedintotheplasmidcarefully.
•Step5:Theplasmidsaremeticulouslyinsertedintothecorrespondingyeastcells.
•Step6:Yeastisallowedtogrowviafermentation.Inthismannerthecells
reproduceandgeneratemorequantumofsurfaceprotein.
•Step7:Afteradurationof48hoursthecorrespondingyeastcellsarerupturedto
freetheensuing‘surfaceprotein’.Theresultingmixtureisdulyprocessedsoasto
extractthepurify
•thesurfaceprotein.
•Step8:Alargeamountofsurfaceproteinparticles,initspurestform,are
obtainedwhich
•ultimatelyprovokeanimmuneresponseeffectively.
•Step9:Theresultingsurfaceproteinsareadequatelymixedwithappropriate
preservationstogetherwithotheringredientstoobtainthevaccine.

131. Humatrope® [Growth Hormone]
•Growthhormoneisananabolichormonesecretedby
theanteriorpituitarywhichstimulatestissuegrowth
andanabolism.Itisfoundtoaffectfat,carbohydrate
andmineralmetabolism.
•Humatrope(somatropin):
ItisapolypeptidehormoneofrDNAorigin.
ManufacturedbyEliLillyandCompany,itisusedto
stimulatelineargrowthinpediatricpatientswholack
adequatenormalhumangrowthhormone.Ithas191
aminoacidresiduesandamolecularweightof
22,125daltons.

132. •Itsaminoacidsequenceisidenticaltothatofhumangrowth
hormoneofpituitaryorigin(anteriorlobe).Humatropeis
synthesizedinastrainofE.colimodifiedbytheadditionofa
geneforhumangrowthhormone.
•OtherhumangrowthhormoneproducedfromrDNAs
include;Omnitrope(Sandoz),Nutropin,Norditropin,Genotrop
in(Pfizer).
•Units:OneAmpouleoftheFirstInternationalStandard(1987)
:4.4unitsofthehumangrowthhormone(somatropin)are
containedin1.75mgoffreeze-driedpurifiedhumangrowth
hormone,with20mgofglycine,2mgofmannitol,2mgof
lactose,and2mgofsodiumbicarbonate.

133. Pharmacokinetics:
•Somatropiniswell-absorbedaftersubcutaneousorIM
injection.
•AfterIVinjectionithasahalf-lifeofabout20-30minutes;
however,aftersubcutaneousorIMadministrationthe
prevailingserumconcentrationsusuallydeclinehavingahalf-
lifeof3-5hours,onaccountoftherelativelymoreprolonged
releasefromthesiteofinjection.Itisfoundtobemetabolised
intheliverandexcretedinbile.

134. Uses and Mechanism of Action :
•Somatropinisasynthetichumangrowthhormone;and
Somatremisitscorrespondingmethionylanalogue.The‘drug’
promotesthegrowthofmuscular,skeletal,andothertissues,
stimulatesproteinanabolism;andalsoaffectsfatandmineral
metabolism.
•Thehormoneexhibitsadiabetogenicactionuponthe
carbohydratemetabolismspecifically.
•Thesecretionisobservedtobepulsatileandsolelydepends
upontheneuralandhormonalinfluences,suchas:(a)
hypothalamicrelease-inhibitinghormonee.g.,somatostatin,
and(b)hypothalamicreleasinghormonee.g.,somatorelin.In
fact,therearecertainphysicalandphysiologicalfactorsthat
largelyinfluenceanenhancedsecretionofthegrowth
hormone,suchas:sleep,emotionalstress,and
hypoglycaemia.