MOLECULARBASISOFINHERITANCE

DNA:StructureofPolynucleotideChain

DNAPolymerofdeoxyribonucleotides
Nucleoside=Nitrogenousbase+Pentosesugar(linkedthrough
N
glycosidicbond
)
Exampleadenosine,deoxyadenosine,cytidine,etc.
Nucleotide=Nucleoside+Phosphategroup(linkedthrough
phosphodiesterbond
)
Manynucleotideslinktogetherthrough3

5
phosphodiesterbond
toformpolynucleotidechain(asinDNAandRNA).

Incourseofformationofpolynucleotidechain,aphosphatemoiety
remainsfreeat5endofribosesugar(5endofpolymerchain)and
oneOHgroupremainsfreeat3endofribose(3endofpolymer
chain).

DoubleHelixModelfortheStructureofDNA
Scientistsinvolved
FriedrichMeischer
FirstidentifiedDNAasanacidic
substancepresentinnucleusandnameditasNuclein
WilkinsandFranklin
ProducedXraydiffractiondatafor
DNAstructure
WatsonandCrick
Proposeddoublehelixstructuremodelfor
DNAbasedonXraydiffractiondata
ErwinChargaff
ProposedthatindsDNA,ratiosA:TandC:G
remainsameandareequaltoone
FeaturesofdoublehelixstructureofDNA:

 InaDNA,twopolynucleotidechainsarecoiledtoformahelix.
Sugarphosphateformsbackboneofthishelixwhilebasesprojectin
wardstoeachother.

Complementarybasespairwitheachotherthroughhydrogenbond.
Purinesalwayspairwiththeircorrespondingpyrimidines.Adenine
pairswiththyminethroughtwohydrogenbondswhileguaninepairs
withcytosinethroughthreehydrogenbonds.

Thehelixisrighthanded.
Pitch3.4nm
10bpineachturn
Theplaneofonebasepairstacksovertheotherinadouble
helix.Thisprovidesstabilitytothehelixalongwithhydrogen
bonding.
PackagingofDNAHelix

PackagingofDNAHelix
DistancebetweentwoconsecutivebasepairsinaDNA=0.34nm=
9
0.3410
m
9
TotalnumberofbasepairsinahumanDNA=6.610
bp
9
9
TotallengthofhumanDNA=0.3410
6.610

=~2.2m
6
2.2mistoolargetobeaccommodatedinthenucleus(10
m).

OrganisationofDNAinprokaryotes:
Theydonothavenucleus.DNAisscattered.
Incertainregionscallednucleoids,DNA(negativelycharged)is
organisedinlargeloopsandisheldbysomeproteins(positively
charged).
OrganisationofDNAineukaryotes:
Theyhavepositivelychargedbasicproteinscalledhistones
(positiveandbasicduetopresenceofpositiveandbasicamino
acidresidues,lysineandarginine).
HistoneoctamerUnitofeightmoleculesofhistone
DNA(negativelycharged)windsaroundhistoneoctamer
(positivelycharged)toformnucleosome.

1nucleosomehasapprox.200bpofDNA.
Nucleosomesinachromatinresemblebeadspresentonstrings.
Beadsonstringstructureinchromatinarefurtherpackagedto
formchromatinfibres,whichfurthercoilandcondensetoform
chromosomesduringmetaphase.

 NonhistonechromosomalproteinsAdditionalsetofproteins
requiredforpackagingofchromatinathigherlevel

Transformingprinciple,HersheyandChaseexperiments,&
Propertiesofgeneticmaterial

DiscoveryofDNAasaGeneticMaterial
Thoughprinciplesofinheritanceanddiscoveryofchromosomesin
nucleuswereachievedlongtimeback,therewasconfusionabout
whichmoleculeactedasgeneticmaterial.
TransformingPrinciple
Griffithperformedexperimentswiththebacteria
Streptococcus
pneumoniae
.ThisbacteriumhastwostrainsSstrainandRstrain.
SstrainBacteria
Producesmoothcolonieson
cultureplate
Haveapolysaccharidecoat

RstrainBacteria
Produceroughcolonieson
cultureplate
Donothavea
polysaccharidecoat

Virulent(causes
pneumonia)
Griffithsexperiment

Nonvirulent(doesnotcause
pneumonia)


LiveRstraininthepresenceofheatkilledSstrainproducevirulence
becausesomehowRstrainbacteriaistransformedbyheatkilledS
strainbacteria.Hence,itwasconcludedthattheremustbetransferof
geneticmaterial.
BiochemicalNatureofTransformingMaterial
Avery,McLeod,andMcCarthyworkedtodeterminethebiochemical
natureofgeneticmaterialresponsiblefortransformation.

ThissuggeststhatDNAhastobethegeneticmaterial.
HersheyandChaseExperimenttoConfirmDNAastheGenetic
Material

 HersheyandChaseworkedonbacteriophages(virusesthatinfect
bacteria).
Whenabacteriophageinfectsabacterium,theviralgeneticmaterial
getsattachedwiththebacterialgeneticmaterialandbacteriathen
treatstheviralgeneticmaterialasitsowntosynthesisemoreviral
particles.
HersheyandChaseworkedtodiscoverwhetheritwasaproteinor
DNAthatenteredthebacteriafromvirus.
Theylabelledsomephageswithradioactivesulphurandtheothers
withradioactivephosphorus.
Theseradioactivephageswereusedtoinfect
E.coli
.

E.coli
wasthenblendedandcentrifugedtoremoveviralparticles.

ItwasobservedthatbacteriawithradioactiveDNAwereradioactive
whilethosewithradioactiveproteinslosttheirradioactivity.
ThisshowedthatitistheDNAthatentersthebacteriafromviruses
andnotproteins.Hence,itwasconcludedthatDNAisthegenetic
material.

PropertiesoftheGeneticMaterial
Itshouldbeabletoreplicate(duplicatetoproduceitsidenticalcopy).
Itshouldbechemicallyandstructurallystable.
Itshouldhavescopeforchangesthatareessentialforevolution.
ItshouldfollowtheMendelianprinciplesofinheritance.
DifferencebetweenDNAandRNA:
DNA
Hasdeoxyribose

RNA
Hasribosesugar

sugar
5methyluracil

Uracilispresentinplaceof

(thymine)ispresent.
MostlyDNAactsas
thegeneticmaterial.

thymine.
RNAactsasamessengerand
adaptor.Itactsasagenetic
materialinsomeviruses.

DNAisstable.

Presenceof2OHgroupatevery
nucleotidemakesRNAlabileand
easilybiodegradable.

Chemicallyless

MutationinRNAisfaster.

reactive,mutates
slowly
DNArequiresRNAfor

RNAdirectlycodesforproteins.

proteinsynthesis.
DNA

RNA

Protein
WhyDNAismorestablethanRNA?
InRNA,a2OHgroupispresentateverynucleotide.ThismakesRNA
unstableanddegradable.
Presenceofthymineinplaceofuracilconfersadditionalstabilityto
DNA.

 RNAbeingabiocatalystismorereactive.
DNAisdoublestrandedhavingcomplementarystrand,whichresists
thechangesbyrepairmechanism.
DNAReplicationwithExperimentalProofMachineryandEnzymes
Involved
WhatisDNAReplication?
DNAreplicationisthephenomenoninwhichaduplicatecopyofDNAis
synthesised.
Inreplication,twostrandsoftheDNAhelixseparateandeachstrand
actsasatemplateforsynthesisingnewcomplementarystrands.
Aftercompletionofreplication,thetwocopiessoproducedwillhave
oneparentalandonenewlysynthesisedstrand.Thisschemeof
replicationiscalledsemiconservativereplication.

ExperimenttoProveThatDNAReplicatesSemiConservatively
PerformedbyMesselsonandStahl
15
E.coli
wasgrowninamediumcontainingheavyisotope
Nasthe

nitrogensource.

NwasincorporatedintonewlysynthesisedDNAaswellandtheDNA

15

becameheavyDNA.

 HeavyDNAmoleculecanbedifferentiatedfromnormalDNAbydensity
gradientcentrifugationusingcesiumchlorideasthegradient.
14
Then,cellswereagaintransferredintoamediumwith
Nasnitrogen

source.SamplesweretakenfromthismediaandtheirDNAwas
extracted.
E.coli
dividesevery20minutes.Therefore,theDNAextractedafter20
minuteshadahybriddensity.

DNAextractedafter40minuteshadequalamountofhybridandlight
intensities.
ThisimpliesthatthenewlysynthesisedDNAobtainedoneofits
strandsfromtheparent.Thus,replicationissemiconservative.

MechanismofDNAReplication
ReplicationoccursinSphaseofcellcycle.
EnzymeinvolvedDNApolymerase(DNAdependentDNApolymerase)
Replicationrequiresenergy.
SourceofenergyDeoxyribonucleosidetriphosphates(DNTPs)
DNTPshavedualpurposeActassubstratesandprovideenergyalso
ReplicationinitiatesatspecificregionsinDNAcalledoriginof
replication.
DNApolymerasepolymerisesalargenumberofnucleotidesinavery
shorttime.
Duringthecourseofreplication,twoparentstrandsdonotcompletely

open,butasmallopeningformsinwhichreplicationoccurs.Thissmall
openingformsareplicationfork.
DNApolymerasecanpolymeriseonlyinonedirectionthatis
Therefore,replicationoccurssmoothlyat

to

'

endofDNA.

(continuousreplication,butoccursdiscontinuouslyat

to

end)

ThediscontinuousfragmentssoformedarejoinedbyDNAligase.

TranscriptionUnitStructureanditsRelationshipwithaGene
Transcription
TranscriptionistheprocessofformationofRNAmoleculesfromthe
DNA.
Duringtranscription,onlyasegmentofDNAfromonlyoneofthe
strandsparticipates.
Bothstrandsarenotcopiedduringtranscriptionbecause:
Ifbothstrandsgettranscribedatthesametimesincethe
sequencesofaminoacidwouldbedifferentinboth(dueto
complementarity),thentwoRNAmoleculeswithdifferent
sequenceswillbeformed,whichinturngiverisetotwodifferent
proteins.Therefore,oneDNAwouldendupgivingrisetotwo
differentproteins.
TwoRNAmoleculessoformedwillbecomplementarytoeach
other,hencewouldendupformingadoublestrandedRNA
leavingtheentireprocessoftranscriptionfutile.

TranscriptionalUnit
Atranscriptionalunithasprimarilythreeregions:
PromoterMarksthebeginningoftranscriptionRNA
polymerasebindshere
StructuralgenePartoftheDNAthatisactuallytranscribed
TerminatorMarkstheendoftranscription
TemplateStrandandCodingStrand
Enzymeinvolvedintranscription,RNApolymerase(DNAdependent
RNApolymerase),catalysesinonlyonedirectioni.e.,5to3.
Therefore,thestrandwithpolarity3

5actsasatemplate
(TemplateStrand).

Thestrandwithpolarity5

3actsascodingstrand(whichisa

misnomersinceitdoesnotcodeforanything).Codingstrandhas
sequencesimilartoRNAformedaftertranscriptionexceptforthe
changethatthymineispresentinsteadofuracil.

Gene
TheDNAsequencewhichcodesfortRNAorrRNAmoleculedefinesa
gene.
CistronSegmentofDNAthatcontainsthegeneticcodeforasingle
polypeptide
Thestructuralgenescouldbeoftwotypes:
Monocistronic(mostlyineukaryotes)
Polycistronic(mostlyinprokaryotes)
Monocistronicgeneshavetwoparts:
ExonSequencesthatcodeforaparticularcharacterandis
expressedinamaturedandprocessedmRNA
IntronInterruptingsequencesthatdonotappearinamature

andprocessedmRNA
RegulatorygenesSequencesthatdonotcodeforanything,buthave
regulatoryfunctions

TypesofRNA&TranscriptionProcess

TypesofRNA
mRNA(messengerRNA)Itservesasatemplateforprotein
synthesis.DNAistranscribedtoformanmRNA,whichinturnis
translatedtoformprotein.[Centraldogmaofmolecularbiology]
tRNA(transferRNA)Itbringsaminoacidsduringtranslationand
readsthegeneticcode.
rRNA(ribosomalRNA)Thesearetheworkbenchesoftranslation.
Theyplayastructuralandcatalyticroleduringtranslation.
TranscriptionProcess
Transcriptionhasthreestepsinitiation,elongation,andtermination.
Initiation:
RNApolymerasebindswiththepromotertoinitiatetheprocess
oftranscription.
Associationwithinitiationfactor()altersthespecificityofRNA
polymerasetoinitiatethetranscription.
Elongation
:
RNApolymeraseusesnucleotidetriphosphateassubstrate,and
polymerisationoccursaccordingtocomplementarity.
Termination:
Terminationoccurswhenterminationfactor(P)altersthe
specificityofRNApolymerasetoterminatethetranscription.
AstheRNApolymeraseproceedstoperformelongation,ashort
stretchofRNAremainsboundtotheenzyme.Astheenzyme
reachestheterminationregion,thisnascentRNAfallsoffand
transcriptionis
terminated.


ComplexitiesAssociatedwithTranscription
Inprokaryotes:
Thereisnocleardemarcationbetweencytosolandnucleus.
Therefore,translationcanbeginevenbeforetranscriptionis
completed.Thus,inprokaryotes,transcriptionandtranslation
arecoupled.
Ineukaryotes:
ThreedifferentkindsofRNApolymerasesarepresent.
RNApolymeraseItranscribesrRNA.
RNApolymeraseIItranscribeshnRNA(mRNAprecursor).
RNApolymeraseIIItranscribestRNA,snRNA,andsrRNA.
TheprecursorofmRNA,i.e.hnRNA,containsbothintronsand
exons.Intronsareremovedandexonsarejoinedbyaprocess
calledsplicing.
CappingInthis,methylguanosinetriphosphateisaddedto
the5endofhnRNA.
TailingInthis,adenylateresiduesareaddedtothe3endof
hnRNA.
WhenhnRNAisfullyprocessed,itisknownasmRNA,whichis

transportedoutofthenucleustogettranslated.

GeneticCodeandStudyofMutations
GeneticCode
Geneticcodedirectsthesequenceofaminoacidsduringthesynthesis
ofproteins.
GeorgeGamowproposedthatif20aminoacidsaretobecodedby4
3
bases,thenthecodeshouldbemadeupofthreenucleotides.4
=64
2
(4
=16),whichislessthan20so,thecodonwasproposedtobe

triplet.
HarGobindKhoranadevelopedachemicalmethodtosynthesiseRNA
moleculeswithdefinedcombinationofbases.
Nirenbergdevelopedcellfreesystemsforproteinsynthesis,which
helpedthecodetobedeciphered.
TheenzymeknownasSeveroOchoaenzyme(polynucleotide
phosphorylase)helpedtopolymeriseRNAwithdefinedsequencesina
templateindependentmanner.
Itfinallygaverisetothecheckerboardforgeneticcode.


Salientfeaturesofgeneticcode:
3
Codonistriplet.4
=64(61codonscodeforaminoacidswhile3

arestopcodons)
Onecodoncodesforasinglespecificaminoacid.Codonsare
unambiguous.
Codonsaredegeneratesincesomeaminoacidsarecodedby
morethanonecodon.
Geneticcodeisuniversal.1codoncodesforsameaminoacidin
allspecies.
Codonsarereadcontinuous.Theylackpunctuations.
AUGhasdualfunctionsCodesforMethionineandactsasa
startcodon
EffectsofMutationsonGeneticCode
Mutationsincludeinsertions,deletions,andrearrangements.
Mutationresultsinchangedphenotypeanddiseasessuchassicklecell
anaemia.(ChangeGlu

Valingenecodingforbetaglobinchainof
haemoglobin)Suchmutationsarecalled
jointmutations
.

Insertionordeletionofasinglebasepairdisturbstheentirereading
frameinmRNA.Suchmutationsarecalled
frameshiftmutations
.
Frameshiftmutationsholdtheproofofthefactthatcodonistriplet

becauseifweinsertthreeormultipleofthreebasesfollowedbythe
deletionofsamenumberofbases,thenthereadingframewillremain
unaltered.
StructureoftRNAProcessofTranslationRegulationofGene
Expression

tRNA
tRNAisanadaptermolecule.Ononehand,itreadsthegeneticcode
andontheotherhand,itbindstospecificaminoacids.
tRNAhasan
anticodonloop
thathasbasescomplementarytothe
mRNAcodeandan
aminoacidacceptorend
whereitbindstothe
correspondingaminoacid.
InitiationtRNAThistRNAisessentialforinitiationoftranslationand
hasAUGinanticodonloopandMetinaminoacidacceptorend.
TherearenotRNAsforstopcodons.

Translation
ThemRNAcontainsthegeneticinformation,whichistranslatedinto
theaminoacidsequencewithhelpoftRNA.Aminoacidsare
polymerisedtoformapolypeptide.
Aminoacidsarejoinedbypeptidebond.
Firstofall,chargingoftRNA(aminoacylationoftRNA)takesplace.In
this,aminoacidsareactivatedinthepresenceofATPandarelinkedto

theircorrespondingtRNA.
Ribosomesaretheworkbenchesfortranslation.Ribosomeshave2
subunits:alargesubunitandasmallsubunit.
SmallersubunitcomesincontactwithmRNAtoinitiatetheprocessof
translation.
TranslationalunitinanmRNAistheregionflankedbystartcodonand
stopcodon.
Untranslatedregions(UTR)aretheregionsonmRNAthatarenot
themselvestranslated,butarerequiredforefficienttranslation
process.Theymaybepresentbeforestartcodon(5UTR)orafter
stopcodon(3UTR).
InitiatortRNArecognisesthestartcodon.(Initiation)
ThentRNAaminoacidcomplexesbindtotheircorrespondingcodon
onthemRNAandbasepairingoccursbetweencodononmRNAand
tRNAanticodon.
tRNAmovesfromcodontocodononthemRNAandaminoacidsare
addedonebyone.(Elongation)
Releasefactorbindstostopcodontoterminatethetranslation.
(Termination)
RegulationofGeneExpression
Regulationofgeneexpressioncouldbeexertedatfollowinglevels.
Transcriptionallevel(followingofprimarytranscripts)
Processinglevel(splicing)
TransportofmRNAfromnucleustocytoplasm
Translationallevel
Inaddition,metabolic,physiological,orenvironmentalconditions
regulatetheexpressionofgenes.
Expressionofgenescodingforenzymesisrequiredonlywhen
substrateforthatenzymeisavailable.
Forexample:
Lactose

Glucose+Galactose

E.coli
synthesisesbetagalactosidase,onlywhenlactoseisavailable.

 Regulationinprokaryotes
Geneexpressionisregulatedbycontrollingtherateof
transcriptionalinitiation.
TheactivityofRNApolymeraseatagivenpromoterisregulated
byaccessoryproteins.Theaccessoryproteinsaffecttheabilityof
apromotertorecognisestartsites.
Aregulatoryproteincouldbeactivatororrepressor.
Accessibilityofpromoterisalsoaffectedbyoperators.Operator
istheregionlocatedadjacenttopromoter.
Eachoperonhasaspecificoperatorandaspecificrepressor.
Usuallyoperatorbindstoarepressorprotein.

RegulationofLacOperon
Lac
Operon
OperonAnarrangementwhereapolycistronicgeneisregulatedbya
commonpromoterandregulatorygenes
Lac
operon,
trp
operon,
his
operon,
val
operonaretheexamplesof
suchsystems.

Theelucidationof
lac
operonasatranscriptionallyactivesystemwas
firstdonebygeneticistJacobandbiochemistMonod.

Genesconstituting
lac
operon:
Gene

Nature

Function

i
gene

Inhibitor

Itcodesforrepressorof
lac
operon.

Structural

Itcodesforgalactosidase.

gene

Lactose

y
gene

Structural

Galactose+Glucose

Itcodesforpermease,whichincreasesthe
permeabilityofcelltogalactosidase.

Structural

Itcodesfortransacetylase.

gene
Allgenesinvolvedin
lac
operonarerequiredformetabolismoflactose.
Inducer
Lactoseactsasaninducerfor
lac
operonsinceitregulates
theswitchingonandoffoftheoperon.

Iflactoseisprovidedtothegrowthmediaofbacteriainabsenceofany
othercarbonsource,thenitistransportedinsidethecellsby
permease.
Forpermeasetobepresentandlactosetoenterinsidethecells,low
levelofexpressionof
lac
operonmustbepresentallthetime.
RegulationinAbsenceofInducer
Inabsenceofinducer,
i
genetranscribestosynthesiserepressor
mRNA,whichtranslatestoformrepressor.

Thisrepressorbindswiththeoperatorregionofoperonandprevents
RNApolymerasetotranscribegenes
z
,
y
,and
a
(negative
regulation).

Therefore,inabsenceoftheproductsofthesegenes,metabolismof
lactoseceases.

RegulationinPresenceofInducer
Inducerbindswiththeproteinproductofgene
i
(repressor)and
inactivatesit.

ThisinactivatedrepressorisunabletoinactivateRNApolymerase
enzymeand
z
,
y
,and
a
genessynthesisetheirrespectivemRNA,which
inturngetstranslatedtoformgalactosidase,permease,and
transacetylase.

 Inpresenceofalltheseenzymes,themetabolismoflactoseproceeds
inanormalmanner.

HumanGenomeProject(HGP)
JointventureofUSdepartmentofenergyandNationalInstituteof
Health(NIH)laterjoinedbyWelcomeTrust(UK)
Launchedin1990,completedin2003
ThisprojectworkedtowardsthedeterminationofcompleteDNA
sequenceofhumans.
DNAisthestorehouseofgeneticinformationanddeterminingits
sequenceofbasepairscansolvemanymedical,agricultural,
environmental,andevolutionarymysteries.
RelationshipofHGPwithBioinformatics
Humangenome(genomereferstothetotalityofgenesthatare
9
presentinahumanbeing)contains310
basepairs.

Costofsequencing1bp=US$3
9
Costofsequencing310
bp=US$9billion

Enormoussequencedatasogeneratedwouldhaverequired3300
bookscontaining1000pageseachjustforahumangenome.
Hence,forstoring,retrieving,andanalysingthisenormousdata,a
newbranchofbiologyhasbeendevelopedknownasbioinformatics.
Genomesofmanynonhumanmodelssuchasbacteria,yeast,
Caenorhabditiselegans
,
Drosophila
,plants(riceand
Arabidopsis
)have
alsobeensequenced.

MethodstoIdentifyGenes

 TwomethodsidentifyingESTs(ExpressedsequenceTags)and
sequenceannotation
ESTsAsthenamesuggests,thisreferstothepartofDNAthatis
expressed,i.e.transcribed,asmRNAandtranslatedintoproteins
thereafter.Itbasicallyfocusesonsequencingthepartdenotinga
gene.
AnnotationInthisapproach,entiregenome(coding+noncoding)
issequencedandlateronfunctionisassignedtoeachregioninthe
genome.
GenomeSequencing
DNAfromthecellsisisolatedandisrandomlybrokenintofragments
ofsmallersizes.
Thesefragmentsareclonedintosuitablehostusingvectors.
Clonedfragmentsamplifyinthehost.Amplificationfacilitatesaneasy
sequencing.
CommonvectorsusedBAC(Bacterialartificialchromosomes)and
YAC(Yeastartificialchromosomes)
CommonhostsBacteriaandyeasts
Automatedsequencersareusedtosequencethesesmallerfragments
(Sangersequencing).
Thesequencessoobtainedarearrangedbasedonoverlappingregions
withinthem(alignment).
Alignmentofthesequencesisalsodoneautomaticallybycomputer
programs.
Thenthesesequencesareannotatedandassignedtoeach
chromosome.
PreparationofGeneticandphysicalmapsonGenome
2methodsareusedrestrictionpolymorphismandmicrosatellites
RestrictionpolymorphismSpecializedenzymescalledrestriction
endonucleasesareusedtocutthegenomeatspecializedsitescalled
restrictionendonucleaserecognitionsiteandmapsarepreparedbased
onit.

 MicrosatellitesThesearerepetitiveDNAsequences.
ObservationsfromHGP
9
Humangenomecontains310
(3164.7million)nucleotidebases.

Anaveragegeneconsistsof3000bases.However,thesizeofgenes
varies.Largestgeneisdystrophin(2.4mbases).
Totalnumberofgenesinhumangenome30,000
Over50%ofthediscoveredgeneshaveunknownfunctions.
Lessthan2%ofgenomeiscoding.
Largeportionofgenomeconsistsofrepeatingsequences.
Repetitivesequenceshavenocodingfunction.Theyarerepeatedover
hundredtothousandtimes.Theymayhavearoleinevolution,
chromosomestructure,anddynamics.
ChromosomewithmostgenesChromosome1(2968)
ChromosomewithfewestgenesChromosomesY(231)
SNPs(singlenucleotidepolymorphism)occuratabout1.4million
locationsinhumanDNA.Theyarebelievedtohavesignificancein
explainingdiseasesandevolutionaryhistoryofhumanbeings.
DNAFingerprinting
Introduction
DNAfingerprintingisamethodforcomparingtheDNAsequencesof
anytwoindividuals.
99.9%ofthebasesequencesinallhumanbeingsareidentical.Itis
theremaining0.1%thatmakeseveryindividualunique.
Itisareallydifficultandtimeconsumingtasktosequenceand
9
compareall310
basesintwoindividuals.So,insteadof

consideringtheentiregenome,certainspecificregionscalledrepetitive
DNAsequencesareusedforcomparativestudy.
BasisofDNAFingerprinting
RepetitiveDNAisseparatedfrombulkgenomicDNAsinceitappears
asadistinctpeakduringdensitygradientcentrifugation.
Majorpeak:FormedbybulkDNA

Smallerpeak:SatelliteDNA
Satellitesareoftwotypesmicrosatellitesandminisatellites,
dependinguponthebasecomposition,lengthofsegmentandthe
numberofrepetitiveunits.
Satellitesdonotcodeforproteins,buthaveamajorroletoplayin
DNAfingerprinting.
Polymorphismisactuallyaresultofmutation.Agermcellmutation
(whichcanpassontothenextgenerationthroughsexual
reproduction)givesrisetopolymorphisminpopulations.
Inotherwords,aninheritablemutationifobservedinhigher
frequenciesinapopulationisknownaspolymorphism.
Polymorphismsarisenormallyinnoncodingsequencesbecause
mutationsinnoncodingsequencesdonotaffectanindividuals
reproductiveability.
MethodologyofDNAfingerprinting
VNTR(variablenumberoftandemrepeats)aresatelliteDNAsthat
showhighdegreeofpolymorphism.
VNTRsareusedasprobesinDNAfingerprinting.
Firstofall,DNAfromanindividualisisolatedandcutwithrestriction
endonucleases.
Fragmentsareseparatedaccordingtotheirsizeandmolecularweight
ongelelectrophoresis.
Fragmentsseparatedonelectrophoresisgelareblotted(immobilised)
onasyntheticmembranesuchasnylonornitrocellulose.
ImmobilisedfragmentsarehybridisedwithaVNTRprobe.
HybridisedDNAfragmentscanbedetectedbyautoradiography.
VNTRsvaryinsizefrom0.1to20kb.
Hence,intheautoradiogram,bandofdifferentsizeswillbeobtained.
Thesebandsarecharacteristicforanindividual.Theyaredifferentin
eachindividual,exceptidenticaltwins.


ApplicationsofDNAFingerprinting
DNAfingerprintingiswidelyusedinforensicssinceeveryDNAofevery
tissuefromanindividualhasthesamedegreeofpolymorphism.
DNAfingerprintingformsthebasisofpaternitytestingsinceachild
inheritspolymorphismfrombothitsparents.
Itcanbeusedforstudyinggeneticdiversityinapopulationand
evolution.