VNTR Analysis:
The Science Behind DNA Fingerprinting
Student Materials
Introduction..................................................................................................................………2
Pre-LabQuestions.........................................................................................................………6
LabProtocol………………………………………………………………………………………………………………….………7
DataCollectionWorksheet……………………………………………………………………………………………………9
Post-LabQuestionsandAnalysis…………………………………………………………………………………..…….10
Lastupdated:3/15/2017
VNTR Analysis
Introduction
WeallknowthatnotwopeoplehavethesameDNA(withtheexceptionofidenticaltwins)butdidyou
knowthatevenpeoplethatappearverydifferent,areactuallyverysimilaratthegeneticlevel,sharing
99.9%ofthesameDNA.ThissimilaritymakesusingDNAtoidentifypeoplealittletricky,butnot
impossible.AlthoughDNAfromdifferentindividualsisnearlyidentical,thereareregionsonevery
chromosomethatcontainsmalldifferencesorpolymorphismsthatmakeeachofusunique.For
example,inanygivensequenceofDNAasinglenucleotidemayvarybetweentwoindividuals(knownas
singlenucleotidepolymorphismsorSNPs).Geneticvariationcanalsobecausedbydifferencesinthe
numberofrepeatedsequencesofDNAatspecificlocations(loci)inthegenome.Theserepeated
sequencescomeinvarioussizes,someareshort–2to6basepairs(bp)long–andproduceShort
TandemRepeats(STRs).Otherrepeatedsequencescanbelonger–10to100bplong–andproduce
longerrepetitivesegmentsknownasVariableNumberofTandemRepeats(VNTRs).(SeeFigure1below
forexamplesofSTRsandVNTRs.)PolymorphismssuchasSTRsandVNTRsgenerallyoccurwithinthe
estimated90%ofhumangenomethatdoesnotencodeforproteins;however,somepolymorphisms
maydisruptageneandresultinadiseaseoralteredphenotype.
Figure1.ShortTandemRepeats(STRs)andVariableNumberofTandemRepeats(VNTRs).
OnecopyofSTR:
ATTCGAGGTACCGGA ATCG TTGGCCAATATA
TAAGCTCCATGGCCT
TAGC AACCGGTTACAC
FourcopiesofSTR:
ATTCGAGGTACCGGA ATCG ATCG ATCG ATCG ATTCGAGGTACCGGA
TAGC TAGC TAGC TAAGCTCCATGGCCT
TAAGCTCCATGGCCTTAGC
OnecopyofVNTR:
ATTTGGCCA GGGGCTTAATTTCCCC ATTTGG
TAAACCGGTCCCCGAATTAAAGGGG
TAAACC
FourcopiesofVNTR:
ATTTGGCCA GGGGCTTAATTTCCCC GGGGCTTAATTTCCCC GGGGCTTAATTTCCCC GGGGCTTAATTTCCCC ATTTGG
TAAACCGGTCCCCGAATTAAAGGGG
CCCCGAATTAAAGGGG CCCCGAATTAAAGGGG CCCCGAATTAAAGGGGTAAACC
Whethertherepeatedsequenceisshortorlong,thenumberoftimesthesequenceisrepeatedwillvary
frompersontoperson.Everypersonhasonlytwocopies(alleles)ofeachrepeatedregion:onecopyon
thechromosomethatisinheritedfromtheirmotherandonecopyonthehomologouschromosome
fromtheirfather.BecauseSTRandVNTRregionsareinheritedalleles,theyareperfectforforensicor
familialtesting,determiningculturalorethnicheritage,identifyingimmigrationpatternsofdifferent
populations,andarcheologicaldiscoveries.
Let’sconsideraVNTRfoundonchromosome1thatiscommonlyusedinhumanidentificationcalled
D1S80.AttheD1S80locus(location)29differentalleleshavebeenidentifiedinhumanpopulations
carryingbetween14and41repeatsofa16bprepeatsequence.Usingprimersspecifictotheregions
2
flankingtheD1S80locus,scientistscanperformPCR(polymerasechainreaction)toamplifyasingle
copytherepeatregiontogeneratemultiplecopies(seeFigure2).
Figure2.ForwardandreverseprimersaredesignedtoflanktheD1S80locusandamplifythatregionon
thematernalandthepaternalchromosomes1.
Forward
Primer
Allele D14 (14 repeats)
3’
5’
5’
3’
Reverse
Primer
Forward
Primer
Allele D18 (18 repeats)
3’
5’
5’
3’
Reverse
Primer
BillionsofcopiesofeachofthetwooriginalD1S80allelesinanyindividual’sDNAcanbesynthesized
usingPCR.ThesecopiescontainthesamenumberofVNTRspresentintheoriginalDNAandcanbe
studiedtodetermineanindividual’sgenotypeorDNAprofile.Let’slookathowPCRworksusingthe
diagramsbelow.
First.ThetemplateDNAmustbeseparatedintosingle-strandedDNAmoleculesbyincreasingthe
temperatureto94°C.Thehightemperaturedisruptsthehydrogenbondsbetweenthecomplementary
basepairs.
Second.Thereactiontemperatureisdecreasedtoatemperaturebetween50and70°C.Thistemperature,
calledtheannealingtemperature,isdeterminedbythesequenceoftheprimersandissettooptimize
primersbinding(annealing)tothecomplementarysequencesonthetemplateDNA.Becauseprimerswillbe
incorporatedintoeachnewmolecule,theprimersarepresentinvastexcess.Thehighconcentrationalso
increasesbindingofprimerstothetemplate.
64 °C
3
Third.ThereactiontemperatureisincreasedtotheoptimaltemperaturefortheDNApolymerasebeing
usedinthereaction.ThereareseveralthermostableDNApolymerasesusedinPCRandeachhasa
slightlydifferentoptimaltemperature.Atthistemperature,theDNApolymeraseaddsnucleotidesto
the3’endoftheprimersinamannerthatiscomplementarytothetemplatestrand.Inareaction
mixture,thereisanabundanceofpolymerasesoallthetemplatestrandsarecopiedsimultaneously
PolymeraseChainReactionGraphicsfromhttps://commons.wikimedia.org/wiki/File:PCR_Steps.JPG
ThesethreestepsconstituteonePCRcycle.AtypicalPCRamplificationrepeatsthethreestepcycle2535times.ThetargetDNA(theDNAflankedbytheprimers)isdoubledduringeachcycle.Beginningwith
asingleDNAmolecule,20cyclescanproduceoveramillioncopiesontheD1S80region.Eachcopy
containsthesamenumberofVNTRrepeatspresentintheoriginalDNA(seeFigure3).
ThefollowingschematicshowsthefirstfourcyclesofPCRamplification.Noticethatasthenumberof
cyclesincreasesthepercentageofPCRproductsofthedesiredlengthalsoincreases.
Figure3.PCRAmplificationofaVNTR.
Polymerase Chain Reaction image from
https://commons.wikimedia.org/wiki/File:PCR_basic_principle1.jpg
4
BycomparingthePCRproductswithsizestandardsthatcorrespondtotheknownD1S80alleles,the
sizesofthePCRproductscanbedetermined,aswellasthegenotypeoftheindividualtested(Figure4).
HomozygousindividualswillhavethesamenumberofrepeatsattheD1S80locusonthehomologous
chromosomesandwilldisplayasingleband.Moreoften,anindividualwillbeheterozygousforthe
D1S80locus.Heterozygousindividualshavedifferentnumberofrepeatsontheirchromosome1sand
thus,twodistinctbandsofdifferentlengths.
Figure4.GelElectrophoresisofPCRProducts.Differentallelesappearasdistinctbandseachcomposed
ofbillionsofcopiesoftheamplifiedallele.
Ladder
Heterozygous
Homozygous D18
Homozygous D14
- electrode
à
+ electrode
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VNTR Analysis
Pre-LabQuestions
Directions:AfterreadingthroughtheintroductionandprotocolfortheVNTRlab,answerthe
questionsbelow.
1. WhatisthedifferencebetweenanSTRandaVNTR?
2. HowmanytimesdoyouexpecttofindtheD1S80alleleinyourgenome?Explainyouranswer.
3. WhydoyouhavetodoPCRonaDNAsamplebeforeyouanalyzetheVNTRs?
4. StudytheVNTRgelimagebelowandanswerthefollowingquestions.
1
2
3
4
5
6
a. Labelthepositiveandnegativeendsofthegelsanddrawanarrowshowingthe
directioninwhichtheDNAmoved.
b. InLane2circlethebandthatrepresentsthelongerVNTRallele.
c. Lane4hasonlyoneband.Why?
d. Thisgelrepresentsasingleblendedfamily–Lane2Dad,Lane3MomandLanes4-6
children.Whichofthechildrenisfromthemother’sfirstmarriage?Howdoyouknow?
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VNTR Analysis
LabProtocol
Intoday’slab,youwillbeanalyzingasingleVNTRlocus(VariableNumberofTandemRepeats)inseveral
differentsubjects.Priortotoday’sexperiment,labtechnicianshavealreadycollectedDNAsamples
fromoursubjectsandusedPCR(polymerasechainreaction)tomakebillionsofcopiesoftheD1S80
locusthatweinterestedinstudying.TheprimersusedforthePCRrecognizeDNAsequencesthatflank
theD1S80locusandproducePCRproductsofvaryingsizesdependingontheallele.Forexample,ifthe
D1S80VNTRisrepeated35timestheproductwillbelongerthanonethatisrepeated14times.Youwill
beusingagarosegelelectrophoresistoseparatethePCRproducts.BycomparingthePCRproductstoa
DNAladderonthegelyouwillbeabletodetermineeachsubject’sgenotype.
Materials:checkyourworkstationstomakesureallsuppliesarepresentbeforebeginningthelab.
StudentWorkstation
CommonWorkstation
•
•
•
•
•
•
•
•
•
1p20micropipetteandpipettetips
1microcentrifugetuberack
1tubewith12µL100bpQuickLoadDNAladder
4tubeswith12µLsubjectDNAlabeled–SubA,
SubB,SubC…etc.*
1agarosegel(2.0%)withDNAstainincluded
1gelelectrophoresisunitwithpowersupply
UVorbluelighttransilluminator
centrifuge(optional)
1Xelectrophoresisbuffer
*specificlabelswillvarybasedonyourassignedsubjects
Procedures:
1. FindthetubescontainingDNAfromthefoursubjectsassignedtoyou(labeledSubA,SubB…)and
thetubeof100bpDNAladder(labeledLadder).LoadingDyehasalreadybeenaddedtothesamples
andtheladder.
2. Usingacentrifuge,quicklyspinthetubestogetallthesamplesandtheladdertothebottom.Ifyou
donothaveacentrifuge,youcangentlytapthetubesonthelabbenchtocollectthecontentsin
thebottomofthetube.
3. Prepareorsourcea2.0%agarosegelwithDNAstainand1Xelectrophoresisbufferasinstructedby
yourteacher.
4. Assemblethegelboxandpositionitwhereitwillrun.
Reminder:Checktomakesurethatthegeltrayisinthecorrectionorientationwiththewells
closesttothenegativeelectrode.
5. Usingyourp20micropipette,load10µLoftheladderintoawellinthegelandrecordthelocation
ofthewellinTable1onthenextpage.
7
6. Usingyourp20micropipetteandacleantip,load10µLofeachsampleintoaseparatewellofthe
gelandrecordthecontentsofthewellinTable1below.
Reminder:Don’tforgettouseanewtipforeachsample.
Table1.Locationofsamplesinthegel
Lane
1
SampleI.D. 2
3
4
5
6
7
8
7. Runyoursamplesasinstructedbyyourteacheroruntilthedyefrontistwo-thirdsofthewaydown
thegel.
8. WhilethegelisrunningcalculatethesizeofthePCRproductsthatyouexpecttoseeforsixofthe
morecommonallelesandrecordtheminTable2below.
9. Onceyourgelisfinishedrunningexaminethelocationofthebandsandcompletethedata
collectionworksheet.
Table2.AllelesandPCRProductSize
Remember–TheD1S80isa16bpsegmentthatisusuallyrepeated14to41timesinthegenome.Thereisa146bp
constantwithineachproduct:23bp(forwardprimer)+23bp(reverseprimer)+100bpflankingprimerregions.
Forward
Primer
(23bp)
Variable region
Reverse
Primer
(23bp)
Constant
Constant
#basepairsinVNTR+#basepairsinprimerregions=PCRproductlength
Forexample:D14=(14x16)+146=370bp
Allele
NumberofRepeats
SizeofPCRProduct(bp)
D14
14
370
D16
16
D29
29
D35
35
D38
38
D41
41
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VNTR Analysis
DataCollectionWorksheet
Directions:AftercompletingtheVNTRAnalysislab,answerthequestionsbelow.
1. Ontheimagebelowdrawwhatyouseeaftergelelectrophoresis:
100bp
Ladder
−electrode
1517
1200
1000
900
800
700
600
500
400
300
200
100
+electrode
2. Determinethegenotypesofeachofyoursubjectsandwritethembelow.Thenshareyourdatawith
theclass.
Subject
EstimatedBandLengths
Genotype
Isthesubjecthomozygousor
(bp)
heterozygous?
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VNTR Analysis
Post-LabQuestionsandAnalysis
Directions:AftercompletingtheVNTRlab,answerthequestionsbelow.
1. WhatarethetwotechniquesusedtocreateaDNAprofileinthisexperiment?Whatfunctiondoes
eachperform?
2. WhatisaDNAladder?Whatisitsfunction?
3. TheD1S80VNTRthatweusedforanalysishas29identifiedalleleswithinthegenome.Howmany
differentallelesdidyouseewithinthepopulationthatclassstudied?Howmightyouexplainyour
findings?
4. Analyzethegelimagebelowrepresentativeofacrimesceneanalysis.Thefirstlane(L)representsan
alleleladder.IdentifythegenotypeoftheDNAsamplesgivenanddeterminewhichsuspect(s)canor
cannotberuledoutoftheinvestigation.
Lane Sample
15
10
7
5
4
3
2
1
Genotype
CS
CrimeScene
V
Victim
S1
Suspect1
S2
Suspect2
S3
Suspect3
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