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HonorsBiology DueTuesdayJanuary24th!Unit4:Genetics
ModifiedfromBethanyLau©2014
Name:____________________________________________________Period:______
Lab 13 : DNA F ingerpr int ing Introduct ion : GelelectrophoresisisalaboratorytoolthatallowsscientiststocompareDNAfromdifferentindividuals.GelelectrophoresisusesanelectriccurrenttoseparatefragmentsofDNAthathavebeencutbyrestrictionenzymes.Everyindividual’sDNAfragmentswillbeslightlydifferent,andthereforethepatternofcutDNAthatiscreatedbygelelectrophoresiswillbeuniquetoeachperson.Thisiswhygelelectrophoresisiscalled“DNAfingerprinting”becauseeveryone’sfragmentpatternwillbedifferentjustlikeeveryone’sfingerprints.The Three Major Steps for DNA F ingerpr int ing :
1. PrepareDNASamples:DNAisextractedfromcellsandcutintofragmentsusingarestrictionenzyme.Thefragmentsthatwillbecreatedwillbedifferentdependingonwhichrestrictionenzymeisused.
2. LoadDNASamples:Gelelectrophoresisusesagarosegel.Agarosegelisaporous,jelly-likesubstance.Forgelelectrophoresis,meltedagarosegelisputintoaformthatmakesitintoarectangularshape.AcombisinsertedtocreatesmallslotsorwellswheretheDNAtobe“loaded”usingasmallpipette.Observethediagrambelowtoseewhatthislookslike.
3. SeparateDNASamples:Therectangulargelissubmersedinaelectrophoresischamberfilledwithaspecialsalterwaterthatcanconductelectricity.Anelectricalcurrentisappliedtothesaltwaterwiththegelinit.TheelectricalcurrentwillcausetheDNAfragmentstomove.DNAisanegativelychargedmoleculethatwillmovetowardthepositivelychargedcathode.Asthefragmentsmovesmallerfragmentswillmovefasterandmoreeasilythroughthethickmatrixormeshofthegel,whilelargerfragmentswillmoveslowerthroughthegel.Thiswillresultinabandingpatternthatisuniquetoaspecificindividual.To“see”theDNA,thegelwillbestainedwithabluedyetostainthebandssotheyarevisibletothenakedeye.
DiagramofGelElectrophoresisSetup
ElectrophoresisChamber
Anode(NegativeCharge)
Cathode(PositiveCharge)
PowerSupply
12345Wells
Lanes:DNASamples1234101005001000#of
Nucleotides
12345
1000
100
50
10
#ofBases
HonorsBiology DueTuesdayJanuary24th!Unit4:Genetics
ModifiedfromBethanyLau©2014
Lab Part A: Cutt ing DNA with Restr ict ion Enzymes Introduction:RestrictionenzymesarespecialenzymesharvestedfromuniquebacteriathatscientistsusetocutDNA.Restrictionenzymesarelikelittle“molecularscissors”!TheycutDNAbutonlyatspecificsequences.ExaminetheDNAsequencebelowandfindthesequenceGAATTCinthetopDNAstrand.Circleitandthebasesbelowinthesequence. UncutDNA:
NoticethatthesequencebelowtheGAATTCsequence(CTTAAG)isidentical,justbackwards!Alotofrestrictionenzymesrecognizesequencescalled“palindromicsequences”.Palindromesarethesamebackwardsasforwards(likethewordsMOMandDADandRACECAR!).
Therestrictionenzymecutsthesequenceintotwopieceswithunpaired
baseshangingontheends.Thecutswouldlooklikethis: Piece1: Piece2:
TheprocedureforperformingRestrictionEnzymeAnalysisisshownbelow: RestrictionEnzymeAnalysisQuestions:
1. Whydoscientistsuserestrictionenzymes?
___________________________________________________________________________________________
___________________________________________________________________________________________
2. WhatwouldscientistsusetoseetheirnewlycutfragmentsofDNA?
___________________________________________________________________________________________
___________________________________________________________________________________________
3. Lookatthesequencesbelow.Ifyouaddarestrictionenzyme(thatcutsatGAATTC)totheuncutDNA,howmanyDNAfragmentswillresult?Circlethefragments.UncutDNA1:
Numberoffragments:_____Lengthoffragments:__________basepairs(ignoreendswithunpairedbases)
HonorsBiology DueTuesdayJanuary24th!Unit4:Genetics
ModifiedfromBethanyLau©2014
UncutDNA2:
Numberoffragments:_____Lengthoffragments:__________basepairs(ignoreendswithunpairedbases)
UncutDNA3:
Numberoffragments:_____Lengthoffragments:__________basepairs(ignoreendswithunpairedbases)
4. YouaregivenanewsampleofDNAthatmatchesoneoftheuncutsamplesabove(1,2,or3).Howcouldyouuserestrictionenzymeanalysistomatchyourunknownsampletooneoftheknownsamplesabove?Explain!___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
Lab Part B : App l icat ions of Ge l E lectrophores is GelelectrophoresiscanbeusedtocompareDNAfromdifferentindividuals,thuswhyitiscalledDNAfingerprinting.DNAiscutusingrestrictionenzymesandthenrunthroughgelelectrophoresis(seefrontpageforhowthisworks).The“banding”patternsinthegelareuniquetoeachperson.Gelelectrophoresiscanhelpdeterminefamilialrelationships.Forexample,itcanbeusedtodeterminetheparentsorrelativesofanindividuallikeinapaternitytest.Gelelectrophoresiscanalsobeappliedinforensics.ItcanbeusedtocompareDNAfoundacrimescenetotheDNAofpossiblesuspects.Lastly,gelelectrophoresiscanbeusedtoisolateaspecificgenethathasseparatedinanindividualband.Ascientistcancutoutaparticularbandcontainingagenetoperformfurtherexperiments.AnalyzingDNABandPatterns:
Example1:
Sample# #ofBasesinSampleFragments Analysis
1(Lane2) 1000bases&300bases
1hastwofragmentsthatare1000and300baseslong
2(Lane3) 500bases 2hasonlyonefragmentthatis500baseslong
3(Lane4) 2000bases,800bases,&300bases
3hasthreefragmentsthatare2000,800,and300baseslong
4(Lane5)1000bases&100bases
4hastwofragmentsthatare1000and100baseslong
12345
2000
800
#ofBases
1000
500
300
100
Control
(Forcom
parison
)
Sample#1
Sample#2
Sample#3
Sample#4
Inthisexample,acontrollanehasbeensetuptocomparebandsizesofthefoursamples.Inthetablebelow,thebandsizesbasedonthenumberofbasesornucleotidesaregiven.
HonorsBiology DueTuesdayJanuary24th!Unit4:Genetics
ModifiedfromBethanyLau©2014
Example2:
Observation&AnalysisofClassDemoGelElectrophoresis:
Mrs.Hjust“loooves”whenkidschewguminherclass.Especiallywhenshefindsitonthefloor.Inthiscrime,Mrs.Hfoundgumunderadesk.ShetookitforaDNAsample.Shechosethreesuspectsthataremosttypicallychewingguminclasstoseeifanyofthemaretheculpritinthisgrossgumcrime.Inthegeldiagramprovided,drawthebandsthatyouobserveinthegelprovidedtoyou.Besuretolabelthelaneswiththeappropriatesamplename!Thencompletethetabletoanalyzethesebandingpatterns.Thenanswerthequestionsthatfollow.
Sample Analysis
CrimeSceneSample#1
Samplematchesvictim’sDNA
CrimeSceneSample#2
SamplematchessuspectBDNA,thereforesuspectBwaspresentatthescene
Victim’sDNA Victim’sDNAmatchescrimescenesample#1
SuspectADNASuspectADNAdoesnotmatcheithersample,suspect
AwasnotatcrimesceneordidnotleaveDNA
SuspectBDNA SuspectADNAmatchescrimescenesample#2,thereforesuspectBwaspresentatthescene
Sample Analysis
1:DNAfromGum
2:SuspectA
3:SuspectB
4:SuspectC
12345
Crim
eScen
eSample#1
Victim
’sDNA
SuspectA
DNA
SuspectB
DNA
Crim
eScen
eSample#1
Inthisexample,twounknownsamplesfromacrimesceneareinlanes1and2.Lane3containsthebandingpatternofthevictim’sDNA.Lanes4and5containthebandingpatternoftwodifferentsuspects.Thetablebelowgivestheanalysisofthisgelelectrophoresisresult.Notethatinthisexample,thenumberofbasesineachfragmentisn’tthefocus,butrathertheuniquebandingpatternfoundineachindividual.
123456
Lane6:LeftBlank
HonorsBiology DueTuesdayJanuary24th!Unit4:Genetics
ModifiedfromBethanyLau©2014
1. Whichsuspectismostlikelythepersonwholeftthegumonthefloor?_________________________________
2. Whatisyourevidencetosupportyourhypothesisfromquestion1?____________________________________
___________________________________________________________________________________________
3. Iftwoofthesuspectsweregeneticallyidenticaltwins,whatdoyouthinkthebandingpatternsmightlooklike
forthesetwosuspects?Explain!________________________________________________________________
___________________________________________________________________________________________
4. WhatcausestheDNAfragmentstomovewithinthegel?____________________________________________
__________________________________________________________________________________________
5. WhichDNAfragmentsmovefasteringelelectrophoresis?Why?Whichmoveslower?Why?______________
__________________________________________________________________________________________
6. Whataretwopossibleusesforgelelectrophoresis?________________________________________________
___________________________________________________________________________________________
GelAnalysisProblems:1. Usethegelbelowtodeterminethefragmentsizesforeachsample.Thelengthsarewrittenontheleftofthegel.Ifa
particularbandisnotexactlylinedupwithoneofthegivenlengths,estimatethelength.Forexample,ifabandisapproximatelyhalfwaybetweenlength100basesandlength50bases,thentheunknownbandisapproximately75baseslong.
Sample FragmentSizesinBases/Nucleotides
1
2
3
4
5
6
123456
DNASamples123456
20001000
800
500
300
100
HonorsBiology DueTuesdayJanuary24th!Unit4:Genetics
ModifiedfromBethanyLau©2014
2. Youworkatasandwichshop.Somecustomershavecomplainedthatworkershavebeensneezingintheirsandwiches!Gross.Youdecidetofigureoutwhoarethesegrosspeople.Youtakeasampleofthesneezejuices(mucusandsaliva)fromtwosandwichesandsamplesfromfourcoworkers.Giventhetablewiththefragmentsizes,drawthebandsthatyouwouldobserveinthisgelelectrophoresis.Putaboxorboxesaroundthebandsoftheculprit(s)whohavebeensandwichsneezing!
SampleFragmentSizesinBases/Nucleotides
Sandwich1 1000,700,250
Sandwich2 950,400
Gregory 800,600,250,100
Tuione 950,400
Rossi 1000,700,250
Henderson 1000,700,100
PartA
Score: 1 2 3 4
Description: Someornoquestionsareansweredareattempted.
Allquestionsareanswered,maynotbethorough,andat
least50%accurate.
Allquestionsareansweredthoroughlyandatleast90%accurate.Demonstrates
understandingofinformation.
Allquestionsareansweredthoroughlyand100%
accurate.Demonstratesunderstandingofinformation.
PartB
Score: 1 2 3 4
Description:
Mostproblemshavebeencompletedandanalyzed
thoroughlyandthoughtfully.Allquestionsansweredbut
lackaccuracy.Questionsarenotanswered
incompletesentences.
Allproblemshavebeencompletedandanalyzed
thoroughlyandthoughtfully.Allquestionsansweredwith50%accuracyandthoroughly.Mostquestionshavebeenansweredincomplete
sentences.
Allproblemshavebeencompletedandanalyzed
thoroughlyandthoughtfully.Allquestionsansweredwith90%accuracyandthoroughly.
Questionshavebeenansweredincomplete
sentences.
Allproblemshavebeencompletedandanalyzed
thoroughlyandthoughtfully.Allquestionsansweredwith
100%accuracyandthoroughly.
Questionshavebeenansweredincomplete
sentences.
Sand
wichSample1
1000
800
500
300
100
123456
Sand
wichSample2
Coworker:Grego
ry
Coworker:Tuion
e
Coworker:Rossi
Coworker:Hen
derson
HonorsBiology DueTuesdayJanuary24th!Unit4:Genetics
ModifiedfromBethanyLau©2014
SetUp:Prepagarosefor12gels(willprovide1gelperlabgroup[maxof9perclass]plusthreeextra)PrepTAEbufferPrepstain&traysPrepdestain(diwater)&traysUsed:Cat130.used130A-E.