Honors Biology Due Tuesday January 24 th ! Unit 4: Genetics Modified from Bethany Lau © 2014 Name: ____________________________________________________ Period: ______ Lab 13: DNA Fingerprinting Introduction: Gel electrophoresis is a laboratory tool that allows scientists to compare DNA from different individuals. Gel electrophoresis uses an electric current to separate fragments of DNA that have been cut by restriction enzymes. Every individual’s DNA fragments will be slightly different, and therefore the pattern of cut DNA that is created by gel electrophoresis will be unique to each person. This is why gel electrophoresis is called “DNA fingerprinting” because everyone’s fragment pattern will be different just like everyone’s fingerprints. The Three Major Steps for DNA Fingerprinting: 1. Prepare DNA Samples: DNA is extracted from cells and cut into fragments using a restriction enzyme. The fragments that will be created will be different depending on which restriction enzyme is used. 2. Load DNA Samples: Gel electrophoresis uses agarose gel. Agarose gel is a porous, jelly-like substance. For gel electrophoresis, melted agarose gel is put into a form that makes it into a rectangular shape. A comb is inserted to create small slots or wells where the DNA to be “loaded” using a small pipette. Observe the diagram below to see what this looks like. 3. Separate DNA Samples: The rectangular gel is submersed in a electrophoresis chamber filled with a special salter water that can conduct electricity. An electrical current is applied to the salt water with the gel in it. The electrical current will cause the DNA fragments to move. DNA is a negatively charged molecule that will move toward the positively charged cathode. As the fragments move smaller fragments will move faster and more easily through the thick matrix or mesh of the gel, while larger fragments will move slower through the gel. This will result in a banding pattern that is unique to a specific individual. To “see” the DNA, the gel will be stained with a blue dye to stain the bands so they are visible to the naked eye. Diagram of Gel Electrophoresis Setup Electrophoresis Chamber Anode (Negative Charge) Cathode (Positive Charge) Power Supply 1 2 3 4 5 Wells Lanes: DNA Samples 1 2 3 4 10 100 500 1000 # of Nucleotides 1 2 3 4 5 1000 100 50 10 # of Bases
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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.
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