PCR Alupv92 09

PCRKit
POLYMERASECHAINREACTION:
AmplificationofAluPV92
Adaptedfrom:
Ah,Lou!Therereallyaredifferencesbetweenus!
MariaAbilock,BayAreaBiotechnologyEducationConsortium
FrankH.Stephenson,AppliedBiosystems
PartnershipforBiotechnologyandGenomicsEducation
BarbaraSoots
LindaCurro
EducationCoordinator
AssistantEducationCoordinator
UniversityofCaliforniaDavis
UniversityofCaliforniaDavis
5307526552
5307526613
5307544410(fax)
5307544410(fax)
besoots@ucdavis.edu
llcurro@ucdavis.edu
Thisprogramismadepossiblethroughthegeneroussupportof:
POLYMERASE CHAIN REACTION
Teacher
Information
AmplificationofAluPV92Insert
Introduction
Materials
Severalrecentstudiessuggestthatasmallpiece
ofDNA,calledanAluelement,founditsway
intothegenomelessthanonemillionyears
ago.Inagivengroupofindividuals,somecarry
this300basepairinsertionwhileothersdonot.
ForEachStudent
gloves
safetygoggles*
disposabletransferpipet
two1.5mlmicrotubes
200lof5%Chelexina1.5mltube
0.2mlPCRtubewithlid
microtubecaplock
papercupwith10mlof0.9%NaCl*
ForEachLabGroup
20lmicropipet
100lmicropipet
boxof20200ltips
microtuberack
PCRtuberack
waterproofpen
80lmastermix
80lprimermix
30l0.9%NaCl*
cupofcrushedice*
Duringthislaboratory,studentswillextract
theirownDNAfromacellsampletakenfrom
theliningoftheirmouth.Theywillthenusea
powerfulmolecularbiologytechnique,called
thePolymeraseChainReaction,toamplifythis
Aluregionsothatitcanbevisualizedusinggel
electrophoresis.
Attachedreadingsandactivitieswillprovide
moreinformationabouttheAluregionandhelp
illustratetheprocessofPCRanditsimportance
inmoderndaybiology.
Objectives
1.
IsolateDNAfromcheekcellsandpreparea
reactionforPCRamplification.
2.
Listandexplaintheimportanceofeach
componentofPCR.
3.
ElectrophoresetheAlurepeatto
determineyourgenotypefortheAlu
insertion.
4.
Understandthedifferencebetween
heterozygousandhomozygous.
5.
Calculatealleleandgenotypefrequencies.
CommonMaterials
twodualgelboxes
powersupply
carboywith1XTBEsolution
2.0%agarosegel
microcentrifuge
heatblock&thermometer
thermalcycler&96wellPCRtray
UVtransilluminator
MiniVisionarysystem
PCRgrid
gelelectrophoresisgrid
BiotechnologyintheClassroom2009
updated9/23/2009
InstructorUse
1000lmicropipet
100lmicropipet
20lmicropipet
boxof1000ltips
boxof20200ltips
100bpladder
controlDNA
loadingdyewithSYBRGreen
sterilewater
labtape
PCRandmicrocentrifugetuberack
4geltrays
410wellcombs
412wellcombs
DAY2:
1.
Buy/preparecrushediceforeachlab
station.
ItisveryimportanttokeeptheMaster
andPrimermixcoldwhilestudentsare
preparingtheirPCRreactions.
2.
3.
*Materialsprovidedbyinstructor
AdvancePreparation
DAY1
1.
Mix4.5gofnoniodizedtablesaltina500
mlbottleofdrinkingwatertocreatea
0.9%NaClsolution.
SincestudentswillbeusingtheNaCl
solutionasamouthwash,besuretouse
containersandmeasuringequipmentthat
isdesignatedforusewithedibleonly
experiments.
2.
Foreachstudent,aliquot200l5%Chelex
ina1.5mltube.
Usea1000lmicropipetsothebeadswill
notclogthetipandswirlthetube
frequentlytoensureeachstudentgetsa
5%solution.
3.
4.
5.
Prepareapositiveandnegativecontrol
solution.
a) PositiveControl:
20lMasterMix
20lPrimerMix
10lcontrolDNA
b) NegativeControl:
20lMasterMix
20lPrimerMix
10lsterilewater
4.
PlacethepaperPCRgridcopiedfromlab
manualnexttothethermalcycler.
Makesurethe96welltrayisinthe
themalcycler.
5.
Followthestartupinstructionsonthe
thermalcyclerlid.
DAY3:
1.
Makea1XTBEsolutionfrom10Xstockand
placeincarboy.
Figureonabout1Lperclass:
M1V1=M2V2
(10X)(x)=(1X)(1,000ml)
x=100ml
Use100mlof(10X)bufferandadd
distilledwatertobringthefinal
concentrationto1,000ml
2.
Pourfour2%agarosegels.Coverbench
withlabpaperpriortopouringgels.
Foreachlabgroup,aliquot100lof0.9%
NaClsolution.Youmayusesomeofthe
leftover0.9%NaClsolutionfromStep1.
Setheatblockto99C(5.5onthehighdial)
Foreachstudent,aliquot10mlof0.9%
NaClsolutioninapapercup
Foreachlabgroup,aliquot:
a) 80lMasterMix(keeponice)
b) 80lPrimerMix(keeponice)
updated9/23/2009
3.
Setupagelrunareawithtwodualgel
boxesandonepowersupply.Tapethe
papergridincludedinthispacketnear
eachbox.Putgeltraysinbox.
Aftertheruniscomplete,tubeswillbeheldat
4Cuntilyouquittheprocedure.Besuretoend
theprocedurepriortoturningthepoweroff.
4.
Add5ulofloadingdyetoeachofthe
studentsampletubes.Besuretochange
tipsforeverytube.
RemembertheSYBRGreenDNAstainisin
theloadingdye.
DAYFOUR:
Retrievegelsfromrefrigerator.
2.
SetupMiniVisionaryphotosystem
accordingtoinstructionsincludedwith
systeminthekitbox.
USEOFSYBRGREEN:
SYBRGreenisafluorescentdyeusedtostain
DNAandRNA.Testshavefoundittobeless
mutagenicthanethidiumbromide.Thisdyehas
beenpreaddedtotheloadingdyestudentsadd
totheDNAsampleandwillfluorescewhen
exposedtoUVlight.
1.
Instructionsforoperatingthethermalcyclerare
tapedtothemachine.Allcyclingprotocolsthat
youwillneedarepreprogrammedintothe
machine.
Thischemicalshouldneverbehandledwithout
gloves.Usedgels,tips,tubes,andTBEbuffer
shouldbereturnedtotheBiotechnologyinthe
Classroomprogramforproperdisposal.
TeachingTips
Inthisexperiment,theagarisnevertouched
withoutgloves.Pleaseseeenclosedsafety
binderformoreinformationaboutSYBR
Green.
CHELEX
Chelexisaresincontainingorganicchemicals
thatremovemetalionsthatmightdegradethe
DNAduringboiling.
USEOFPOWERSUPPLIES:
USEOFMICROPIPETTES:
Thepowersupplyproducesavoltagethatis
highenoughtocausesevereelectricalshockif
handledimproperly.
Theseextremelypreciseandexpensive
instrumentsneedtobehandledgently.
DoNOTplugpowersupplyintowall
receptacleuntilthesafetycoveris
positionedonthecellandallother
electricalconnectionsareproperlymade.
Donotwindthemicropipetdialpastthe
maximumamountindicatedonthetopof
theplunger.
Alwaysholdthemicropipetinavertical
position.
USEOFTHEUVTRANSILLUMINATOR:
Alwayschangetipsbetweenaliquotsto
reduceriskofcontamination.
USEOFTHERMALCYCLER
Makesureyouhaveatrayinstalledinthe
heatblockofthethermalcycler.Students
tubesshouldbeinthistray.Withouta
tray,thetubesmaymelt!
Whenstudentsarereadytolookattheirgels,
viewthemonlythroughtheUVblockingcover.
Donotattempttooperatethetransilluminator
withtheUVblockingcoverraised.Anyattempt
tooverridethesafetyinterlockandviewthegel
directlycouldresultinseriouseyeandskin
damage.
DonotusethetransilluminatoriftheUV
blockingcoverbecomescrackedorbroken.
updated9/23/2009
BUFFERSOLUTION:
Tris/Borate/EDTA(TBE)bufferiscommonly
usedinelectrophoresissystems.Thissalt
solutionbothconductstheelectriccurrentand
controlsthepHofthesolutionduring
separationofDNAfragments.Oncethegels
haverun,theinstructorshouldpourtheused
TBEsolutiondownthedrain.
Mineralsinregulartapwaterwillquicklystain
equipment.Pleaserinseandairdryboththegel
traysandgelboxesindistilledwater.Becareful
nottodislodgethewiringatthebaseofthegel
box.
USEOFTHEMINIVISIONARYSYSTEM
Pleasereviewtheenclosedinstructionmanual
forsetupoftheMiniVisionarycables.Notethat
thecamerahoodshouldremainonthecamera
andthattheSYBRgreenfilterisinplace.
Donotremovethehoodfromthecamera
orthefilterfromthehood.
Followtheseinstructionswhentakingapicture
ofyourgelelectrophoresisresults:
1. Pluginthepowercordstoboththe
controllerandtheprinter.Turnbothpieces
ofequipmenton.
Settheintegrationtimeto100usingthe
buttonsontopofthecontroller.
3.
WiththetransilluminatorOFF,placethe
geltraydirectlyontheplatform.Thereis
noneedtoremovethegelfromthetray.
Thecamerahoodisstrictlyfor
photographicpurposes.Donot
attempttousethecamerahoodto
viewsubjectsonthe
transilluminator.Seriousskinoreye
injuriesmayresultfromimproper
use.
updated9/23/2009
Turnonthetransilluminator.
5.
Printtheimagebyusingthelargeprint
buttononthefrontpanelofthecontroller.
Donotusethesmallbuttononthefront
panelofthethermalprinter.
6.
Turnoffthetransilluminator.Removeand
disposeofthegelsintheregulartrash.
DISTILLEDWATER:
2.
4.
AnswerstoStudentActivity
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Answerswillvarywithclassdata.
Answerswillvarywithclassdata
Yes,theresultsshouldadduptoone.
Answerswillvarywithclassdata.
68
0.7
0.8
0.42
Laboratory
BackgroundReading
ALUREGION
OnlythreetotenpercentofthenearlysixbillionbasepairsofDNAinyourbodyisactuallyusedto
directlycodeforproteins.Theproteinencodingregionsarescatteredthroughoutthegenomeandthe
genesmaybeseparatedbymanythousandsofbasepairs.Furthermore,mostgenesarethemselves
brokenintosmallerproteinencodingsegmentscalledexonsandinterveningregionswithanilldefined
ornonessentialrolecalledintrons.Whatevertheirfunction,examinationoftheseintronshasrevealed
thepresenceofuniquegeneticelementsthatcanbefoundinanumberofdifferentlocationswithinthe
genome.OneofthefirstsuchrepeatingelementsidentifiedisAlu.
Alurepeatsareapproximately300basepairsinlength.Sonamedbecausemostcarrywithinthemthe
basesequenceAGCT,therecognitionsitefortheAluIrestrictionendonuclease,over500,000ofthese
repeatsarescatteredthroughoutthehumangenome.Onaverage,onecanbefoundevery4,000base
pairsalongahumanDNAmolecule.Howtheyaroseisstillamatterofspeculationbutevidencesuggests
thatthefirstonemayhaveappearedinthegenomeofhigherprimatesabout60millionyearsago.
Approximatelyevery100yearssincethen,anewAlurepeathasinserteditselfinanadditionallocation
inthehumangenome.Alurepeatsareinheritedinastablemanner;theycomeintactintheDNAyour
motherandfathercontributedtoyourgenome.
SomeAlurepeatsarefixedinapopulation,meaningallhumanshavethatparticularAlurepeat.Others
aresaidtobedimorphic;differentindividualsmayormaynotcarryaparticularAlusequenceata
particularchromosomallocation.InthislabwewillbeanalyzeadimorphicAluregion.
PV92isahumanspecificAluinsertiononchromosome16.ThePV92geneticsystemhasonlytwoalleles
indicatingthepresence(+)orabsence()oftheAlutransposableelementoneachofthepaired
chromosomes.ThisresultsinthreePV92genotypes(++,+,or).The+andallelescanbeseparatedby
sizeusinggelelectrophoresis.
POLYMERASECHAINREACTION
Inordertoanalyzeaparticulargeneoutofthetensorhundredsofthousandsofgenesinagenomic
DNAsample,sensitiveandsequencespecifictechniquesareneeded.Onesuchmethodology,
PolymeraseChainReaction(PCR),hasbeendevelopedtoamplifyspecificDNAsequencesinaDNA
sample,evenwithminuteamountsofstartingmaterial.ThePCRtechniqueiscommonlyusedinthe
molecularanalysisofgenes,butithasalsobeenusedtoamplify,andsubsequentlyanalyze,theDNA
frommanysourcesoncethoughtimpossible.Forexample,usingPCRscientistshaveanalyzedDNAfrom
asinglesperm,museumspecimensofextinctanimalsandmillionsofyearoldplantandanimalfossils.
Inaddition,PCRcanbeusedtoanalyzetheDNAofabloodorskinsamplethatmightbeevidenceina
criminalinvestigation.FollowingisastepbystepdescriptionofthePCRprocess.
ThehypotheticalDNAsegmentbelowwillbeamplifiedthroughPCR.The????????maybeaverylongor
shortsetofdeoxyribonucleotidesitdoesntmatter.
AATTGCCCCGGGAAATTT????????????????????AAATTTGGGCCCAA
TTAACGGGGCCCTTTAAA????????????????????TTTAAACCCGGGTT
updated9/23/2009

STEP1SYNTHESIZEPRIMERS
Synthesizeprimersofabout20deoxyribonucleotideslong.AprimerisasmallsegmentofDNAthat
matchesaknownseriesofbasesonthetargetstrand.Thisstepisaccomplishedbyusingamachine
calledaDNAsynthesizer.
Inthisexample,wewillmakeoneprimerexactlylikethelowerrighthandsequence,andoneprimer
exactlyliketheupperlefthandsequence.
TargetDNAstrand:
???????????????????AAATTTGGGCCCAA
AATTGCCCCGGGAAATTT
TTAACGGGGCCCTTTAAA???????????????????TTTAAACCCGGGTT
Primer1:
Primer2
AATTGCCCCGGGAAATTT
TTTAAACCCGGGTT
STEP2DENATUREDNA
TargetDNAisheated,whichcausesthepairedstrandstoseparate.Thesinglestrandsarenow
accessibletoprimers.
STEP3ANNEALING
Addtheprimersyoucreatedearlier.TheamountshouldbelargerelativetotheamountofDNAbeing
amplified.Coolthereactionmixturetoallowdoublestrandstoformagain.Becauseofthelargeexcess
ofprimers,thetwostrandswillalwaysbindtotheprimers,insteadofwitheachother.
Primer2
Primer1
updated9/23/2009
STEP4EXTENSION
Addthefourdeoxyribonucleotidesandanenzymethatcanreadtheopposingstrand'ssentenceand
extendtheprimer'ssentencebyhookingletterstogetherintheorderinwhichtheypairacrossfromone
another.ThisparticularenzymeiscalledaDNApolymerase.OnesuchenzymeusedinPCRiscalledTaq
polymerase(originallyisolatedfromabacteriumthatcanliveinhotsprings).Thisenzymecanwithstand
thehightemperaturenecessaryforDNAstrandseparationandcanbeleftinthereaction.
TAAA???????????????????
???????????????????AAATT
TTAACGGGGCCCTTTAAA???????????????????TTTAAACCCGGGTT
Ifyoulookatthisarrangement,youcanseethatprimer2pairedtotheupperstrandisextendedtothe
leftinthedirectionofthearrowandprimer1pairedtothelowerstrandandisbeingextendedtothe
rightinthedirectionofthearrow,Rememberthatthe???alsorepresentdeoxyribonucleotides.
Therearenowfourstrands,whereoriginallytherewereonlytwo.Ifoneleaveseverythinginthetest
tubeandrepeatstheprocedure,therewillbeeightstrands.Doitagainandtherewillbe16,etc.
Therefore,about20cycleswilltheoreticallyproduceapproximatelyonemillioncopiesoftheoriginal
sequences.
Withthisamplificationpotential,thereisenoughDNAinonetenthofonemillionthofaliter(0.1
microliter)ofhumansalivatousethePCRsystemtoidentifyageneticsequenceashavingcomefroma
humanbeing.
Inthefollowinglaboratory,youwillusePCRtoamplifyadimorphicAlurepeat(designatedPV92)found
onyournumber16chromosome.YouwilluseyourownDNAastemplate.AfteryouamplifytheAlu
repeatregionusingPCR,youwilldeterminewhetherornotyoucarrythisparticularAlusequenceon
oneorbothofyournumber16chromosomes.ThiswillbeaccomplishedbyrunningyourPCRproduct
onanagarosegel.
AttachedreadingsexplainthePCRprocessandthesignificanceofAluregionsinmoredetail.Inaddition,
anexcellentanimatedtutorialshowingthestepsofPCRisavailableattheColdSpringHarborwebsite:
http://vector.cshl.org/shockwave/pcranwhole.htm
updated9/23/2009
Objectives
1.
IsolateDNAfromcheekcellsandpreparea
reactionforPCRamplification.
2.
Listandexplaintheimportanceofeach
componentofPCR.
3.
ElectrophoresetheAlurepeatto
determineyourgenotypefortheAlu
insertion.
4.
Understandthedifferencebetween
heterozygousandhomozygous.
5.
Calculatealleleandgenotypefrequencies.
Materials
ForEachStudent
gloves
safetygoggles*
disposabletransferpipet
two1.5mlmicrotubes
200lof5%Chelexina1.5mltube
0.2mlPCRtubewithlid
microtubecaplock
papercupwith10mlof0.9%NaCl*
ForEachLabGroup
20lmicropipet
100lmicropipet
boxof20200ltips
microtuberack
PCRtuberack
waterproofpen
80lmastermix
80lprimermix
30l0.9%NaCl*
cupofcrushedice*
updated9/23/2009
CommonMaterials
twodualgelboxes
powersupply
carboywith1XTBEsolution
2.0%agarosegel
microcentrifuge
heatblock&thermometer
thermalcycler&96wellPCRtray
UVtransilluminator
MiniVisionarysystem
PCRgrid
gelelectrophoresisgrid
InstructorUse
1000lmicropipet
100lmicropipet
20lmicropipet
boxof1000ltips
boxof20200ltips
100bpladder
controlDNA
loadingdyewithSYBRGreen
sterilewater
labtape
PCRandmicrocentrifugetuberack
4geltrays
410wellcombs
412wellcombs
*Materialsprovidedbyinstructor
Precautions
TheSYBRGreenstainfoundinthe
loadingdyeisahazardouschemical.
Althoughitispresentinanextremelylow
concentration,itstillshouldnotbe
touched.Afterelectrophoresis,thegels
shouldonlybehandledwithgloves.
Alwaysleavegelinitstray.
Whenusingthemicrocentrifuge,always
makesurethetubesareloadedinthe
rotorsymmetrically;eachtubeshouldbe
balancedbyanothertubedirectly
oppositeit.Neverrunthecentrifugewith
thelidopenortherotormissing.Never
puthandsintherotorareaunlessthe
rotoriscompletelystopped.
Ultraviolet(UV)radiationcancause
severeeyeandskindamage!Thecamera
hoodisstrictlyforphotographicpurposes.
Donotattempttousethecamerahoodto
viewsamplesonthetransilluminator.
Makesurethatthehoodisseated
correctlyandtherearenoraisededges
thatwouldallowUVlighttoleakout.
Ifyouchoosetoviewthegelpriorto
makingaphotograph,besuretheUV
blockingcoverisdownbeforeturningthe
poweron.
Followyourteachersinstructions
regardingmicropipethandling.
Thepowersupplyproducesavoltagethat
ishighenoughtocausesevereelectrical
shockifhandledimproperly.Assurethat
youarethoroughlydrilledinthe
proceduresregardingtheuseofthisunit,
makingelectricalconnections,andthat
theteacherdirectlysupervisesyou.
updated9/23/2009
10
Procedure
DNAPREPARATIONUSINGASALINEMOUTHWASH
1.
2.
3.
4.
5.
6.
7.
8.
9.
Gatherallrequiredlabsuppliesandthinkofa4digitidentificationnumber(PIN)towritedown
onyourlabsheet.
Getapapercupwith10mlof0.9%NaClsolutionfromyourinstructor.Swirlthesolutionin
yourmouthfor30seconds.
ExpeltheNaClsolutionbackintothecupandswirltomixthecells.
Labelthecapofa1.5mlmicrotubewithyourPIN.Usingadisposablepipet,transfer1000lof
thesaliva&NaClsolutionintothetube.Closethecaptightly.Discardcup.
Inabalancedmicrocentrifuge,spinsamplefor1minutetopelletthecells.
Observeyourcellpelletatthebottomofthetube.Pouroffanddiscardthesupernatant,being
carefulnottoloseyourcellpellet.
Note:Itisokayifsomesupernatantisleftinthetube.
Resuspendyourcellpelletin30lof0.9%NaCl.Makesuretheentirecellpelletisthoroughly
mixedbypipettingupanddownseveraltimesorrackingyourtube.
Note:Torackyoursample,besurethetopofthetubeisclosed,holdtubefirmlyat
thetop,andpullitacrossamicrofugerack23times.
Labela1.5mltubeof5%ChelexwithyourPINnumber.
Withdraw30lofthecheekcellsuspensionandaddittotheChelextube.
Note:DonotpipetupanddownatthissteporelseyouwillclogthetipwithChelex.
10. Secureyourtubewithacaplockandplaceitinthe99Cheatblockfor10minutes.
11. Removetubefromheatblockandtakeoffyourcaplock.Shakeyourtubewellandthenplaceit
inabalancedcentrifuge.Spinfor1minute.
12. Withdraw60lofsupernatant(noChelexbeads)toacleantube,labeledwithyourPIN.
Discardallothertubes.
Note:ThisstoredsampleisyourDNAtube.
13. Cleanupyourlabstationandwashyourhands.
STOPPOINT:PlaceyourDNAtubeintheclassmicrotuberacksothatyourteachercan
refrigerateyourisolatedDNAuntilyouarereadytoprepareyourPCRamplification.
updated9/23/2009
11
POLYMERASECHAINREACTION
1.
2.
3.
4.
5.
6.
7.
8.
GatherallrequiredlabsuppliesandretrieveyourDNAtubefromtheclassmicrotuberack.
Labela200lPCRtubewithyour4digitPIN.
Putanewtiponyour20lmicropipetanddispense20lofMasterMixintoyourPCRtube.
Changeyourpipettipandadd20lofPrimerMixintoyourPCRtube.
Changeyourpipettipandadd10lofyourpurifiedDNAintoyourPCRtube.
Note:Slowlypipetupanddownseveraltimestomixallthereagentsinyourreaction
tube.
YourinstructorhaspreparedtwoPCRcontroltubes:
PositiveControl:
20ulMasterMix
20ulPrimerMix
10ulControlDNA
NegativeControl
20ulMasterMix
20ulPrimerMix
10ulsterilewater
Placeyourreactiontubeintothethermalcyclerandrecordthelocationofyourtubeonthe
gridprovidedbyyourteacher.
Yourinstructorwillbeginthethermalcycler.ThecyclingprotocolforamplificationofthisAlu
regionis:
95C10minutes
94C30seconds
60C30seconds
72C2minutes
Repeatfor30cycles
72C10minutes
4Chold
Note:Thisprocesswilltakelongerthanyourclassperiodsoyourinstructorwillremove
thetubesfromthemachineuponcompletion.
STOPPOINT:InstructorThethermalcyclermaybeleftonovernight.Uponremovalfromthe
machine,placetheamplifiedDNAtubesintheclassPCRtuberack.Refrigeratethesamples
untilyouarereadytorunyouragarosegels
updated9/23/2009
12
ELECTROPHORESISOFAMPLIFIEDDNA
1.
2.
3.
4.
5.
6.
7.
Gathernecessarylabsuppliesandputonglovesandsafetygoggles.
RetrieveyourPCRtubeandtapitlightlyonthecountertobringtheliquidtothebottomofthe
reactiontube.
Confirmthatyourinstructorhasalreadyloaded5lofloadingdyetoyourPCRtube.
Carefullyload1520lofyourreactionintoawellononeofthegelsyourteacherhas
prepared.Onthegridnexttothegelbox,writedownyourPINintheappropriatelanenumber.
Note:Avoidpokingthepipettetipthroughoutthebottomofthegelorspillingsample
overthesidesofthewell.Useanewtipforeachsample.
Yourinstructorwillload5lofthe100bpladderintooneofthewellsofeachgel.
Yourinstructorwillloadonelaneineachgelwith10lofapositivecontrolandonelanewith
10lofanegativecontrol.
Whenallsamplesareloaded,yourinstructorwillattachtheelectrodesfromthegelboxtothe
powersupplyandelectrophoreseyoursamplesat125voltsfor4550minutes.
STOPPOINT:InstructorGelsmaybestoredinaTupperwarecontainerfilledwith70%ethanol
orkeptintheTBEbufferforonedaybeforephotographing
updated9/23/2009
13
PHOTOGRAPHINGAGAROSEGELS
1.
2.
3.
4.
5.
6.
7.
8.
Putonglovesandsafetyglasses.Retrieveyourgeltrayfromyourinstructor
MakesurethepowertotheUVtransilluminatorisOFF.LifttheUVblockingcoverandplacegel
trayontheblackplatform.Closethelidcompletelybeforeturningmachineon.UVradiation
cancausesevereeyeandskindamage.
Brieflyobservethenumber,placementandrelativebrightnessoftheDNAbands.Turnthe
transilluminatorOFF.
WiththetransilluminatorOFF,yourinstructorwillplacethehoodedCCDcameraovertheblack
platform.Makesurethatthehoodisseatedcorrectlyandtherearenoraisededgesthatwould
allowUVlighttoleakout.
TurnONthetransilluminator.Yourinstructorwillprintanimageforeachmemberofyourlab
group.
TurnOFFthetransilluminator.
RemovetheCCDcamera/hoodfromthetransilluminator.
Followyourteachersinstructionsforcleanup.Discardallgelsintotheplasticdisposalbags.Do
nottouchthegel!Gelboxesandplatesmustberinsedindistilledordeionizedwaterandair
dried.Pleasetakegreatcarewiththeequipmentsomanymorestudentsmayenjoythese
experiments.
9. Washhandsandcleanlabstation.
10. Analyzeyourresultsandcompletetheattachedstudentactivitysheet.
updated9/23/2009
14
ANALYZINGRESULTS
Byexaminingthephotographofyouragarosegel,youwilldeterminewhetherornotyoucarrythe
Alurepeatonone,both,ornoneofyournumber16chromosomes.PCRamplificationofthisAlusite
willgeneratea415bpfragmentiftherepeatisnotpresent.Iftherepeatispresent,an715bp
fragmentwillbemade.
DNA region amplified by
415 bp
Chromosome without Alu repeat
Alu
Chromosome with Alu repeat
715 bp
Whenyouexaminethephotographofyourgel,itshouldbereadilyapparentthatthereare
differencesbetweenpeopleattheleveloftheirDNA.Eventhoughyouamplifiedonlyonesite,asite
thateveryonehasintheirDNA,youwillnoticethatnotallstudentshavethesamepatternof
bands.Somestudentswillhaveonlyoneband,whileotherswillhavetwo.
Weusethetermalleletodescribe
differentformsofageneorgenetic
site.ForthosewhohavetheAlu
repeat,wecansaythattheyare
positivefortheinsertionanddenote
thatalleleconfigurationwitha+
sign.IftheAlurepeatisabsent,we
assignaalleledesignation.Ifa
studenthasasingleband,whetherit
isasingle415bpbandorasingle715
bpband,thenboththeirnumber16
chromosomesmustbethesamein
regardstotheAluinsertion.Theyare
saidtobehomozygousandcanbe
designatedwiththesymbols+/+or
/,respectively.IfastudentsDNA
generatesa415bpbandandan715
bpbandduringPCR,thestudentsis
saidtobeheterozygousatthissiteand
thedesignation+/isassigned.A
personsparticularcombinationof
allelesiscalledtheirgenotype.
updated9/23/2009
15
Student
Activity
PIN#:________
Vocabulary
check one
_____+/+_____+/_____/
YourGenotype:
Allele:Oneofthevariantformsofageneataparticularlocus,orlocation,ona
chromosome.Inthislaboratory,weareinvestigatingalocuswithtwopossible
alleles:(+)or().
Genotype:Thegeneticidentityofanindividualthatdoesnotshowasoutward
characteristics.Thischaracteristicisbasedontheallelespresentatagivenlocus.
Inthislaboratory,therearethreepossiblegenotypes:(+/+),(+/),or(/).
AlleleFrequency
Withinyourclass,howuniqueisyourparticularcombinationofAlualleles?Anallele
frequencyisthepercentageofaparticularallelewithinapopulationofalleles.Itis
expressedasadecimal.YoucancalculateanallelefrequencyfortheAluPV92insertion
inyourclassbycombiningallyourdata.
Example1:Imaginethatthereare100studentsinyourclassandthegenotypedistribution
withintheclassisasfollows:
Genotype
+/+
+/
/
Total
Students
20
50
30
100
+alleles
40
50
0
90
alleles
0
50
60
110
Sinceeachpersoninyourclasshastwonumber16chromosomes,theremust
betwiceasmanytotalallelesastherearepeople.Therefore,inthissampleof
100students,thereare200hundredalleles,90(+)allelesand110()alleles.
Thefrequencyofthe(+)alleleinthisclassis:
90(+)alleles= 0.45
200totalalleles
Thefrequencyofthe()alleleinthisclassis:
Iftheallelefrequencies
donotaddupto1.0then
youhavemadeanerror
inthemath.
110()alleles= 0.55
200totalalleles
updated9/23/2009
16
1. Fillinthechartbelow.
Genotype
Students
+alleles
alleles
+/+
+/
Total
2. Calculatethe(+)allelefrequency=___________________youwillneedthisnumberlater
3. Calculatethe()allelefrequency=___________________youwillneedthisnumberlater
4. Dotheseallelefrequenciesaddupto1.00?_____________
GenotypeFrequencies
Agenotypefrequencyisthepercentageofindividualswithinapopulationhavingaparticular
genotype.Youcancalculatethefrequencyofeachgenotypeinyourclassbycountinghowmany
studentshaveaparticulargenotypeanddividingthatnumberbythetotalnumberofstudents.
Example2:Inaclassof100students,20studentshavethe(+/+)genotype.Thegenotype
frequencyforthe(+/+)genotypeinthisclassis20/100=0.2.
Usethegenotypeinformationinthechartabovetocalculatethegenotypefrequenciesforyour
class.
3. (+/+)frequency=_____________________________
4. (+/)frequency=_____________________________
5. (/)frequency=______________________________
updated9/23/2009
17

ExpectedGenotypeFrequencies(HardyWeinbergLaw)
Ifwithinaninfinitelylargepopulationnomutationsareacquired,nogenotypesarelostor
gained,matingisrandom,andallgenotypesareequallyviable,thenthatpopulationissaidtobe
inHardyWeinbergequilibrium.Insuchpopulations,theallelefrequencieswillremainconstant
generationaftergeneration.Genotypefrequencieswithinthispopulationcanthenbe
calculatedfromallelefrequenciesbyusingtheequation:
p2+2pq=q2=1.0p&qareallelefrequenciesforthetwoformsofageneticsite
Thegenotypefrequencyofthehomozygousconditioniseitherp2orq2dependingonwhich
alleleyouassigntopandwhichtoq.Theheterozygousgenotypefrequencyis2pq.
Example3:Useourfictitiousclasstocalculateexpectedgenotypefrequencies.Inexample1
wedeterminedthefollowingallelefrequencies.Wewillassignptothe(+)
alleleandqtothe()allele.
p=0.45
q=0.55
Thegenotypefrequencyfor(+/+)isequaltop2:
p2=(0.45)2=0.2025
Thefrequencyforthe+/genotypeis2pq:
2pq=2(0.45)(0.55)=0.495
Thefrequencyforthe/genotypeisq2:
q2=(0.55)2=0.3025
Toconvertthesedecimalnumbersintonumbersofstudents,wemultiplyeach
bythetotalnumberofstudents.Sincethereare100studentsinthisfictitious
class,thenumberofstudentsintheclassexpectedtohavethe(+/+)genotype
is:
100x0.2025=20.25
Thenumberofstudentswhoshouldbe(+/)is
100x0.495=49.5
Thenumberofstudentswhoshouldbe(/)is
100x0.3025=30.25
Inourexample,theexpectednumbersofstudentswithaspecificgenotype
matchestheobservedveryclosely.Thisisnotalwaysthecase.
updated9/23/2009
18
6. Usetheallelefrequenciesyoucalculatedearlierinquestions2&3todeterminethe
expectedclassgenotypefrequencies.Followingthat,recordtheactualgenotype
frequencies:
p(+allele)=_______________
q(allele)=_________________
Genotype
Actual
Genotype
Frequency
(questions
57)
Expected
Genotype
Frequency
Total#of
Students
Expected#of
Studentswith
SpecificGenotype
Actual#of
Studentswith
specific
genotype
+/+
p2=
+/
2pq=
q2=
ReviewQuestions
7. AclassislookingatadimorphicAluinsertonchromosome#3.Howmanytotalallelesare
thereinaclassof34studentsforthisAlusite?
8. Theallelefrequencyfortheclassis0.3,Whatisthe+allelefrequency?
9. AclassinHardyWeinbergequilibriumhasa+/+genotypefrequencyof0.64.Whatisthe
+allelefrequency?
10. The+/+genotypefrequencyforaclassis0.49andthegenotypefrequencyis0.09.
Whatisthe+/genotypefrequencyiftheclassisinHardyWeinbergequilibrium?
updated9/23/2009
19

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