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PR037
Polymerase Chain Reaction
(PCR)

Teachers Guidebook
(Cat. # BE305)
Page2of16
MATERIALSINCLUDED.......................................................................................................3
SPECIALHANDLINGINSTRUCTIONS...................................................................................3
ADDITIONALEQUIPMENTREQUIRED................................................................................3
TIMEREQUIRED.................................................................................................................3
OBJECTIVES........................................................................................................................4
BACKGROUND...................................................................................................................4
TEACHERSPREEXPERIMENTSETUP................................................................................8
GENOMICISOLATION....................................................................................................8
PREPAREPCRREAGENTS...............................................................................................8
PROGRAMTHERMOCYCLER...........................................................................................9
VISUALIZATIONOFPCRPRODUCTS...............................................................................9
MATERIALSFOREACHGROUP........................................................................................10
PROCEDURE.....................................................................................................................11
ISOLATIONOFGENOMICDNA.....................................................................................11
POLYMERASECHAINREACTION..................................................................................13
RESULTS,ANALYSIS&ASSESSMENT................................................................................15


MATERIALS INCLUDED
Thiskithasenoughmaterialsandreagentsfor24students(sixgroupsoffourstudents).
3bottlesDNAReleaseBuffer
2bottlesPrecipitationSolution
1bottleDNASaltSolution
1vialProtease:DryProtease
30CytologyBrushes
120CentrifugeTubes(1.5ml)
1vialPCR:5GenomicPrimer
1vialPCR:3GenomicPrimer
1vial10XPCRBuffer(Mg
2+
plus)
2vialsPCR:Deoxynucleotides(dNTPs)
1vialTaqDNApolymerase
2vialsSterileWater
2vialsPCR:MineralOil
24PCR:PCRtubes
SPECIAL HANDLING INSTRUCTIONS
Store5and3GenomicPrimers,TaqReactionBuffer,dNTPs,andTaqDNA
polymeraseat20C
Allotherreagentscanbestoredatroomtemperature.
ADDITIONAL EQUIPMENT REQUIRED
15mlCentrifugeTube
Waterbathorbeakerandthermometer
PCRMachine(Thermocycler)
AgaroseElectrophoresisEquipment
TIME REQUIRED
Day1:3hours
Day2:23hours
Page3of16
OBJECTIVES
IsolateyourownDNAgenome.
Amplifyaspecificgenefromyourgenome.
Understandtheprinciplesofthepolymerasechainreaction.
BACKGROUND
ThePolymerasechainreaction(PCR),firstenvisagedin1984byKaryMullis,has
revolutionizedlifesciencesandhasbecomeanessentialtechniqueinmanyaspectsof
science,includingclinicaldiagnostics,forensicsandgeneticengineering.KaryMullis
eventuallyreceivedtheNobelPrizeinChemistryin1993.
PCRallowsscientisttomakeunlimitedcopiesofDNAfragmentsandgenesfromasingle
copyofinitialDNA.Eachcycleofthepolymerasechainreactiondoublesthenumberof
copiesofthegeneofinterest,soforthisexperiment,whichhas33cycles,over17billion
copiesofyourgeneofinterestwillbemadeforeachstartingtemplate(seefigure1).

Geneofinterest
Template
DNA
Cycle 33
Cycle 1 Cycle 2 Cycle 3 Cycle 4
Number
Figure1:Theexponentialcopyingofageneofinterestduringthepolymerasechain
reaction.
PCRutilizesthenaturalfunctionofpolymeraseenzymes.Inanormaldividingcell,the
copyingofthegenesrequiresaseriesofenzymemediatedreactions:
1. TheDNAstrandsareunwound(denatured)byenzymestoformtwosinglestrands.
2
2
2
3
2
4
2
5
2
34
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2. ARNApolymerasebindsandsynthesizesashortcomplementarypieceofRNAon
theDNAstrandattheinitiationsiteofreplication.
3. ThisDNA/RNAheteroduplexactsasaprimingsitefortheDNApolymerasethat
bindsandproducesthecomplementarystrand.
Thekeytothepolymerasechainreactionwasfirstdiscoveredin1976.Thekeyisthe
TaqpolymerasethatwaspurifiedfromthethermophileThermusaquaticus.A
thermophileisanorganismthatgrowsatextremetemperature(>100C).The
importanceoftheTaqpolymerasebeingpurifiedfromathermophileisthattheenzyme
willnotbedestroyedathightemperaturesrequiredtodenaturetheDNAandallowPCR
tobegin.
AschematicofthePCRreactionisshowninfigure2andarepresentationofthecritical
temperaturecyclesisshowninthegraphinfigure3.

Figure2:SchematicrepresentationofthePolymeraseChainReaction
Page5of16
TherearethreebasicstepsinPCR(Figure2).First,thetemplateDNAorgeneticmaterial
isdenatured;thestrandsofitshelixareunwoundandseparatedbyheatingto9096C.
InanormalcelltheDNAisunwoundbyspecificenzymes.
Thesecondstepishybridizationorannealing.TheTaqpolymeraserequiresashort
pieceofRNAtoinitiateDNAreplication,whichinanormalcellissynthesizedbytheRNA
polymerase.InthePCRreaction,shortcomplimentarydoublestrandedoligosare
addedthatbindthedenaturedDNAandactasoriginsofreplications.Thesedouble
strandedoligosareknownasprimersandarecomplimentarytosequencesupand
downstreamofthegeneofinterest.Twoprimersareused,oneforeachstrandofDNA.
Followingdenaturation,thereactionmixtureisrapidlycooledtoatemperaturebelow
themeltingpointofthespecificprimers(~55C),belowthistemperaturetheprimers
bindtotheircomplementarybasesonthenowsinglestrandedDNA.
Inthethirdstep,thetemperatureofthereactionisraisedtotheoptimaltemperature
forthepolymerase(6872C).ThepolymerasesynthesizesnewDNA,startingfromthe
primer,thepolymerasereadsatemplatestrandandgeneratescomplementary
nucleotidesveryquickly.Theresultistwonewhelixesinplaceofthefirst,each
composedofoneoftheoriginalstrandsplusitsnewlyassembledcomplementary
strand.
40
50
60
70
80
90
100
0 5 10 15 20 25 30
Time (min)
T
e
m
p
e
r
a
t
u
r
e

(
C
)
CYCLE 1 CYCLE 2 CYCLE 3 CYCLE 4 CYCLE 5 CYCLE 6
Denature Denature Denature Denature Denature
Anneal Anneal Anneal Anneal Anneal Anneal
Denature
Extend Extend Extend Extend
Extend
Extend

Figure3:ThetemperaturecyclesusedduringthePCRreaction.Thegraphdepictsthe
changesintemperatureandtheresultingeffectontheDNA.Aschematicofthese
effectsisshowntotherightofthegraph.
Page6of16
Thepolymerasechainreactionisabletoproducelargecopiesofthegenesofinterestas
theabovecyclecanberepeatednumeroustimesleadingtoanexponentialincreasein
thenumberofnewcopies(figure1).
Thethermocycleristhemostimportantpieceoftechnologyforresearcherswantingto
usePCR.Athermocyclertightlyregulatesthetemperaturechangesrequiredfor
denaturation,annealingandextension.Italsocontrolsthenumberofcycles.Todays
thermocyclersarefullyprogrammableandallowforrapidheatingandcoolingand
thereforetightercontrolofthePCR.
Inthisexperiment,studentswillamplifyanucleotidesequencefromchromosome16to
lookfortheinsertionofashortDNAsequencecalledAluwithinthePV92locus.DNA
fromdifferentindividualscontainmanyregionsthatexhibitagreatdealofdiversityand
theseregionsareknownaspolymorphic(manyforms)andprovidethebasisforgenetic
diseasediagnosis,forensicidentificationandpaternitytesting*.
TheAlufamilyofshortinterspersedrepeatedDNAelements(orSINEs)aredistributed
throughoutprimategenomesandoverthepast65millionyearstheAlusequencehas
beenamplifiedtoacopynumberofabout500,000compromisinganestimated5%of
thehumangenome.TheAluelementsareapproximately300bpinlengthandderive
theirnamefromasinglerecognitionsiteoftheAluIendonucleaselocatedinthemiddle
oftheAlusequence.Inthisexperiment,theAluelementbeingamplifiedisdimorphic,
meaningthatitispresentinsomeindividualsbutnotothers(Figure4).
Thiskitisdesignedtoteachthebasicsofthepolymerasechainreactionandallows
studentstoamplifyagenefromtheirowngenome.

Figure4:PossibledistributionofAluinsertinPV92locusandPCRfragmentsizes.
*TheAluallelesareinheritedfromparentsandcanpotentiallyrevealinformationabout
familyrelationshipsandasaresultwecautionagainstgeneratingdatafrommultiple
familymembers.Ifthisisaconcern,werecommendmixingupthestudentsamplesafter
thecellshavebeencollectedtoensureanonymity.
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TEACHERS PRE EXPERIMENT SET UP
GenomicIsolation
1. Priortothecommencementoftheexperiment,add0.5mlDNAReleaseBufferto
thevialofdryproteasetorehydrate.Mixbyinvertingthevialseveraltimesuntila
clearsolutionisvisible.Thissolutioncanbestoredfrozenforlateruse.
Instructstudentstostopeatinganddrinkingatleastonehourbefore
experimentation.Thishelpspreventloosecellsbeingwashedaway.
2. PreparetheDNAwashsolution.Labela15mltubewithDNAWash.Transfer
10.5mlPrecipitationSolutionand4.5mlultrapurewatertothelabeledtube.
Invert56timestomix.
3. Prepareawaterbathorheatingblockat5060C.Lowertemperaturescanbe
used,downtoroomtemperature,howeverlongerdigestiontimeswillberequired.
4. Tuberacksorfloatsarealsorequired.
PreparePCRreagents
1. Storeallreagentsonicethroughouttheexperiment.
Allcomponentsusedinthepolymerasechainreactionshouldbekepton
ice.Thestudentsexperimentsshouldallbecarriedoutonice.
2. Transfer150lSterileWatertothe5GenomicPrimertube.Resuspendtheprimer
bygentlypipettingupanddown.
3. Labelsixtubeswith5Primer.Transfer25l5GenomicPrimerfromstep2to
eachtube.Supplyeachgroupwithasingletube.
4. Transfer150lSterileWatertothe3GenomicPrimertube.Resuspendtheprimer
bygentlypipettingupanddown.
5. Labelsixtubeswith3Primer.Transfer25l3GenomicPrimerfromstep4to
eachtube.Supplyeachgroupwithasingletube.
6. LabelsixtubeswithTaqBuffer.Transfer40l10XPCRBuffer(Mg
2+
plus)toeach
tube.Supplyeachgroupwithasingletube.
7. Transfer126lSterileWatertoeachvialofdNTPs.ResuspendthedNTPsbygently
pipettingupanddown.
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8. LabelsixtubeswithdNTP.Transfer40ldeoxynucleotidestoeachtube.Supply
eachgroupwithasingletube.
9. LabelsixtubeswithTaq.Add45lSterileWatertothevialofTaqDNA
Polymerase.Transfer10lTaqDNApolymerasetoeachtube.Supplyeachgroup
withasingletube.
10. LabelsixtubeswithH
2
O.Transfer0.5mlSterileWatertoeachtube.Supplyeach
groupwithasingletube.
ProgramThermocycler
NOTE:ThiskitisdesignedsothateachstudentsetsuptheirownPCRreaction,a
totalof24.Ifyourthermocyclerisunabletoaccommodate24samplestheneitherallow
everystudenttosetupthereactionandrunaselectionorhavestudentsworkinpairs
generating12samples.Adjustthevolumesaliquotedaccordingly.
1. Followthemanufacturersinstructionsforyourthermocycler.Ifthethermocycler
hasaheatedlidthenthemineraloilisnotrequired.Youwillneedenoughspace
forthenumberofstudentsintheclass(maximum24).
2. Programthefollowingprogram:
A. 1Cycleof96Cfor2minutes.
B. 33Cycles94Cfor1min,55Cfor1min,68Cfor2min.
C. 1Cycle68Cfor7mins.
D. 1Cycleof4Cforever.
VisualizationofPCRproducts
1. InordertovisualizethePCRproductsa2%agarosegelwillneedtoberun.Each
studentrequires1wellandadditionalwellsarerequiredforreferencemarkers.
YoumayuseyourownequipmentandsuppliesoruseGBiosciencesIntroduction
toAgaroseElectrophoresiskit(Cat.#BE304).
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MATERIALS FOR EACH GROUP
Supplyeachgroupwiththefollowingcomponents.Severalcomponentsaresharedby
thewholeclassandshouldbekeptonacommunaltable.
3bottlesDNAReleaseBuffer(sharedwithclass)
1vialprotease(sharedwithclass)
1bottleDNAWash(sharedwithclass)
1bottlePrecipitationSolution(sharedwithclass)
1bottleDNASaltSolution(sharedwithclass)
4CytologyBrushes
8centrifugetubes(1.5ml)
1vial5Primer
1vial3Primer
1vialTaqReactionBuffer
1vialdeoxynucleotides(dNTPs)
1vialTaqDNApolymerase
1vialSterileWater
1vialmineraloil(sharedwithclass)
4PCRtubes

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PROCEDURE
IsolationofgenomicDNA
1. Labeltwo1.5mlCentrifugeTubeswithyourname.
2. Add0.4mlDNAReleaseBuffertoalabeled1.5mlTube.Thissolutioncontainsa
detergentthatdisruptsthecellstructureandreleasesthegenomicDNAintothe
solution.
3. CollectCheekCells:Usethecytologybrushtocollectyourcheekcellsbyscraping
theinsideofyourcheek.Scrapetheinsideofeachcheek;workontheareaaround
thegumline20times,whilsttwirlingthebrushbetweenyourfingers.
Instructstudentsnottooscrapetoovigorously.Thebestplaceforcollectingthe
cellsisatthegumline.
4. PlacethebrushintotheDNAReleaseBufferandleaveinthesolutionfor1minute
periodicallytwirlingthebrush.
5. Removethebrushfromthesolution,ensuringthatyouthoroughlyscrapethe
brushonthesideofthetube,releasingthecheekcellsintotheDNARelease
Buffer.
Thesolutionshouldbeslightlycloudyandviscous.
6. Add0.02mlProteasetothetubetodigestandremovethecellularmaterialand
proteinfromthegenomicDNA.
7. Closethecap.Brieflymixbyinvertingthetube56timesandthenplaceina50
60Cwaterbathorheatingblockfor2060mins.
8. After1hour,add90lDNASaltSolutiontothetubeandmixbyinvertingthetube
severaltimes.ThesaltsolutionaidsintheprecipitationoftheDNA.
9. Centrifugethetubefor5minutesat10,000xgtopelletthecelldebris.Transferthe
supernatanttoyourotherlabeledtube.
10. Slowly,add0.95mlPrecipitationSolution,closethetubeand,whilstwatching,
slowlyinvertthetubeseveraltimestomix.WhiteDNAstrandsmayappear.
ThelackofwhitestrandsdoesnotindicatealackofDNA.Onlyatinyamountof
DNAisneededforPCRamplification.
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11. TocollecttheDNAcentrifugethetubeat10,000xgfor10minutes.Atightwhite
pelletshouldbevisualizedinthebottomofthetube.Discardthesupernatantand
keepthepelletofDNA.
Thewhitepelletmaybeveryhardtovisualize.Toaidinlocatingthepellet,makea
noteofthetubesorientationwithinthecentrifuge.
12. Add0.2mlDNAWashtothepelletandcentrifugeat10,000xgfor10minutes.
TheDNAWashisamixtureofalcoholandwaterthatallowsfortheremovalof
contaminatingsalts,withoutdissolvingtheDNA.
13. RemovealltheDNAWashwithapipettetip.Add50lSterileWatertotheDNA
pellet.Incubateatroomtemperaturefor1015minuteswithperiodicmixing.
Ifnecessary,thisisaconvenientstoppingpoint.StoretheDNAat20Cina
freezer.TheDNAisstableforuptoamonth.
14. Optional:TovisualizethegenomicDNA,remove10lgenomicDNAandvisualizeon
a1%agarosegel.

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PolymeraseChainReaction
AllthecomponentsofthePCRreactionandthesettingupthereactionshouldbe
doneonice.
1. LabelthesideofaPCRtubewithyourinitialsandplacethetubeonice.
2. AsagroupprepareaMasterMixofthePCRreactioncomponents.Ina2ml
centrifugetube,addthefollowingcomponents.Useadifferentpipettetipforeach
component.Pipetteeachcomponentintothebottomofthetube.Onadditionof
eachcomponent,gentlymixbypipettingupanddown.
20l5GenomicPrimer
20l3GenomicPrimer
40lTaqReactionBuffer
40ldNTPs
10lTaqpolymerase
250lSterileWater
3. Transfer5lofyourdilutedgenomicDNA(fromstep13)toyourlabeledPCRtube.
4. Add95lMasterMixfromstep2toyourPCRtube.Gentlymixbypipettingup
anddown45times.
5. Ifusingathermocyclerwithoutaheatedlid,add50lMineraloiltoprevent
evaporation.
Informyourstudentsiftheyrequiremineraloil.
6. TheThermocyclershouldbeprogrammedasfollows
1Cycleof96Cfor2minutes
33Cycles94Cfor1min,55Cfor1min,68Cfor2min
1Cycle68Cfor7mins
1Cycleof4Cforever.
7. Thefollowingday,afterthereactionhasfinished,removethetubesandremove
30ltobeanalyzedbyagaroseelectrophoresis.
ThePCRreactioncanbestoredat20Cuntilrequired.
Seepreexperimentsetupforinformationonagaroseelectrophoresis.ThePCRreaction
shouldberunona2%agarosegelasthegeneis415andor715bp.
Page13of16
STOPthegelwhenthebluedyefrontismorethan3cmfromthegelbottom.Ifrun
furtherthePCRproductwillbelostasitrunsaheadofthedyefront.

Page14of16
Page15of16
RESULTS, ANALYSIS & ASSESSMENT
1. WhatwasthesizeofthePCRproductthatwasvisualizedontheagarosegel?
415bp,715bporboth.
2. ExplainyourresultbelowintermsofyourAluelements?
Ifasingle415bpbandwasseenthentheindividualishomozygousnegative,
meaningtheydonothavetheAluelement.
Ifasingle715bpbandwasseenthentheindividualishomozygouspositive,
meaningtheyhavetheAluelementonbothDNAstrands.
Ifbotha415and715bpbandwasseenthentheindividualisheterozygous,
meaningthatoneDNAstrandhastheAluelementandtheotherdoesnot.
3. ExplainthevariousstepsofthePCRprogram,explainingtherelevanceofthe
temperaturesandtimes.
Thefirstlong96CcycleistoallowcompletedenaturationofthegenomicDNA
The33cyclesarethemainpartofthePCRreactionandtheseconsistofashort
denaturationstageat94C,followedbyanannealingstep.Thelowtemperature
allowstheprimerstoattachtothedenaturedDNA,highertemperaturesmay
preventannealing.The68Ctemperatureisanoptimaltemperatureforthe
polymerasetosynthesizenewcopiesoftheDNA.Thelongertimesandincreasing
incubationtimesallowthepolymerasetosynthesizethecompletegene.Longer
geneswouldrequirelongertimes.
4. Discusstheroleofeachcomponentinthereactionmixture.
Primers:BindtothedenaturedDNAandactastheoriginsofreplicationfortheTaq
polymerasetobindto.
dNTPs:Asourceofnucleotides(adenosine,guanine,cytosine,thymidine)
triphosphates(NTP)requiredforthegenerationofnewDNA.
GenomicDNA:Actsasthetemplatefornewgenesynthesis.
Taqpolymerase:CrucialenzymerequiredforDNAsynthesis.
Lastsaved:9/5/2012CMH

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