Polymerasechainreaction(PCR)hasrapidlybecomeoneofthemostwidelyusedtechniquesinmolecularBIOLOGyandforgoodreason:itisarapid,inexpensiveandsimplemeansofproducingrelativelylargenumbersofcopiesofDNAmoleculesfromminutequantitiesofsourceDNAmaterial--evenwhenthesourceDNAisofrelativelypoorquality.

PCRinvolvespreparationofthesample,themastermixandtheprimers,followedbydetectionandanalysisofthereactionproducts.Thesestepsarediscussedbelow.

PCRisveryversatile.Manytypesofsamplescanbeanalyzedfornucleicacids.MostPCRusesDNAasatarget,ratherthanRNA,becauseofthestABIlityoftheDNAmoleculeandtheeasewithwhichDNAcanbeisolated.Byfollowingafewbasicrules,problemscanbeavoidedinthepreparationofDNAforthePCR.TheessentialcriteriaforanyDNAsamplearethatitcontainatleastoneintactDNAstrandencompassingtheregiontobeamplifiedandthatanyimpuritiesaresufficientlydilutedsoasnottoinhibitthepolymerizationstepofthePCRreaction.

AlthoughanyprotocolisacceptableforPCRpurposes,itisoftenbesttousethefeweststepspossIBLeinDNApreparationinordertopreventaccidentalcontaminationwithunwantedDNA.Usuallya1:5dilutionofthesamplewithwaterissufficienttodiluteoutanyimpuritieswhichmayresultfromthepurifyingprotocol.

ThesimplestmethodofisolatingDNAfromcellsisasfollows:

1. Cellscanbeobtainedbyusingatoothpicktoscrapeunderthefingernails,swabbingtheinsideofthemouthorfromtherootsofpluckedhairs.Regardlessofsource,cellsareresUSPendedin20ulofwater.Skiptostepfour.

2. Ifyouareusingcellssuspendedinmedia,centrifugeat1200-1500Xgfor5minutes.Resuspendthecellpelletin1mlofphosphatebufferedsaline(PBS)andrepelletbyspinningat1200-1500Xgfor5minutes.Repeat.ThesePBSwashesremovemedium,anditsinhibitoryfactors,fromthesurfaceofthecells.Afterthelastwashresuspendthecellpelletin20ulofdistilledwater.BeawarethattoomuchcelldebriscaninhibitthePCRreaction.Ifthishappens,itmaybenecessarytofurtherdilutetheDNAsample.Gotostepfour.

3. Forbacterialsamplestakeatoothpickandscrapetheteeth,orswabthethroat,earsorbetweenthetoes.Resuspendmaterialin500ulofwater.Freezeandthawsamplethreetimeswithvigorousshakingorvortexingbetweenrepetitionstobreakthebacterialcellwall.AlthoughnotallDNAwillbereleasedfromthecells,therewillbeasufficientquantityforPCR.Gotostepfour.

4. Placethesampleina95oCheatingblock,orinboilingwater,for5minutes.ThisstepinactivatestheDNasemoleculesthatarefoundinthesamplepreparation.Ifleftintact,DNasecouldclipthedesiredDNAtemplatemoleculeintofragmentswhichwouldbeunsuitableforPCR.IfthereisverylittleDNAinthesamplepreparation,theDNAcanbeconcentratedbyethanolprecipitation.ThesampleisnowreadyforPCR.
DNAsamplesforPCR--regardlessofpreparationmethod--aregenerallyruninduplicateinordertoprovideacontrolfortherelativequalityandpurityoftheoriginalsample.AddingasmallamountofDNAtothecontroljustafterthemastermixstepallowsthedetectionofanythinginthecompletedsampleprepwhichwouldinhibitthePCRreaction.

TheMasterMixcontainsallofthecomponentsnecessarytomakenewstrandsofDNAinthePCRprocess.TheMasterMixreagentsinclude:

FinalComponentPurposeConc.Water1XBufferkeepsthemastermixattheproperpHsothePCRreactionwilltakeplace.200uMDeoxynu-provideboththeenergyandnucleosidesforthecleotidessynthesisofDNA.Itisimportanttoaddequalamountsofeachnucleotide(dATP,dTTP,dCTP,dGTP)tothemastermixtopreventmismatchesofbases.0.2-1.0uMPrimersShortpiecesofDNA(20-30bases)thatbindtotheDNAtemplateallowingTaqDNApolymeraseenzymetoinitiateincorporationofthedeoxynucleotides.Bothspecificanduniversalprimerscanbeused.2.5U/100ulAmpliTaqAheatstableenzymethataddsthepolymerasedeoxynucleotidestotheDNAtemplate.0.05-1.0ugTemplateTheDNAwhichwillbeamplifiedbythePCRDNAreaction.

TheMastermixbufferisoftenstoredasa10Xstocksolution(100mMTris-HCL,pH8.3,500mMKCL,1.5mMMgCl2)whichisdilutedto1Xforuse.BoththeMastermixbufferandthepurifiedwatercanbestoredatroomtemperature.Storedeoxynucleotides,primersandTaqDNApolymeraseenzymeat-20oC.

Although100ulofmastermixperreactionisgenerallyused,itispossibletouseaslittleas25or50ultosaveoncostofreagents.Regardlessofthetotalvolume,becertaintokeepthefinalconcentrationsofreagentsconstant.

Mastermixreagentscanbeoptainedfromavarietyofcompanies.Oftentheinitialconcentrationofthereagentwilldifferdependingonwhichcompanyproducedit.Itiseasytofigureouthowmuchstockreagenttousebyfollowingasimpleformula:

(initialconcentration)X(volumeneeded)=(finalconcentration)X(volumeofsample)

Forexample:Ihave10Xbuffer,10mMofeachnucleotide,0.5mMprimersandTaqDNApolymeraseat5Units/ul.Iwanttomakeone50ulreaction.Calculationsareasfollows:

10Xbuffer:(10X)X(5ul)=(1X)X(50ul)Nucleotides:(10,000uM)X(1ul)=(200uM)X(50ul)(10mM=10,000uM)primers(500uM)X(O.1ul)=(1.0uM)X(50ul)Sinceitisimpossibletopipet0.1ulaccurately,adilutionneedstobemadefirst.Add10ulofstockprimersolutionto990ulofwatertoget5uMconcentrationofprimers.Thisnewprimerdilutioncanbestoredat4oC.Calculationfor5uMstock:(5uM)X(10ul)=(1.0uM)X(50ul)TaqDNApolymerase(5Units/ul)X(0.25ul)=(.025Units/ul)X(50ul)2.5Units/100ul=Sinceitisimpossibletopipet0.25ulaccurately,a.025Units/uldilutionneedstobemadefirst.Add1.25ulstockto3.75ulwatertogeta1.25Units/ulconcentration.Discardandmakefreshwitheachuse.Calculationfor1.25Units/ulstock:(1.25Units/ul)X(1ul)=(.025Units/ul)X(50ul)

Tomakethemastermixforonereactionadd:
• 5ul10Xbuffer
• 4ulEachnucleotide(1uleachofdATP,dCTP,dGTP,dTTP))
• 20ulEachprimer(10ulofeach)
• 1ulTaqDNApolymerase(Totalvolume=30ul)
• add15ulofwater
• 5uloftemplate(Totalvolume=50ul)
Ifwanttomake3reactions,3X50ul=150ul.Usethisnumberintheformulafor"volumeofsample."

AprimerisashortsegmentofnucleotideswhichiscomplementarytoasectionoftheDNAwhichistobeamplifiedinthePCRreaction.PrimersareannealedtothedenaturedDNAtemplatetoprovideaninitiationsitefortheelongationofthenewDNAmolecule.PrimerscaneitherbespecifictoaparticularDNAnucleotidesequenceortheycanbe"universal."UniversalprimersarecomplementarytonucleotidesequenceswhichareverycommoninaparticularsetofDNAmolecules.Thus,theyareabletobindtoawidevarietyofDNAtemplates.

BacterialribosomalDNAgenescontainnucleotidesequencesthatarecommontoallbacteria.Thus,bacterialuniversalprimerscanbemadebycreatingprimerswhicharecomplementarytothesesequences.Examplesofbacteriauniversalprimersequencesare:Forward5"GATCCTGGCTCAGGATGAAC3"(20mer)Reverse5"GGACTACCAGGGTATCTAATC3"(21mer)

Animalcelllinescontainaparticularsequenceknownasthe"alugene".Thereareapproximately900,000copiesofthealugenedistributedthroughoutthehumangenome,andmultiplecopiesdistributedthroughthegenomeofotheranimalcells,aswell.Thus,thealugeneprovidesthesequenceforauniversalprimerforanimalcelllines.Thealuprimerisespeciallyusefulinthatitbindsinbothforwardandreversedirections.Thealuuniversalprimerseqeunceisasfollows:5"GTGGATCACCTGAGGTCAGGAGTTTC3"(26mer)

Whenusinguniversalprimerstheannealingtemperatureonthethermalcyclerisloweredto40-55degreesC.

SometimesprimerunitsarelistedinopticaldensityreADIng(OD).Ifthisisaproblemyouwillneedtoconverttomolarityusingthefollowingequations:Changeopticaldensityreadingofprimertomolarity(uMunits)-

1. N=#ofprimerbases
2. SIGMA260=~10,000XN/mXcm
3. Molecularweight=~330XN
4. OD₂₆₀/SIGMA260X10⁶=Concentration(uM)
Forexample-primeris20baseslong/OD₂₆₀=10.
1. N=20
2. SIGMA260=~10,000X20/mXcm=20,000/mXcm
3. molecularweight=~330X20=6,600
4. 10OD₂₆₀/20,000m^-1cm^-1X10⁶=50uM
ThePCRproductshouldbeafragmentorfragmentsofDNAofdefinedlength.Thesimplestwaytocheckforthepresenceofthesefragmentsistoloadasampletakenfromthereactionproduct,alongwithappropriatemolecular-weightMarkers,ontoanagarosegelwhichcontains0.8-4.0%ethidiumbromide.DNAbandsonthegelcanthenbevisualizedunderultraviolettrans-Illumination.Bycomparingproductbandswithbandsfromtheknownmolecular-weightmarkers,youshouldbeabletoidentifyanyproductfragmentswhichareoftheappropriatemolecularweight.

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