PrimerBankisapublicresourceforPCRprimers.Theseprimersaredesignedforgeneexpressiondetectionorquantification(real-timePCR).Thereareseveralwaystosearchforprimers:byGenBankAccession,NCBIproteinaccession,LocusLinkID,PrimerBankIDorKeyword(genedescription).PrimerBankcontainsabout180,000primerscoveringmostknownhumanandmousegenes.

PolymeraseChainReactionAmplification(PCR)isoneofthemostactivelyusedtechniquesinmolecularBIOLOGy.Inrecentyears,PCRhasbeenincreasinglyusedforgeneexpressiondetectionorquantification.Itisamoreconvenientmethodingeneexpressionstudiescomparingtoothertechniques,suchasNorthernBlot.OnecommonprobleminPCRisthenon-specificamplificationsofothergeneproductsbecauseCDNAslibrariesofthousandofgenesareoftenusedasPCRtemplates.Therefore,weneedtocarefullydesignPCRprimersthatspecificallyamplifythegenesofinterest.Unfortunately,mostavailableprimerdesignprogramsonlyfocusonprimerchemicalproperties,suchasmeltingtemperature,GCcontent,secondarystructure,etc.Littleemphasisisgiventoprimermisprimingtoothergenes.Incontrast,allprimersinPrimerBankwerecarefullydesignedtoensuregenespecificity.

Theprimerselectionalgorithmhasbeenextensivelytestedbyreal-timePCRexperimentsforPCRspecificityandefficiency.Uptonow,wehavetestedoverathousandprimersandthedesignsuccessrateis>99%.

YoumaysearchPrimerBankbyGenBankAccession,NCBIproteinaccession,LocusLinkID,orPrimerBankID.

Searchtermsareautomaticallycombinediftheyareseparatedbyspace.Forexample,search"kinases6"returnsallrecordswithbothkeywords"kinase"AND"s6".ANDandORBooleanoperatorsmayalsobeused.Hereareafewexamples:

Currently,onlyalimitedsetofkeywords(keywordsdefinedintheproteindefinitions)canbesearched.Forexample,youcannotsearchbygenesymbols.Ifyoudidnotfindyourgenes,pleaselookupthecorrespondinggeneIDs(DNAaccessions,proteinsaccessions,orLocusLinkIDs)andsearchbyIDs.

BecauseofthesequenceredundancyinGenBank,eachgeneisusuallyrepresentedbymorethanoneNCBIrecord.PrimerBanksometimesusestheNCBILocusLinkindexfiletomapmultiplesequencerecordstothesamegenelocus.Asaresult,adifferentaccessionnumberotherthanoriginallysubmittedmayberetrieved.However,bothaccessionsrepresentthesamegene.IfaretrievedrecordhasadifferentGenBankaccession,aLocuLinkIDwillappearinthegenedescriptionfield.

Youmayuseyourbrowser"ssavefunctiontosavethewebpageinyourlocalcomputer.Othersavingoptionswillbeimplementedlater.

AlltheprimersinPrimerBankweredesignedusingaprogramcalleduPrimer.Greatcarehasbeengiventoavoidprimermisprimingtootherknowngenesinagenome.Hereisalistofcriteriaforgenespecificprimerdesign:

1. Theprimerlengthrange:19-23nt,withtheoptimallengthat21nt.
2. TheprimerGCpercentagerange:35%-65%.
3. ThedeltaGvalueforthefive3’end-basesisatleast-9kcal/mol.
4. TheprimerTmrange:60-63°C,determinedbytheNearestNeighborMethod.
5. ThePCRproductlengthrangeis100-250bp.Ifthisrequirementcannotbesatisfied,alternativerangeswillbeused.
6. Thedefaultnumberofprimerpairsdesignedforeachsequenceis3.
7. Noprimerisdesignedfromlow-complexityregions.
8. Aprimerdoesnotcontain6ormorecontiguoussamenucleotides.
9. Aprimerdoesnotcontainanyambiguousnucleotide.
10. Norepetitive15-merfromothergenesequencesinthegenome(forbothstrands)anywhereinaprimer.
11. Norepetitive13-merfromnon-codingRNAsequences(forbothstrands)anywhereinaprimer.
12. TheglobalBLASTscoreforanyprimerislessthan30(equivalentto15-merperfectmatch).
13. ThemaximumTmforthe3’endperfectmatchtoothergenesequencesdoesnotexceed46°C;doesnotexceed42°Cwhencomparedtonon-codingRNAsequences(TmdeterminedbytheNearestNeighborMethod).
14. Forprimersecondarystructure(theprimer-primerself-annealing)
    1. Norepetitive5-merisallowedanywherewhenaprimersequenceiscomparedtoitscomplementarystrand.
    2. Thefour3’-endbasesshouldbeuniquewhencomparedtotheprimer’scomplementarystrand.
15. Theforwardandreverseprimersshouldnotannealtoeachother.Thefiltersettingisthesameasintheprimersecondarystructurefilter.
16. Forsequencesecondarystructure(theprimer-sequenceself-annealing)
    1. Norepetitive9-merisallowedwhenaprimersequenceiscomparedtothecomplementarystrandofitscognatesequence.
    2. TheBLASTscoreislessthan18whenaprimersequenceiscomparedtothecomplementarystrandofitscognatesequence.

AlltheprimersinPrimerBankhavemeltingtemperatures(Tm)of60-63°C.Ahigherannealingtemperatureresultsinmorespecificpriming.PreviousstudiesindicatedsufficientprimingshouldoccuratprimerTm.Therefore,anannealingtemperatureof60°CisrecommendedforallPrimerBankprimers.Non-specificPCRproductsarelikelytooccuratlowerannealingtemperature.Wehavetestedafewhundredprimersunder60°CannealingtemperatureandthePCRexperimentsworkedverywell.

TheprimerTmvaluesarecalculatedusingtheNearestNeighborMethodwiththeup-to-dateThermodynamicparameters.Tmvaluesarealsodependentonprimerandsaltconcentrations.HigherconcentrationsofprimerandsaltusuallyleadtohigherTm.TheTmvaluesincludedinPrimerBankarefor0.25uMprimer,1.5mMMg²⁺,50mMNa⁺,and0.8mMdNTP,whicharetypicallyusedinPCRexperiments.OthercommonPCRconditionsonlyaffecttheTmslightly.

ToguaranteePCRefficiency,smallPCRproductsarerecommended.MostPrimerBankprimersleadtoampliconsinthesizerangeof100-250bp.PCRefficiencyiscloseto100%inthisrange(supportedbyreal-timePCRexperiments).However,PCRefficiencymaybereducedformuchlargerPCRproducts..Lessthan1%ofPrimerBankprimersleadto>400bpamplicons.PleasebecautiousaboutPCRefficiencywhentheseprimershavetobeused.

Sometimesitisdesirabletoselectaprimerpairthatspansintron.Inthisway,genomicDNAcontaminationcanbecloselymonitored.UsuallyaPrimerBankprimerpairspansintronbecauseatypicalexonisquitesmall.Theprimerintron-spanninginformationwillbeincludedinthenextversionupdate.Ifitisimportantforyoutobesurethataprimerpairspansintron,youmaysimplydoaBLASTsearchoftheprimerpairsequences,orbettertheampliconsequences(listedintheprimerdetailpage)againstthegenomicsequences.

AgarosegelelectrophoresisormeltingcurveanalysismaynotalwaysreliablyreflectPCRspecificity.Fromourexperience,bimodalmeltingcurvesareoccasionallyobservedforlongampliconsevenwhenthePCRsarespecific.Theobservedheterogeneityinmeltingtemperaturewasduetointernalsequenceinhomogeneity(e.g.independentlymeltingblocksofhighandlowGCcontent)ratherthanampliconcontamination.Ontheotherhand,forshortampliconsveryweakbandsmigratingaheadofthemajorspecificbandsareoccasionallyobservedonagarosegel.Theseweakbandsaresuper-structuredorsingle-strandedversionofthespecificampliconsinequilibriumstate.Althoughgelelectrophoresisormeltingcurveanalysisalonemaynotbe100%reliable,thecombinationofbothcanalwaysrevealPCRspecificityinourexperience.

AlthoughallPrimerBankprimersaredesignedtobegenespecific,westillneedtobeverycarefulaboutPCRconditions.

Non-specificprimerextensionofonlyafewbasesatlowtemperaturebyDNApolymerasecaneasilyleadtonon-specificPCRamplifications.Therefore,hot-startPCRisSTRONGLYrecommended.Infact,Ionlydohot-startPCRinmyexperiments.IroutinelyuseAmpliTaqGoldpolymerase(AppliedBiosystems)forhot-startPCR.

TheannealingtemperaturemayaffectPCRspecificity.Toavoidnon-specificPCRproducts,ahighannealingtemperature(thesmalleroneofthetwoTmvaluesfromtheprimerpair)andashortannealingtimearerecommended.

Ifyoustillseenon-specificbands,itcouldmeantheprimerpairinuseistheproblem.AlthoughgreatcarehasbeengiventodesignPrimerBankprimers,thesuccessrateisnot100%.Lessthan1%oftheprimersmayhavedesignproblems(seeourpaperfordetaileddiscussion).Inthiscase,pleasetryadifferentprimerpairforthesamegene.Clickhereifyouwouldliketoreportprimerproblems.

PoorqualityofPCRtemplates,primers,orreagentsmayleadtoPCRfailures.First,pleaseincludeappropriatecontrolstoeliminatethesepossibilities.

Somegenesareexpressedonlyincertaintissues.PleasefirstreadliteraturetomakesureyourgenesareincludedinthecDNAtemplates.Inourexperience,thisisthemostlikelycausefornegativePCRresults.Ifyouaresurethegenesareexpressed,thentryloweringtheannealingtemperature(toTm-5°C)andincreasingtheannealingtimetoensuresufficientprimerannealing.

IfyoustillcouldnotseeanyPCRband,itcouldmeantheprimerpairinuseistheproblem.AlthoughgreatcarehasbeengiventodesignPrimerBankprimers,thesuccessrateisnot100%.Lessthan1%oftheprimersmayhavedesignproblems(seeourpaperfordetaileddiscussion).Inthiscase,pleasetryadifferentprimerpairforthesamegene.Clickhereifyouwouldliketoreportprimerproblems.

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