PrimerBLAST操作说明.ppt

GeXPGeneExpressionMultiplexDesign UsingNCBIPrimer BLASTtodesignhighperformance gene specificprimersforanXP PCRmultiplex Updated March07 2011 TwoDesignStrategies UseAccessionNumbersorProprietaryGenestocreategene specificprimersforamplicons105 350nucleotidesinlength Theshotgunapproachisanefficientmethodtousewhenlittleornogenomeinformationisavailablefortheorganismofinterest Littleornohomology pseudogeneortranscriptvariantinformationNoSNPinformationNointron exonboundaries Thestrategicapproachisrecommendedwhenamodelorganismisusedand orthereissufficientgenomicdataavailableinNCBIforPrimer BLASTtoscanprimersfor Homologyand ortranscriptvariantsSNPIntron exonboundariesareknown 1 2 AccessionNumbers StrategicApproach Pre designconsiderationsChoosingthegeneandaccessionnumberUseonlymRNAaccessionnumbersAmpliconlengthandspacebetweenpeaksDesignusingNCBIPrimer BLASTAmpliconlengthsDesignprimerstodetectalltranscriptvariants ifnotspecified orauniquetranscriptvariant ifspecified DesignprimerstoavoidamplifyingtranscribedpseudogenesDesignintron spanningprimerswhenpossibleExcludeSNPsfromprimers StrategicApproach AdvancedPrimerDesignWorkflow ObtainmRNAaccessionnumbersorusegenesequence Checkgenefortranscriptvariants pseudogenesandintroninformation EntrezGene SeeSpecificDesignConsiderations DesignprimersusingPrimerBLAST Enterprimername fragmentsize genenameintomultiplexTDFfiletemplate Determinethedesiredlengthofamplicon withoutuniversaltags 1 ChoosinganAccessionNumberAsinglegenecanberepresentedintheNCBIdatabasebymultipleaccessionnumbersmRNAAlwaysusereferencesequence RefSeq NM XXXXXX fromtheDatabaseUsecautionwithothermRNAaccessionnumbers partialsequences mutations ESTsDonotuseanaccessionnumberforgenomicsequences2 Ensurethefollowingforeachaccessionnumberthatischosen CorrectgeneischosenMultiplenamesandaliasesDifferentgenescanhavesimilarnamesSpeciesofinterestIfnon RefSeqaccessionnumberisusedVerifythatthesequencecontainsonlythelettersA T G CorN Pre DesignConsiderations AmpliconLength Designampliconssuchthateachfragmentisnolessthan5nucleotidesapartfromitsnearestneighbor Thisallowsforvariationinmigrationtomeettheminimumpeakseparationdistanceof3nucleotides Designampliconsbetween105 350nt withoutuniversaltags 142 387ntwithuniversaltagsStartwithsmallfragmentsizeandworktowardlargerfragmentsizeNote ForFFPEsamples designampliconsbetween105 160nt withoutuniversaltags therecommendedtotalnumberoffragmentsinapanelistwentyorless Pre DesignConsiderations Continue GeneInformationIntronandExonInformationTranscriptVariantsInformationPseudogenesInformation Pre DesignConsiderations Continue 1 ChoosetheGenedatabase 2 Entergenenameoraccessionnumber 3 ClickSearch NCBIwebordirectlygotohttp www ncbi nlm nih gov gene Step1 GettingGeneInformation Getthealias es andasummary Example NM 01825 Viewthetranscriptsandreferencesequences ELP2containsasingletranscriptwith22exonsEachgreenbarrepresentanexon ELP2locatedattheChromosome18 Step2 Checkingforhomologyandpseudogenes Performaspecies specificquery Blasthumangenomeifitisahumangene Gotohttp blast ncbi nlm nih gov Blast cgi Entertheaccession here Select referenceonly database Click BeginSearch Click ViewReport ClickViewReporttoviewgeneinformation Onlyonehit there snohighhomologousregionorpseudogenes Clickonthelinktogotothemapviewandintron exoninformation Reportviewpage Blackhashmarksindicateexon exonboundaries NM 018255 Gotohttp blast ncbi nlm nih gov Blast cgiScrolldowntofindtheSpecializedBLAST clickPrimerBLAST Step3 DesignGene SpecificPrimers NCBIPrimer BLAST Thisprogramwillaccomplishthefollowingwhentheparametersaresetproperly Checkforspecificitybothwithinaspeciesandbetweenspecies mustaddspecies IncludeorexcludetranscriptvariantsExcludeSNPsfromtheprimerbindingsitesCreateintron spanningprimersand orexon junctionprimersAllowsfordesigntospecificregionswithinthegeneorusingapre designedprimersequencesAvoidslowcomplexityprimerbindingregionsNote Generallythemorerestrictionsthatareplacedontheprimerselectionprogram thefewerprimersbindingsiteswillbeavailableThisprogramdoesNOT CheckforrepeatsequenceswithintheampliconwhichmayleadtostutterCheckforpseudogenes DesignGene SpecificPrimerswithNCBIPrimer BLAST Primer BLASTcontinued Results PrimerDesigncontinue AddtheuniversetaqsequencetoeachofthegenespecificprimersequenceasthefinalprimersequenceExample ForwardSequencewiththeUniversalTaqSequenceAGGTGACACTATAGAATATGATCGGGTCTTCCTTCATCReverseSequencewithUniversalTaqSequenceGTACGACTCACTATAGGGAGCCATTAAGGCCCTTCTTTCThedesignedfragmentsizeisthegenespecificsizeplus37bpoftheuniversaltaq eg thedesignedsizeis105bp theexpectedproductsizeis142bp OrderprimerswithUniversalTags exceptKanR inbuffers 1 Keeptheallheadercolumnsinformationunchanged 2 Changeanewmultiplexname cellA2 whenthemultiplexinformationwasmodified 3 Primername GeXPproductsize GeneNameandAccessionnumberinformationareessential 4 EntersameinformationforbothGeneNameandAccessionnumbercolumns 5 UsetheactualCEQfragmentsizeintheproductsizewithuniversaltaqcolumn6Row3hastobeempty7 Keepatthelastlineofprimername CreateaMultiplexfromaTDFfiletemplate WhatistheTDFFile TheTDFfilecontainsthemultiplexinformationandthatlinkstheX ProfilerAnalysiswiththeimportedGeXPcsvdata AmultiplexcanbeuploadedintoeXpressProfilerfromadministratoraccountwithavalid TDFfile UniqueMultiplexName cellA2inExcelspreadsheet Row3hastobeemptyKeepallthecolumnheaderunchanged Mandatoryfieldstobefilled PrimerName GeneName AccessionNumber ProductSizew Universals useGeXPfinalproductsizeeasierforbinning EntersameinformationintheGeneNameandAccessionNumber Canbegenenameoraccessionnumber Primernamedonotinclude otherwisethequotationmarkswillcreatedwhenyoumodifiedtheTDF theTDFwillfailtobeimported SpecificPrimerDesignConsiderations TargetedspecifictranscriptionvariantsExcludedpseudogenes Example TargetedPrimerDesignforBIRC5 http www ncbi nlm nih gov gene Targetthe5 exon1 2tocapturethreevariants GotoNucleotidehomehttp www ncbi nlm nih gov nuccore enteraccession BRC5genehasthreetranscriptvariants ClickGraphics GotoNCBINucelotidehomehttp www ncbi nlm nih gov nuccore enteraccession Graphictoviewexoninformation Example NM 001012271 fiveexons Rightclicktheexonbartoviewexonpositions PrimerDesigned Specifictheforwardprimerandreversepositions PrimerDesigned Selected Allowtoamplifysplicevariants Results Primerstargetedthreetranscriptvariants DesignprimerstoregionsuniquetotherealmRNA DesignPrimersexcludingthePseudogenes Whydesignprimersspanninganintron ToavoidgenomicDNAinterferencefromSamplesresistanttoDNaseSamplestoosmalltobetreatedwithDNaseSinglecellanalysisTominimizeoreliminateRTminuscontrolSavingforreagentsandtime Note ThisstepisoptionalorsometimesimpossiblestepforcertainmultiplexpanelOptionalwhenDNasetreatmentwillbeperformedroutinelyImpossiblewhengene variantspecificityisonlyinthe3 UTR nointrons PlexBRTminuscontrol EachprimerpairwasdesignedondifferentexonstoavoidinterferencefromgenomicDNAcontamination Nofalsepositive OtherConsiderations DesigningprimersforgenefamilymembersDesigningprimersforsplicevariantsChoosingqualityreference housekeeping genesXP PCRPrimerSpecifications Designgene specificprimersforgenefamilymembers Whengenefamilymembersareinvolved proprietarysequencesshouldbecreatedusingnon homologoussequences Ifablockofnon homologoussequenceisnotavailable individualprimersetsshouldbedesignedpriortothemultiplexdesign Theprimershouldhaveits3 endlandinnon homologousregion Designgene specificprimersforgenefamilymembers whenusingproprietarysequenceisnotanoption PerformalignmentPickareverseprimerthathasits3 endlandonnon homologregionPastetheprimersequenceintorightprimerwindowPickaforwardprimerthathasits3 endlandonnon homologousregionPastetheprimersequenceintoleftprimerwindowClickExecuteThistaskcanalsobeperformedviaPrimer BLASTathttp www ncbi nlm nih gov tools primer blast index cgi Example designgene specificprimerregionsforCYP6Z1 AlignmentofCYP6Zfamily PrimerDesignforSpliceVariants mRNA Commonreverseprimerandspecificforwardprimersamplifymultipletranscriptvariants Specificreverseandforwardprimersamplifyasingletranscriptvariant 4 ChoosingQualityReferenceGenes Equivalentexpressionofeachreferencegeneoverallsamples tissues treatments timepoints examinedGeXPHumanReferencePlexNopsuedogenespresentortargetauniqueregioninthereferencegeneUsemultiple 3 referencegenesfornormalizationAddmorethanandthenchoosewhichonestouseforstudy Housekeeping Reference GenesPrimersavailable TBP NM 003194 170bp 212bpPSCM4 NM 153001 143bp 271bpHRT1 NM 000194 234bpCCNG1 NM 004060 278bpGUB2 NM 000181 198bpRPLP0 NM 053275 115bp 127bpGAPDH NM 002046 248bpMousemTBP NM 013684 151bpmGUB2 NM 010368 238bpmCCNG1 NM 009831 281bpmGAPDH NM 008084 349bp Human GAPDHhasmanypseudogenes Itisimpossibletodesignprimersaroundallthepseudogenes XP PCRPrimerSpecificationsFeatureminoptimalmaxunitTm 576063oCLength172023ntAmpliconSize105 350nt Thedefaultvaluesfor Tableofthermodynamicparameters and Saltcorrectionformula underadvancedparameters havebeenchangedinNCBIBlasttovaluesrecommendedinprimer3program Tableofthermodynamicparameters ischangedfrom Breslaueretal 1986 to SantaLucia1998 and Saltcorrectionformula ischangedfrom SchildkrautandLifson1965 to SantaLucia1998 Asaresult thedefaultTmvaluesforyourprimerswillbedifferentfrompreviouscalculation Ifyouwouldlikeadifferentvalueforfutureuse youcansaveyourcustomparametersusing Savesearchparameters atthetopofthepagewithTm Breslaueretal 1986 andSaltcorrection SchildkrautandLifson1965 SNPsNomorethan1or2SNPsin5 end noSNPinthe6ntclosestto3 endRepeatsNomorethan6mono ordi nucleotiderepeats orseveralshortedrepeatsbrokenbysinglealternativenucleotide intheamplicon visualcheckorrepeatmaskinampliconBLAST SpecificityPrimersarespecifictointendedtarget considerhomologousgenes transcriptvariantsandpseudogenesSpacingMinimumof5ntdesignedbetweeneachpeakastheymayendupcloserduetoCE Nolessthan3ntACTUALspacebetweenpeaks Intron spanningDesignprimersontwoseparateexonsthatspanalargeintron Thisisoptional butadvisedforsampleswherecompleteDNasedigestionisnotlikely 1 EnterFASTAsequence 2 Enterthedesiredsizerange 3 Click PickPrimers BLASTQCwithNCBIhttp blast ncbi nlm nih gov Blast cgi PasteFASTA ClickBlast CheckingSNP BLASTQCwithNCBIhttp blast ncbi nlm nih gov Blast cgi