甲基化软件

MethylPrimerExpress®Softwarev1.0QuickReferenceCardOverviewMethylPrimerExpress®Softwareallowsyoutodesignprimersforbisulfitesequencing(BSP)and/ormethylation-specificPCR(MSP)—thetwomostcommonlyusedtechniquesformethylationmapping.ThesoftwaresearchesDNAsequencesforCpGislands,simulatesbisulfitemodificationontheCpG-containingsequences,thenrecommendsprimerpairsforMSPorBSP.Primerdesignforeithermethodcanbeadjustedtoaccomodatevariationinexperimentaldesign.Theessentialstepsindesigningsequence-specificDNAmethylationexperimentsaresubstantiallyautomatedwithMethylPrimerExpress.AfteryouinserttheselectedDNAsequenceintothesoftware,thesoftwarefindsallrelevantCpGislands,thenprovidesprimerdesignsbasedonselectedtargetsequences.YoucanreviewallCpGislandsfoundinyoursequence,andallsuggestedprimerpairsbasedonyourtargetsequence,inreportviews.MethylPrimerExpress®SoftwareWorkflowByusingMethylPrimerExpresssoftwaretodesignprimersyouwilllearnto:ImportagDNAsequenceAnnotatethesequenceSearchforCpGislandsSelectaCpGislandofinterestSelectatargetsequenceMethylPrimerExpress®Softwarev1.0Page2StartingtheMethylPrimerExpress®SoftwareTostartMethylPrimerExpress®Softwarev1.0,clickonthedesktop.UsinganExampleSequenceThisQuickReferenceCardusesanexamplehumanDNAsequence(BRCA1gene)toshowyouhowtouseMethylPrimerExpresssoftware.ImportingaSequenceToenterthesequenceintoMethylPrimerExpresssoftware:1.SelectFileImportSequence.2.OpentheGenBankdatabase,thencopytheBRCA1genesequence(U37574).3.PastethesequenceintotheInsertNucleotideSequencebox.IMPORTANT!IfyouimportasequencethathasoneormoreNcalls,thesoftwaredoesnotallowyoutoproceedtothenextstepandanerrormessageisdisplayed.TominimizethechancethatagDNAsequencehasNbasestoconvert,acquirethemostrecentGenbanksequenceyoucanfind.Note:Intheexamplesequence,Nbaseswerepreviouslyconverted.AnnotatingtheSequenceYoucanannotatetheimportedsequencewithoneormoreidentifyingelementssuchas:Auser-specifiedsequencenameThetranscriptionstartpointofthegenesequenceThetranslationstartcodonofthegenesequenceInthisexampleworkflow,youannotatethesequenceonlywiththetranscriptionstartpoint.YoucandeterminethetranscriptionstartpointbyreferringtothemRNAlineintheGenBankrecordofyourimportedgenomicsequence.ThetranscriptionstartpointbeginsatthebeginningofthegenemessengerRNA.Page3AnnotatingtheTranscriptionStartPoint1.IntheFeaturessectionoftheGenBankfilefortheBRCA1gene(fileU37574),findthemRNAstartsequencenumber.2.ClickthepointeranywhereinyourimportedgenomicsequenceandviewitssequencenumberpositionintheBasePositionfieldatthebottomrightcornerofthescreen.3.MovethepointerasyouscrolldownthesequencetomatchtheBasePositionnumbertothemRNAstartnumberintheGenBankfile.(1581intheexample)4.Selectthestartpointbaseplus12ormorebasesafterthestartpoint.Page45.SelectSequenceFeaturesSetTranscriptionBases.Thetranscriptionbasesarehighlightedandunderlinedinblue.Scrolltoseetheannotatedbases.Withthesequenceimportedandannotated,youarereadytohavethesoftwarefindtheCpGislands.FindingCpGIslandsAfterannotatingtheimportedsequence,youcansearchfortargetCpGislandstobegintheprocessofdesigningeffectiveprimers.1.SelectDesignPrimersFindCpGIslands.Page5TheCpGIslandsdialogboxpromptsyoutoreviewthecriteriacurrentlysetforidentifyingCpGislands.2.Ifyouselect:No–ThesoftwareusesdefaultsettingstosearchforCpGislandsandastatuswindowshowstheprogress.Yes–IntheCustomizeSettingsdialogboxthatopens,adjusttheparametervaluessetbyMethylPrimerExpress,thenclickOK.(TheFrommeralgorithmisusedtopredictCpGislands).IMPORTANT!Althoughthedefaultsettingsaretherecommendedparametervaluestorunthesoftware,youcanmodifysettingsaccordingtotheobjectivesofyourexperiment.Formoredetails,seetheMethylPrimerExpress®Softwarev1.0GettingStartedGuide.3.Whenthesearchends,reviewtheCpGReportasdescribednext.ReviewingtheCpGReportIntheCpGReport,CpGsitesareindicatedbypinkverticalbarsalongthehorizontalaxis.Thetranscriptionstartpointisindicatedbyabluebar;thetranslationstartcodonisindicatedbyaredbar(ifset–seetheMethylPrimerExpress®Softwarev1.0GettingStartedGuideforalloptionalstepinstructions);CpGislandsareindicatedbysolidpinkbarsbelowthehorizontalaxis.Scrollthroughthereporttoreviewthesearchresults.Page6SelectingaTargetSequenceAfterreviewingtheCpGislandsfoundinyoursequence,youcanselectaportionofyoursequencetouseasthetargetsequenceforprimerdesign.1.IntheCpGReport,selecttheislandofinterestfromthe“Selectanisland”drop-downlistatthebottomrightofthescreen,thenclickNext.2.SelecttheregionoftheCpGislandthatyouwanttodesignprimersfrom,oracceptthedefaultarea(highlighted),thenclickNext.Note:Ifyouannotatedthetransciptionstartbase,thesoftwareautomaticallyselectsthetargetsequencewithin500basesofthetransciptionalstartpoint.Ifyoudidnotannotatethetranscriptionbase,theCpGislandthatyouseehighlightedisautomaticallysetasthetargetsequence.Tooverwritetheselection,selectanothertargetsequence,thenclickNext.Page7ReviewingtheDNASequenceReportReviewtheDNASequenceReportfortherecommendedtargetsequence.TheDNASequenceReportdisplaystheRecommended,Selected,MethylatedandNon-methylatedsequences