oligo 7 使用介绍.pptx

oligo 7 使用介绍.pptx

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Oligo7PrimerAnalysisSoftware,RulesforPCRPrimerSelectionandPresentationoftheSoftware,2010MolecularBiologyInsights,Inc.,Contents,1.PCRPrimerSelectionCriteriaexplanationofprimerstabilitycalculationswhatmakesagoodPCRprimerotherfactorsbesidesindividualprimercharacteristicsinfluencingPCRreaction2.Oligo7AnalyzeFeaturestheSequencewindowgeneralinformation（theKeyInfo）duplexandhairpinformationdataaboutprimers（CompositionandMeltingTemperatures）falseprimingsitesandhomologyanalysisoligonucleotidestabilitygraph（Tm,DG）internalstabilityanditsimportancesequencefrequencyotherDNAanalysisoptions:

ORF,restrictionsites,DNAcalculator3.SearchOptionssearchforprimersandprobesothersearchesbatchprocessing4.OligonucleotideDatabasemultiplexing5.ConcludingRemarks,PrimerStabilityCalculationsAnintroductiontothenearestneighbormethod,Oneofthemostimportantcharacteristicsofaprimerisitsthemeltingtemperatureandstabilityofvariousitsregions.ThemostaccurateTmcalculationprovidesthenearestneighbormethod.TheprimerTmformulaisquitecomplex:

Tm（C）=DH/（DS+R*lnC）+16.6logK+/（1+0.7*K+）273.15whereDHandDSaretheenthalpyandentropyforhelixformation,respectively,Risthemolargasconstant（1.987cal/C*mol）,andCisconcentrationoftheprimer/probe（thatisalwaysusedinseveral-foldexcessthanthetargetDNA）,butfortunatelycomputerdoesallthework.Tounderstandcomplexityofduplexstabilitycalculationsitiseasiertoexplainitbyshowinghowfreeenergy（DG,anothermeasureofDNAhelixstability）,astheformulaissimpler:

DG=DHTDS（TisthetemperatureinK）.Althoughislookssimpleenough,itisnotsosimpleifyouactuallydoit.HerestheexampleofDGcalculationofa4-merTCGA,hybridizingtoalongerstrandlikethis:

Now,theDGcanbecalculatedlikethissurprisinglylongformula:

DG（TC）+DG（CG）+DG（GA）+initiationDGforT+initiationDGforA+DGof3-danglingendAC+DGof5-danglingendGTThenextslideshowsthecalculationgraphically（datafortemperatureof37）.,DGcalculationexample,TotalDG=1+1+0.3+0.5-1.3-1.3-2.2=-2.0kcal/molThisisararecase,asusuallythedanglingendsarestabilizing.Thisisshowntoalertyouthatsometimesaddingabasemayactuallydecreasemeltingtemperatureofaduplex.Thenextslideshowsanexamplewhereaddingeven2baseshasnoeffectontheTm.,Hereisascreenshotshowingtheselectedprimersandprobes:

TheForwardPrimerTmis56.7,whiletheReversePrimerTmis56.2（notmuchdifferent）.Byaddingonebaseoneachend,andselectingthefragmentsasUpperandLowerOligos,wecanseethattheUpperOligoTmis56.7（nochangeinTm）whiletheLowerOligosTmroseupto61（from56.2）.Withoutconsiderationofthedanglingends,increasingprimerlengthalwaysincreasesitsTm.MorecomprehensiveanalysisdataforthoseparticularoligosyouwillseeontheKeyInfowindowexplanation.,Whatmakesagoodprimer,Highprimingefficiencytoachievethis,agoodprimershouldbeofareasonablyhighTm,withoutdimers,especiallyontheir3-ends（topreventself-extension）,withouthairpinstems,especiallyontheir3-ends（topreventself-priming）,lackingrepetitivesequencestoensurequickandcorrectannealing.allprimers/probesinoneincubationmixtureshouldnotformsignificant3-dimersbetweeneachother.Highspecificitytoachievethis,agoodprimershouldbelongenoughtoincreasespecificity,unique,especiallyatits3-end,toavoidfalsepriming,moderatelystableatits3-end（asopposedtohighlyGC-rich）toensurethataveryshortfragmentwontinitializetheextension（toolow3-endstabilityhurtstheprimingefficiency）.,OtherfactorsbesidesindividualprimercharacteristicsinfluencingPCRreaction,Besidestheprimerpropertieslistedonthepreviouspage,PCRefficiencyalsodependsonthetemplate.RegionswithhighsecondarystructuresandGCislandsamplifypoorly.Oligo7penalizesprimersthatwouldamplifystronghairpinstructures,decreasingoverallscoresforPCRprimersets.Ifyouhavetomakehardtoamplifyproducts,selectprimerswithhighscores（usuallywithhigherTmandGCcontentsthanstandard）,andadjustcompositionofyourPCRmixbyaddingsolvents,additivesorreducing/changingsalts.Increasingprimerconcentrationabove1mmolisnotrecommendedduetounspecificprimer/primerextensions（evenifthesoftwaredoesnotshowany3-dimers）.,Oligo7AnalyzeFeatures:

theSequenceWindow,TheSequenceWindowisthecentralwindowthatshowsseveralfeaturesofthesequenceandselectedprimers.Whenyouopenthetestsequence,providedwiththesoftware,youwillseethisscreen:

Thesequencefilenameisinthetopleft.Inthetopyouseealso3tables.Thefirstontheleftshowsthesequencelength,thereadingframeforthetranslation（proteinsequenceisacolorfulstringofletters,atthebottom）,theCurrentOligo（sequencesurroundedbyagreyrectangle）lengthandposition,anditsTm.Thesecondtablelistspositionsofselectedprimers,oligos（toselect,clickthesquarelocatedontheleftoftheoligoname）,andthePCRproduct（forthis,youneedtoselect2Primers）.Clickthe“i”togetmoreinfoabouttheselectedOligo.Thethirdtablelistssequencefeatures（ifsequenceannotations/featuresarepresent;youmayenterthemmanuallyfromtheEditMenu）.,Oligo7AnalyzeFeatures:

theSequenceWindow,Belowthetablesyouseetheoligonucleotidestabilitygraph（defaultisTm）.ItisaminiaturizedMeltingTemperatureGraphWindow,availablefromtheAnalyzeMenu.BelowtheTmgraphthereareaseriesofhorizontallinesorrows.Eachrowmaycontainagraphicalrepresentationofvariousfeaturesspreadoutintheentiresequence,suchasprimerspositions,PCRproductandothers.Inthisexample,thefirstfilledrowisapatternofredrectanglessymbolizingweakhairpinloopsinyettobeselectedprimers.Thegreenrectanglesbelowtheredrowrepresentpalindromes.Thelastfilledrowshowsthecodingsequenceasablueline,astheFeatureTablehighlights“CDS”.,Thebottompartofthewindowshowstheenlargedpartofthesequence,thatishighlightedinyellowintheTmgraph（topleftofthispicture）.ThecursorpositionandtheTmofanoligothatwouldstartatthecursorpositionisindicatedontheright,justabovethepositionnumbersscale.BelowthescaleyouseetheDNAsequence,wherethepositivestrandisinred.Ifyouselectprimerstheywouldappearaboveandbelowthesequence.Translatedproteinsequenceisdisplayedinvariouscolors.Thecolorssymbolizecodonfrequency（blackisthemostfrequent,seetheOpenReadingFrameswindow）.Therowsbelowandtheircolorssymbolizeidenticalfeatures,butenlarged,tothosedisplayedjustbelowtheTmgraph.Moredetailedexplanationisonthenextslide.,Thegraphincoloredrectangleisexpandedinthezoomarea,WithOligoyoucanselectupto4primersorprobes,ExplanationofothersymbolsintheSequenceWindow,TheKeyInfoWindows,TheKeyInfowindowsdisplaybasicinformationaboutselectedprimersorprobes.Thesearetheoligosdiscussedintheprevioussection,wherealonger13-meroligo（lowerleft）hasthesameTmasitsshorter11-merversion,displayedaboveit.Thetmisameltingtemperaturecalculatedwithoutthedanglingendseffect,andthisoneincreasedbyalmost7.Notethatthecomplementaryoligosofthesamelength,shownontheright,havethesametmasexpected,butdifferentTm,especiallythe13-mers.,MorestatisticsonthoseoligosprovidetheComposition&Tmwindows,aswellastheConcentrationswindow.,TheDuplexFormationWindows,TheDuplexFormationwindowsdisplaythestrongest3-duplexes,themoststableduplexthatisnotnecessarilyatthe3-end,andthestrongesthairpin.TheDGdataaregivenwithconsiderationofthedanglingendsandinitiationDGvalues.TheMixedOligosDuplexeswindowdoesnotdisplayhairpins.,TheHairpinFormationWindows,TheHairpinFormationwindowsdisplayallpossiblehairpinsinagivenoligo,listedfromthestrongestonetotheweakest.Whenagivensegmentisinvolvedinmanydifferentstructures,therealmeltingtemperatureofaparticularhairpinislowerthancalculatedduetocompetitionbetweenthestructures.,TheComposition&TmWindows,TheComposition&Tmwindowsdisplaymeltingtemperaturescalculatedwithdifferentmethodswidelyavailable,thatisthenearestneighbormethod（TdisanormalizedTmin1Msaltconditions,whiletheTmisavariablevalue,thatdependsonsaltandDNAconcentrationsetintheSearchParameterswindow）.TheTmCalculatedwiththe%GCmethoddonotdependontheSearchParameterssettings.Also,thesimplestmethodofTmcalculation（2forAorTand4forCorG）isgiven,followedbytheTmvaluesofRNAandDNA/RNAhybrids.Thesecondtablelistsbasecomposition,andthelasttheTmvaluesinvarioussaltsandformamideconcentrationscalculatedwiththenearestneighbormethod.,TheFalsePrimingSitesWindows,TheFalsePrimingSiteswindowsdisplaypotentialfalseprimingsitesofprimers.Thisparticularexampleshowsanoldcommerciallyavailableuniversalm13reverseprimer.ThesitewithP.E.of244pointsistheonlyrealfalseprimingsiteasconfirmedexperimentally（Steffens,D.L.,Sutter,S.L.,andRoemer,S.C.（1993）“AnAlternateUniversalForwardPrimerforImprovedAutomatedDNASequencingofM13”,BioTechniques15,580-582.）,TheHomologyWindows,TheHomologywindowsdisplayhomologicalsitesforprobes,andliststhesitesfromthehighestsimilaritytothelowest.The3-endisirrelevantinthiscase,andhomologyofaprobe（oraprimer）isalignedwiththeactualDNAstrand,andnotwithitscomplement,asintheFalsePrimingSiteswindow.WhenusingshorterDNAfragmentsinhybridizationexperiments,ahomologybelow90%isusuallynotsignificant,andwontproducefalsesignals.,TheSelectedOligonucleotidesWindow,AfterasearchforPrimers&Probesyoumayopent