oligo 7 使用介绍.pptx
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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,and
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