Deteco deMutaes Genotipagem

Curvasdedesnaturao

UsandoduassondasTaqMan cadaumacomfluorforo diferente

Emparelha fluoresce aps hidrlise

Noemparelhanofluoresce

 Sequencespecific hybridizationprobescan bedesignedthatallowdetectionandanalysis ofPCRproductswithouttheneedforany postPCRsamplemanipulation,allowinghigh throughputgenotyping andproduct quantification.

 Fluorescencemonitoringofamplification usinghybridizationprobesisbasedonthe conceptthatafluorescencesignalis generatediffluorescenceresonanceenergy transfer(FRET)occursbetweentwoadjacent fluorophores,illustratedinFigure1(3).

TransfernciadeEnergiadeRessonnciaporFluorescncia(Fluorescence Resonance Energy Transfer FRET),

Fluorescence from theacceptor probewillonlyoccurwhenboththedonor probeandtheacceptorprobehaveannealedtotheproduct. Thisprocessoftransferringtheenergyfromonefluorescentdye,toasecond fluorescentdyeiscalledFluorescence Resonance Energy Transfer (FRET)

Only one reaction andonesetofprobesarerequiredforgenotyping Amelting curveof hybridizationprobefluorescense caneasilydiscriminateeventhe moststablesinglebasemismatches

Melting curveanalysissimplifies theoptimizationofprobehybridizationwithcontinuous monitoringofprobehybridizationstatusasthetemperature changes. Asinglebasemismatch undertheprobedecreasesthemeltingtemperaturebyaslittleas 3CforG::Tmismatches,toasgreatas710CforA::Cmismatches.

Typically theprobe shouldbedesignedtoproducethegreatesttemperaturechange betweenthewildtypeandmutant melting curves. Figure3demonstrates atypical derivate melting curveforsinglebasegenotyping

Mutant allelesaredistinguishedfromwildtypeby themeltingtemperature(Tm)oftheprobe. Hybridization probe pairsaredesignedthatrecognizeadjacent internalsequenceswithintargetDNA sequences.Bothprobesarefluorescently labeledsuchthat,whenannealedtothe template,thefluorophores areseparated byonebase,allowingastrongFRETsignal.

Fluorescence ismonitoredonceeachcycleatthe endoftheannealingstepandincreases abovebackgroundatacyclenumberthat isdependentontheinitialtemplate concentration

AtwoprobesystemisalsousedforrapidgenotypingofthefactorVLeidenmutation. Asinglebasechange(G1691A)causedbytheLeidenmutationresultsinadecreasein probeTmof7oC. AmorestablesubstitutionintheMTHFRgene(C677T)givesa3oCdifferenceinTm betweenwildtypeandmutantalleles.Bothchangescaneasilybedistinguishedwith the LightCycler,suggesting thatthisfluorescencemethodissuitableforallsinglebase mismatches.

FactorVgenotyping Atypical factorVgenotyping experiment usingtwohybridizationprobesis illustrated. Alonganchorprobe(a36mer)islabeledat the5'endwithLightCycler Red640anda secondshorterprobe(a23mer)islabeled withfluoresceinatthe3'end. Theprobes recognizeadjacentsequenceswiththe shorterprobelyingoverthemutationsite. Thegreaterstabilityoftheanchorprobe meansthatlossoffluorescenceoccursas theshorterprobemeltsoffthetemplate.A singlebasemismatchundertheprobe resultsinaTmshiftof7oC.Meltingcurves areconvertedtomeltingpeaks(Figure5) allowingeasydistinctionofwildtypefrom mutant.

Theprobedesignoftheseexperiments placesbothprobesonthesamestrand,as farfromtheextendingprimeraspossible. Thisdelaysdisplacementoftheprobeby thepolymeraseandallowsmaximum hybridization time.

TheLeidenmutationrepresentsaGto Asubstitutionwithinexon10ofthefactor VgenecreatinganA:Cmismatchbetween thewildtypeprobeandthemutanttemplate. ThismismatchresultsinalargeTm shiftduetoitspoorstabilityandallowsthe mutanttobeeasilydistinguishedfromthe wild type.

EXERCICIO:

If amutantprobewithawildtypetemplateis selected,theprobewillmeltoffthewild typealleleata*_________ Tmthanthe mutantalleleforwhichitisaperfectmatch.

*lower/higher?

blaSHV

Deteco deMutaes nogene

Meltingcurves(C;fluorescenceF3versusT)andmelting peaks (D;plottedasthenegativederivativeoffluorescence F3versusT)ofblaSHV genesinstandardstrains. FluorescenceF3(CandD),generatedbythefluorophore of themutationatcodons238and240 andrecordedbychannel3,revealedmeltingpeaksat threedifferentTs: below50CforstrainsharboringblaSHV1andblaSHV8 (twomismatches), ca.59CforstrainsharboringblaSHV2(onemismatch), and ca.66CforthestrainexpressingblaSHV5(no mismatches).

Comovariaatemperaturadedesnaturao paracadamutanterelativamenteaonmeros denucleotdeos noemparelhados? Asondafoidesenhadaparaquemutante?