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Deteco deMutaes

Genotipagem

Curvasdedesnaturao

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UsandoduassondasTaqMan

cadaumacom fluorforo

diferente

NoemparelhanofluoresceEmparelha

fluoresce aps

hidrlise

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Sequencespecific hybridizationprobescan

bedesigned

that

allow

detection

and

analysis

ofPCRproductswithouttheneedforany

postPCRsamplemanipulation,allowinghigh

throughputgenotypingand

product

quantification.

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TransfernciadeEnergiadeRessonnciaporFluorescncia(Fluorescence

Resonance Energy TransferFRET),

Fluorescence from theacceptor probewillonlyoccurwhenboththedonor

probeandtheacceptorprobehaveannealedtotheproduct.

Thisprocessoftransferringtheenergyfromonefluorescentdye,toasecond

fluorescentdye

is

called

Fluorescence Resonance Energy Transfer (FRET)

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Only one reaction andonesetofprobesarerequiredforgenotyping

Amelting curveof hybridizationprobefluorescense caneasilydiscriminateeventhe

moststablesinglebasemismatches

Melting curveanalysissimplifies theoptimizationofprobehybridizationwithcontinuous

monitoringofprobehybridizationstatusasthetemperature changes.

Asinglebasemismatch undertheprobedecreasesthemeltingtemperaturebyaslittleas

3Cfor

G::T

mismatches,

to

as

great

as

710C

for

A::C

mismatches.

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Typically theprobe shouldbedesignedtoproducethegreatesttemperaturechange

betweenthewildtypeandmutantmelting curves.

Figure3demonstrates a

typicalderivate melting curve

for

single

base

genotyping

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Mutant

allelesare

distinguished

from

wild

type

by

themeltingtemperature(Tm)oftheprobe.

Hybridization probe

pairsare

designed

that

recognize

adjacent

internalsequenceswithintargetDNA

sequences.Bothprobesarefluorescently

labeledsuchthat,whenannealedtothe

template,thefluorophores areseparated

byone

base,

allowing

astrong

FRET

signal.

Fluorescence

ismonitoredonceeachcycleatthe

endof

the

annealing

step

and

increases

abovebackgroundatacyclenumberthat

isdependentontheinitialtemplate

concentration

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AtwoprobesystemisalsousedforrapidgenotypingofthefactorVLeidenmutation.

Asingle

base

change

(G1691A)

caused

by

the

Leiden

mutation

results

in

adecrease

in

probeTmof7oC.

AmorestablesubstitutionintheMTHFRgene(C677T)givesa3oCdifferenceinTm

betweenwildtypeandmutantalleles.Bothchangescaneasilybedistinguishedwith the

LightCycler,suggesting thatthisfluorescencemethodissuitableforallsinglebase

mismatches.

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FactorVgenotypingAtypical factorVgenotyping experiment

usingtwohybridizationprobesis

illustrated.

Alonganchorprobe(a36mer)islabeledat

the5'endwithLightCycler Red640anda

second

shorter

probe

(a

23

mer)

is

labeledwithfluoresceinatthe3'end.

Theprobes

recognizeadjacentsequenceswiththe

shorterprobe

lying

over

the

mutation

site.

Thegreaterstabilityoftheanchorprobe

meansthatlossoffluorescenceoccursas

theshorterprobemeltsoffthetemplate.A

singlebasemismatchundertheprobe

resultsin

aTm

shift

of

7oC.

Melting

curves

areconvertedtomeltingpeaks(Figure5)

allowingeasydistinctionofwildtypefrom

mutant.

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Theprobedesignoftheseexperiments

placesbothprobesonthesamestrand,as

farfromtheextendingprimeraspossible.

Thisdelaysdisplacementoftheprobeby

thepolymeraseandallowsmaximum

hybridization time.

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TheLeiden

mutation

represents

aG

to

Asubstitutionwithinexon10ofthefactor

VgenecreatinganA:Cmismatchbetween

thewildtypeprobeandthemutanttemplate.

ThismismatchresultsinalargeTm

shiftduetoitspoorstabilityandallowsthe

mutanttobeeasilydistinguishedfromthe

wild type.

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EXERCICIO:

If amutantprobewithawildtypetemplateis

selected,theprobewillmeltoffthewild

typealleleata*_________ Tmthanthe

mutantalleleforwhichitisaperfectmatch.

*lower/higher?

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Deteco deMutaes nogeneblaSHV

Meltingcurves

(C;

fluorescence

F3

versus

T)

and

melting

peaks(D;plottedasthenegativederivativeoffluorescenceF3versusT)ofblaSHV genesinstandardstrains.

FluorescenceF3(CandD),generatedbythefluorophore ofthemutationatcodons238and240

andrecordedbychannel3,revealedmeltingpeaksat

three

different

Ts:

below50CforstrainsharboringblaSHV1andblaSHV8(twomismatches),

ca.59CforstrainsharboringblaSHV2(onemismatch),and

ca.66CforthestrainexpressingblaSHV5(nomismatches).

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Comovariaatemperaturadedesnaturao

paracada

mutante

relativamente

ao

nmeros

denucleotdeos noemparelhados?

Asondafoidesenhadaparaquemutante?