GenéticaProf.Doutor José Cabeda

Genética Molecular em Análises Clinicas

TÉCNICAS DE AMPLIFICAÇÃO TÉCNICAS DE AMPLIFICAÇÃO DE ÁCIDOS NUCLEICOSDE ÁCIDOS NUCLEICOS

Técnicas de amplificação de ácidos nucleicos

PCRPCR Real Time PCRReal Time PCR NASBA/TMANASBA/TMA

PCR

OPTIMIZAÇÃO DO PCR

[MgCl[MgCl22]] ThTh [dNTP][dNTP] [primers][primers] [DNA][DNA] [inibidores][inibidores]

Variações

RT-PCRRT-PCR Multiplex PCRMultiplex PCR Nested PCRNested PCR

Técnicas de amplificação de ácidos nucleicos

PCRPCR Real Time PCRReal Time PCR NASBA/TMANASBA/TMA

Genética2001/2002 Prof.Doutor José Cabeda

Principio de funcionamento do Real-Time PCR

http://biochem.roche.com/lightcycler/_vti_bin/shtml.dll/hardds01.htm/map

Detecção dos produtos de PCR

Montar uma reacção

Genética2001/2002 Prof.Doutor José Cabeda

Químicas Utilizáveis

SYBR-Green I

5’3’

5’ 3’

SGExcitation

SG

SG

SG

SG

SYBR-Green I

5’3’5’ 3’

SG

SG

SG

SG

SG

Excitation Emission

Detcção com SybGreen

Sybr-Green Detection

Intercalates to dsDNAIntercalates to dsDNA Inhibits DNA amplification Inhibits DNA amplification

so concentration is criticalso concentration is critical Detects specific and non-Detects specific and non-

specific targetsspecific targets Low starting background Low starting background

with large increase in with large increase in signalsignal

Can run a melt curve to Can run a melt curve to look at product specificitylook at product specificity

Detecção com sondas

Quimicas utilizáveis:sondas taqman

R Q

Sequence specific probes: Dual labeled probes

5’3’5’ 3’

5’3’

5’ 3’

Excitation RQQR QRExcitation

Quimicas utilizáveis:molecular beacons

Molecular Beacons I

10mer

25mer

FAM DabCyl

Molecular Beacons II

R QExcitation

RQ

QR Emission

Molecular Beacons II

R QExcitation

RQ

QR Emission

Can be used to quantitation and Can be used to quantitation and mutation detectionmutation detection

Need beacons for the normal and Need beacons for the normal and mutation sequencemutation sequence

Design is difficult due to nature of Design is difficult due to nature of folding structurefolding structure

Expensive when compared to FRETExpensive when compared to FRET

Molecular Beacons III

Quimicas utilizáveis:sondas FRET

Genética2001/2002 Prof.Doutor José Cabeda

Hyb-ProbesTM

Oligo 1: Fluorescein Oligo 2: Quencher

Excitation Emission

Transfer

Quenched FRET analysis

Two labeled probes, FAM at the 5’ and Two labeled probes, FAM at the 5’ and BH1 on the 3’BH1 on the 3’

When the probes hybridize the FAM When the probes hybridize the FAM energy is quenched by the BH1energy is quenched by the BH1

After the product is amplified run a melt After the product is amplified run a melt curve to determine genotypecurve to determine genotype

Different melt points are seen for normal Different melt points are seen for normal and mutationand mutation

5’ 3’

Quimicas utilizáveis:sondas Eclipse

F Q

F F

MGB

Q

F

MGB

Q Q

F

Genética2001/2002 Prof.Doutor José Cabeda

Aplicações

QuantificaçãoQuantificação

End Point versus Real-Time

Quantificação

Genética2001/2002 Prof.Doutor José Cabeda

Aplicações

Detecção de Mutações / SNPDetecção de Mutações / SNP

Detecção de mutações

Genética2001/2002 Prof.Doutor José Cabeda

Aplicações

Multiplex DetectionMultiplex Detection

Detecção simultânea de vários produtos

Genética2001/2002 Prof.Doutor José Cabeda

Os equipamentos

Light Cycler

Smartcycler

Rotorgene The samples spin continually during The samples spin continually during

a run at 500 rpma run at 500 rpm The samples are heated and cooled The samples are heated and cooled

in a low mass air ovenin a low mass air oven Tubes are illuminated as they pass Tubes are illuminated as they pass

the detectorthe detector On data acquisition energy is On data acquisition energy is

averaged over 20 revolutions to give averaged over 20 revolutions to give the fluorescence of each samplethe fluorescence of each sample

Four ChannelsFour Channels• Ch1: ex 470nm, det 510nm• Ch2: ex 530nm, det 550nm• Ch3: ex 585nm, det 610nm• Ch4: ex 625nm, det 660nm

O Futuro próximo

Detecção com sondas

Detcção com SybGreen

Quantificação

Detecção de mutações

Detecção simultânea de dois produtos

Técnicas de amplificação de ácidos nucleicos

PCRPCR Real Time PCRReal Time PCR NASBA/TMANASBA/TMA

NASBA/NUCLISENS

Amplification: schematic diagram

Primer 1

Reverse Transcriptase

RNase HPrimer 2

Reverse Transcriptase

T7 RNA polymerasePrimer 2

Reverse Transcriptase Reverse Transcriptase

RNase HPrimer 1

• sense RNA• antisense RNA• sense DNA• antisense DNA

Legend:

Técnicas de amplificação de Sinal bDNA (Quantiplex)bDNA (Quantiplex)

bDNA (I)

bDNA (II)

bDNA (III)

bDNA (IV)

bDNA (V)