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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
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 QR
Excitation
Quimicas utilizáveis:molecular beacons
Molecular Beacons I
10mer
25mer
FAM DabCyl
Molecular Beacons II
R QExcitation
R
Q
QR Emission
Molecular Beacons II
R QExcitation
R
Q
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)
