Genética Molecular em Análises Clinicas
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Transcript of Genética Molecular em Análises Clinicas
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GenéticaProf.Doutor José Cabeda
Genética Molecular em Análises Clinicas
TÉCNICAS DE AMPLIFICAÇÃO TÉCNICAS DE AMPLIFICAÇÃO DE ÁCIDOS NUCLEICOSDE ÁCIDOS NUCLEICOS
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Técnicas de amplificação de ácidos nucleicos
PCRPCR Real Time PCRReal Time PCR NASBA/TMANASBA/TMA
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PCR
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OPTIMIZAÇÃO DO PCR
[MgCl[MgCl22]] ThTh [dNTP][dNTP] [primers][primers] [DNA][DNA] [inibidores][inibidores]
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Variações
RT-PCRRT-PCR Multiplex PCRMultiplex PCR Nested PCRNested PCR
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Técnicas de amplificação de ácidos nucleicos
PCRPCR Real Time PCRReal Time PCR NASBA/TMANASBA/TMA
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Genética2001/2002 Prof.Doutor José Cabeda
Principio de funcionamento do Real-Time PCR
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Detecção dos produtos de PCR
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Montar uma reacção
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Genética2001/2002 Prof.Doutor José Cabeda
Químicas Utilizáveis
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SYBR-Green I
5’3’
5’ 3’
SGExcitation
SG
SG
SG
SG
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SYBR-Green I
5’3’5’ 3’
SG
SG
SG
SG
SG
Excitation Emission
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Detcção com SybGreen
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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
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Detecção com sondas
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Quimicas utilizáveis:sondas taqman
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R Q
Sequence specific probes: Dual labeled probes
5’3’5’ 3’
5’3’
5’ 3’
Excitation RQQR QRExcitation
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Quimicas utilizáveis:molecular beacons
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Molecular Beacons I
10mer
25mer
FAM DabCyl
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Molecular Beacons II
R QExcitation
RQ
QR Emission
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Molecular Beacons II
R QExcitation
RQ
QR Emission
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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
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Quimicas utilizáveis:sondas FRET
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Genética2001/2002 Prof.Doutor José Cabeda
Hyb-ProbesTM
Oligo 1: Fluorescein Oligo 2: Quencher
Excitation Emission
Transfer
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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’
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Quimicas utilizáveis:sondas Eclipse
F Q
F F
MGB
Q
F
MGB
Q Q
F
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Genética2001/2002 Prof.Doutor José Cabeda
Aplicações
QuantificaçãoQuantificação
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End Point versus Real-Time
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Quantificação
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Genética2001/2002 Prof.Doutor José Cabeda
Aplicações
Detecção de Mutações / SNPDetecção de Mutações / SNP
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Detecção de mutações
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Genética2001/2002 Prof.Doutor José Cabeda
Aplicações
Multiplex DetectionMultiplex Detection
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Detecção simultânea de vários produtos
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Genética2001/2002 Prof.Doutor José Cabeda
Os equipamentos
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Smartcycler
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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
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O Futuro próximo
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Detecção com sondas
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Detcção com SybGreen
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Quantificação
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Detecção de mutações
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Detecção simultânea de dois produtos
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Técnicas de amplificação de ácidos nucleicos
PCRPCR Real Time PCRReal Time PCR NASBA/TMANASBA/TMA
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NASBA/NUCLISENS
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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:
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Técnicas de amplificação de Sinal bDNA (Quantiplex)bDNA (Quantiplex)
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bDNA (I)
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bDNA (II)
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bDNA (III)
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bDNA (IV)
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bDNA (V)