genética molecular em análises clinicas
DESCRIPTION
Genética Molecular em Análises Clinicas. TÉCNICAS DE AMPLIFICAÇÃO DE ÁCIDOS NUCLEICOS. Técnicas de amplificação de ácidos nucleicos. PCR Real Time PCR NASBA/TMA. PCR. OPTIMIZAÇÃO DO PCR. [MgCl 2 ] Th [dNTP] [primers] [DNA] [inibidores]. Variações. RT-PCR Multiplex PCR Nested PCR. - PowerPoint PPT PresentationTRANSCRIPT
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Genética Molecular em Análises Clinicas
TÉCNICAS DE AMPLIFICAÇÃO DE ÁCIDOS NUCLEICOS
2001/2002 Prof.Doutor José Cabeda
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Técnicas de amplificação de ácidos nucleicos
– PCR
– Real Time PCR
– NASBA/TMA
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PCR
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OPTIMIZAÇÃO DO PCR
• [MgCl2]
• Th• [dNTP]• [primers]• [DNA]• [inibidores]
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Variações
• RT-PCR• Multiplex PCR• Nested PCR
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Técnicas de amplificação de ácidos nucleicos
– PCR
– Real Time PCR
– NASBA/TMA
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Principio de funcionamento do Real-Time PCR
2001/2002 Prof.Doutor José Cabeda
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Detecção dos produtos de PCR
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Montar uma reacção
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Químicas Utilizáveis
2001/2002 Prof.Doutor José Cabeda
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Detcção com SybGreen
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Sybr-Green Detection
• Intercalates to dsDNA• Inhibits DNA
amplification so concentration is critical
• Detects specific and non-specific targets
• Low starting background with large increase in signal
• Can run a melt curve to look at product specificity
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Detecção com sondas
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Quimicas utilizáveis:sondas taqman
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Quimicas utilizáveis:molecular beacons
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Quimicas utilizáveis:sondas FRET
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2001/2002 Prof.Doutor José Cabeda
Hyb-ProbesTM
Oligo 1: Fluorescein Oligo 2: Quencher
Excitation Emission
Transfer
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Quimicas utilizáveis:mgb-probes
F
Q
F F
MGB
Q
F
MGB
Q Q
F
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Aplicações
Quantificação
2001/2002 Prof.Doutor José Cabeda
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End Point versus
Real-Time
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Quantificação
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Aplicações
Detecção de Mutações / SNP
2001/2002 Prof.Doutor José Cabeda
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Detecção de mutações
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Aplicações
Multiplex Detection
2001/2002 Prof.Doutor José Cabeda
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Detecção simultânea de vários produtos
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Os equipamentos
2001/2002 Prof.Doutor José Cabeda
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Smartcycler
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Rotorgene
• The samples spin continually during a run at 500 rpm
• The samples are heated and cooled in a low mass air oven
• Tubes are illuminated as they pass the detector
• On data acquisition energy is averaged over 20 revolutions to give the fluorescence of each sample
• Four 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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Near-patient-testing
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Técnicas de amplificação de ácidos nucleicos
– PCR
– Real Time PCR
– NASBA/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: