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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.

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gen tica molecular em an lises clinicas

Genética Molecular em Análises Clinicas

TÉCNICAS DE AMPLIFICAÇÃO DE ÁCIDOS NUCLEICOS

Prof.Doutor José Cabeda

optimiza o do pcr
OPTIMIZAÇÃO DO PCR
  • [MgCl2]
  • Th
  • [dNTP]
  • [primers]
  • [DNA]
  • [inibidores]
varia es
Variações
  • RT-PCR
  • Multiplex PCR
  • Nested PCR
qu micas utiliz veis

Químicas Utilizáveis

Prof.Doutor José Cabeda

sybr green detection
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
slide19

Transfer

Excitation

Emission

Hyb-ProbesTM

Oligo 1: Fluorescein

Oligo 2: Quencher

Prof.Doutor José Cabeda

aplica es

Aplicações

Quantificação

Prof.Doutor José Cabeda

aplica es1

Aplicações

Detecção de Mutações / SNP

Prof.Doutor José Cabeda

aplica es2

Aplicações

Multiplex Detection

Prof.Doutor José Cabeda

os equipamentos

Os equipamentos

Prof.Doutor José Cabeda

rotorgene
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
amplification schematic diagram
Amplification: schematic diagram

Primer 1

Legend:

Reverse Transcriptase

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

RNase H

Primer 2

Reverse Transcriptase

T7 RNA polymerase

Primer 2

Reverse Transcriptase

Reverse Transcriptase

RNase H

Primer 1