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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




Quimicas utiliz veis molecular beacons
Quimicas utilizáveis:molecular beacons



Transfer

Excitation

Emission

Hyb-ProbesTM

Oligo 1: Fluorescein

Oligo 2: Quencher

Prof.Doutor José Cabeda


Quimicas utiliz veis mgb probes

MGB

MGB

F

F

F

F

F

Q

Q

Q

Q

Quimicas utilizáveis:mgb-probes


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




Nasba nuclisens
NASBA/NUCLISENS


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


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