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Lec16 Realtime PCR

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Lec16 Realtime PCR

  1. 1. Realtime PCR FISH 507a
  2. 2. What is it good for?
  3. 3. Real-Time qPCR Template Taq Fluorescence detection of Cycle 1 5’ 5’ amplification product Taq 2X Template Taq 5’ 5’ Taq Cycle 2 Taq 5’ 5’ Taq 4X Template Linear View Log View 30-40 cycles
  4. 4. Data Analysis Early Exponential CT Phase Log-Linear Phase Linear Ground Phase
  5. 5. Real-Time qPCR Chemistries • Fluorescence-based – After absorbance of certain wavelengths of light (excitation), the fluorophore emits light at a longer wavelength (emission) – Fluorescence proportional to amplified product • Two commonly used chemistries: ® – SYBR Green I – Hybridization Probes: Displacement probes ® Cleavage probes: TaqMan Excite Detect Excitation Fluorescence Emission Fluorophore Wavelength
  6. 6. Real-Time Chemistry: SYBR Green I • SYBR Green I binds dsDNA λ λ • SGI fluorescence increases when bound to dsDNA • As dsDNA accumulates, more λ λ SGI binds the DNA and the PCR fluorescence increase Reaction Progression λ λ λ λ λ λ λ λ λ λ λ λ
  7. 7. SYBR Green Melt Curve Analysis Due to increasing DNA temperature denatured Tm
  8. 8. SYBR Green Melting Curve Analysis • Plot the negative of the rate of change of fluorescence vs. temperature (-dI/dT) • For new amplicons, perform melting curve followed by gel analysis (and sequencing) to validate reaction specificity
  9. 9. SYBR Green I Chemistry • Advantages – Experiment only requires primers – Post-amplification melting curve analysis • Disadvantages – Potential contribution to fluorescence from non-specific products (primer-dimers) – No multiplexing
  10. 10. Actual Research Question- Is increased fish mortality associated with a parasitic worm?
  11. 11. Actual Research Question- Is increased fish mortality associated with a parasitic worm? Biology Extract Genomic DNA Design Primers (NCBI) Run PCR Run PCR on gel or Real-time
  12. 12. Actual Research Question- Is increased fish mortality associated with a parasitic worm? Biology Extract Genomic DNA Design Primers (NCBI) Run PCR Run PCR on gel or Real-time
  13. 13. SYBR Green continued - Monoplexing - Multiplexing - Cost saving - High Specificity - Fast initial screening - High Sensitivity Sybr Green I® Taqman Probe
  14. 14. Hallmarks of a Good qPCR Assay One Specific Product
  15. 15. Primer-Dimer Formation in Non-Optimized Assay 10,000 copies 2,000 400 Primer- 0 (NTC) dimers
  16. 16. Primer Interactions (Primer Dimers) Amplification 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ Stable Interaction Primer Dimers For traditional PCR, primer-dimers are usually tolerated Product Primer-dimers NTC A B C
  17. 17. Primer-Dimers in Real-Time qPCR • Can contribute to reaction fluorescence when using SYBR Green I – Miscalculated Ct values • Amplifying primer-dimers affects reaction efficiency – Lose sensitivity of detection – Poor reproducibility
  18. 18. One Step or Two Step RT-PCR? One Step RT-PCR Two Step RT-PCR (One tube) RNA (Two tubes) 1 target X targets Oligo dT GS primers Random Primers (GS Primers) cDNA Target pool 1 amplicon Amplicon Amplicon X amplicons
  19. 19. One Step or Two Step RT-PCR? One-Step RT-PCR Two-Step RT-PCR • Highly defined conditions to • Separate conditions for cDNA support RT and Taq synthesis & PCR • Requires gene specific primer • Flexible choice of primers • Ideal for quantification of 1 or 2 • Ideal for quantification of messages from a large number multiple genes from a limited of RNA samples number of RNA samples Perfect for: Perfect for: - Lot of samples - Few samples - Small amount of targets - Large amount of targets Two-step RT-PCR is more convenient and cost effective
  20. 20. Real-Time Chemistry: TaqMan ® • Target specific hybridization probe • 5’ reporter and 3’ quencher • Utilizes FRET quenching Light* Light Energy R Reporter R Q Q Quencher * heat for BHQs
  21. 21. TaqMan® Chemistry λ 1. During PCR, probe hybridizes to target sequence R 2. Probe is partially R Q Taq Taq displaced during extension 3. Probe cleaved by 5’- 3’ nuclease activity of polymerase 4. Illuminated free reporter exhibits unquenched fluorescence
  22. 22. TaqMan Primers * equal Tm (58-600 C) * 15-30 bases in length * G+C content 30-80% * no runs of four or more Gs (any nucleotide) * no more than two G+C at the 3’ end * no G at the 5' end * amplicon size 50-150 bp (max 400) * span exon-exon junctions in cDNA ABI Primer Express Software Tutorial (www)
  23. 23. TaqMan® Chemistry • Advantages – Fluorescence is target specific – Multiplexing • Disadvantages – High initial cost – Assay design not trivial
  24. 24. Worksheet
  25. 25. What you need for a good assay Quality RNA NO DNA …. How would you check this? Good Replication An appropriate normalizing target something that does not change
  26. 26. Contributors to Poor Reproducibility • Laboratory technique • Specificity issues – Cross homology of primers – Primer-dimer • Reaction efficiency – Secondary structure of amplicon – Primer-dimers
  27. 27. Improving Reproducibility • Laboratory Techniques – Use clean bench (hood) – Use aerosol resistant tips – Use calibrated micropipettors – Use large volumes (5µL and up) – Pipette into each reaction vessel once
  28. 28. Same Reagents, Different Hands Cycle Cycle Good Technique Poor Technique
  29. 29. MINER