Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Lec16 Realtime PCR

8,611 views

Published on

Published in: Education, Technology

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

×