Tecniche di amplificazione quantitative, Real-Time PCR

1. Tecniche di amplificazione quantitative, Real-Time PCR Mauro Pistello Dipartimento Patologia Sperimentale Università di Pisa

2. Fluorescence (Förster) Resonance Energy Transfer Quencher Reporter Laser Light quenching 5’ 3’ Light emission 3’ 5’

3. Light Absorbance and Emission of Fluorescent Dyes TAMRA Dye Spectra

4. Heating Block Optical Fiber Lens Cap Tube Thermal Cycler Block

5. Fluorescence Resonance Energy Transfer Quencher Reporter Laser Light quenching 5’ 3’ Light emission 3’ 5’

6. Raw Spectra Starting cycle Quencher Reporter Reporter End point Quencher

7. Increment of Fluorescence Positive Sample Fluorescence Intensity Negative Control Quencher emission Reporter emission Wavelength

9. HBV DNA

10. Variability of PCR(96 replicates) C.V. 20 - 50% 2Rn Number of Cycles

11. Variability of PCR(96 replicates) 2Rn C.V. 6 - 12% Number of Cycles

12. Threshold Cycle (CT) DRn CT

13. HBV DNA

14. HBV DNA

15. Efficiency of PCR E = 10(-1/S) – 1 where E = PCR efficiency S = slope

16. HBV DNA E = 0.893

17. TTV DNA E = 0.959

18. Commercial Real-Time Systems

19. Taqman PCR (1) Denaturation Annealing R = Reporter Q = Quencher • Polymerization Q Q R 5’ 5’ 3’ 5’ 3’ 5’

20. Taqman PCR (2) . Cleavage R = Reporter Q = Quencher R Q Q 5’ 5’ 3’ 5’ 3’ 5’

21. Scorpions Double-dye probe held in a hairpin loop configuration by a complementary stem sequence

22. Scorpions

23. Hairpin Primers

24. Molecular Beacons Double-dye probe with a stem-loop structure that changes its conformation when the probe hybridizes to the target

25. Hybridization Probes 1. Probes hybridize in head-to-tail arrangement 2. The green fluorescent light emitted by the Fluorescein excites the LC Red 640 that subsequently emits a red fluorescent light

26. Dye-alone a Double stranded DNA intercalating dyes (e.g. SYBR GreenTM 1) b c

27. Primer-dimer results from extension of one primer using the other one as template, even though no stable annealing between primers is possible Primer 1 5’ 3’ Primer 2 Once such an extension occurs, primer-dimer is amplified with high efficiency

28. Methods for Confirming Specificity of Target Detection in Dye-alone Real-Time PCR • Yield of fluorescence at “plateau” in the growth curve • Tm analysis of the DNA products Tm, temperature at which half the DNA is melted or annealed. It depends on DNA sequence and can be determined by heating the DNA to 95°C and slowly cooling. Double strand DNA-specific dyes intercalate with annealed DNA. Rate of increase in fluorescence Temp

29. Factors for Optimal Probe Performance • Quenching in the intact probe • Hybridization conditions • Cleavage of probe/amplimer hybrids • Length and GC-content of oligonucleotides • Tm probe at least 5° higher than Tm primers • Avoid the G nucleotide at the 3’ end • Avoid secondary structures

30. Real-Time NASBA

31. Advantages of Real-Time Amplification • Test results in short time • Reduced handling, material and labor costs • Quantitation over a 5-6 log range • High throughput • Simultaneous detection of multiple analytes • Long shelf-life of labeled probes • Low risk of contamination

32. Amplicons Content After PCR Aerosol

33. Disadvantages of Real-Time Amplification • Theoretical and real primer and PROBE • performances can be very different • Assay set up longer than conventional PCR • High cost of the real-time instruments • Cost of reagents (patent royalties) • Cost of probe synthesis

34. Ruolo dei microarrays in virologia clinica

35. Processes Involved in Making and Using an Array

36. The DNA Microarray Process Technological needs for DNA microarrays

37. Capture Molecules for Protein Arrays

38. Potential Virus Targets for Blood Testing Chips a No disease association. Petrik, Vox Sanguinis 2001 (mod.)

39. DNA Microarrays Versus Real-Time PCR