1. PE Biosystems Sequence Detection Systems Real time Detection PCR for Nucleic Acids Quantitation

2. PE Biosystems Sequence Detection Systems Outline What is Real Time detection in PCR? What is Real Time detection in PCR? What instrument you need to perform RTD? What instrument you need to perform RTD? Why is RTD better for quantitative analysis? Why is RTD better for quantitative analysis?

3. PE Biosystems Sequence Detection Systems TaqMan™ Fluorogenic Probe Quencher (Rhodamine) Reporter (Fluoresceine) Phosphate Group 3’ 5’ Laser Excitation Energy Transfer Fam Vic Tet

4. PE Biosystems Sequence Detection Systems TaqMan™ Fluorogenic Probe Quencher (Rhodamine) Reporter (Fluoresceine) Phosphate Group 3’ 5’ Laser Excitation Fam Vic Tet Cleaved Probe

5. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry PolymerizationPolymerization 5’ 3’ 5’ 3’ 5’ forward primer R R = Reporter Q = Quencher Denaturation Annealing Q Q

6. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry PolymerizationPolymerization 5’ 3’ 5’ 3’ 5’ forward primer R R = Reporter Q = Quencher Denaturation Annealing Q Q

7. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry PolymerizationPolymerization 5’ 3’ 5’ 3’ 5’ forward primer R R = Reporter Q = Quencher Denaturation Annealing Q Q

8. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry PolymerizationPolymerization 5’ 3’ 5’ 3’ 5’ forward primer R R = Reporter Q = Quencher Denaturation Annealing Q Q

9. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry PolymerizationPolymerization 5’ 3’ 5’ 3’ 5’ forward primer R R = Reporter Q = Quencher Denaturation Annealing Q Q

10. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry PolymerizationPolymerization 5’ 3’ 5’ 3’ 5’ forward primer R R = Reporter Q = Quencher Denaturation Annealing Q Q

11. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry PolymerizationPolymerization 5’ 3’ 5’ 3’ 5’ forward primer R R = Reporter Q = Quencher Denaturation Annealing Q Q

12. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry PolymerizationPolymerization 5’ 3’ 5’ 3’ 5’ forward primer R R = Reporter Q = Quencher Denaturation Annealing Q Q

13. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry R = Reporter Q = Quencher. Strand displacement. Strand displacement 5’ 3’ 5’ 3’ 5’ forward primer reverse primer Q R Q

14. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry 5’ 3’ 5’ 3’ 5’ forward primer R = Reporter Q = Quencher. Cleavage R Q Q

15. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry 5’ 3’ 5’ 3’ 5’ forward primer R = Reporter Q = Quencher. Cleavage R Q Q

16. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry 5’ 3’ 5’ 3’ 5’ forward primer R = Reporter Q = Quencher. Cleavage R Q Q

17. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry 5’ 3’ 5’ 3’ 5’ forward primer R = Reporter Q = Quencher. Cleavage R Q Q

18. PE Biosystems Sequence Detection Systems Taqman PCR Chemistry 5’ 3’ 5’ 3’ 5’ forward primer R = Reporter Q = Quencher R Q Q Polymerization completed.

19. PE Biosystems Sequence Detection Systems Polymerization completed. 5’ 3’ forward primer R Q Q 5’ 3’ 5’ Taqman PCR Chemistry R = Reporter Q = Quencher R

20. PE Biosystems Sequence Detection Systems ß-Actin ∆R n vs Cycle Number 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 010203040 cycle number ∆R n no template 20000 10000 5000 2000 1000 500 100

21. PE Biosystems Sequence Detection Systems Why Real Time Detection allows Accurate Quantitation?

22. PE Biosystems Sequence Detection Systems Problems with End-Point Quantitation 96 replicates Cycle Number Fluor (delta Rn) C t 30

23. PE Biosystems Sequence Detection Systems Real-Time Quantitation: PCR Uniformity in Log Phase Threshold = 0.05 96 replicates Cycle Number Fluor (Delta Rn) geometric

24. PE Biosystems Sequence Detection Systems AccuracyAccuracy How well a measured sample matches the true value = How well a measured sample matches the true value = the measure of exactness. Important for absolute quantitation.

25. PE Biosystems Sequence Detection Systems PrecisionPrecision The degree that multiple samplings of the same homogenous source give similar values = degree of agreement. Precision is critical for quatification.