Real time Detection PCR for Nucleic Acids Quantitation

1. Real time Detection PCRforNucleic Acids Quantitation

2. Outline • What is Real Time detection in PCR? • What instrument you need to perform RTD? • Why is RTD better for quantitative analysis?

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

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

5. Taqman PCR Chemistry Denaturation Annealing R = Reporter Q = Quencher • Polymerization Q Q R forward primer 5’ 5’ 3’ 5’ 3’ 5’

6. Taqman PCR Chemistry Denaturation Annealing R = Reporter Q = Quencher • Polymerization Q Q R forward primer 5’ 5’ 3’ 5’ 3’ 5’

7. Taqman PCR Chemistry Denaturation Annealing R = Reporter Q = Quencher • Polymerization Q Q R forward primer 5’ 5’ 3’ 5’ 3’ 5’

8. Taqman PCR Chemistry Denaturation Annealing R = Reporter Q = Quencher • Polymerization Q Q R forward primer 5’ 5’ 3’ 5’ 3’ 5’

9. Taqman PCR Chemistry Denaturation Annealing R = Reporter Q = Quencher • Polymerization Q Q R forward primer 5’ 5’ 3’ 5’ 3’ 5’

10. Taqman PCR Chemistry Denaturation Annealing R = Reporter Q = Quencher • Polymerization Q Q R forward primer 5’ 5’ 3’ 5’ 3’ 5’

11. Taqman PCR Chemistry Denaturation Annealing R = Reporter Q = Quencher • Polymerization Q Q R forward primer 5’ 5’ 3’ 5’ 3’ 5’

12. Taqman PCR Chemistry Denaturation Annealing R = Reporter Q = Quencher • Polymerization Q Q R forward primer 5’ 5’ 3’ 5’ 3’ 5’

13. Q Q R . Strand displacement R = Reporter Q = Quencher Taqman PCR Chemistry forward primer 5’ 5’ 3’ 5’ 3’ 5’ reverse primer

14. Q Q Taqman PCR Chemistry . Cleavage R = Reporter Q = Quencher R forward primer 5’ 5’ 3’ 5’ 3’ 5’

15. R Q Q Taqman PCR Chemistry . Cleavage R = Reporter Q = Quencher forward primer 5’ 5’ 3’ 5’ 3’ 5’

16. R Taqman PCR Chemistry . Cleavage R = Reporter Q = Quencher Q Q forward primer 5’ 5’ 3’ 5’ 3’ 5’

17. R Taqman PCR Chemistry . Cleavage R = Reporter Q = Quencher Q Q forward primer 5’ 5’ 3’ 5’ 3’ 5’

18. R Taqman PCR Chemistry . Polymerization completed R = Reporter Q = Quencher Q Q forward primer 5’ 5’ 3’ 5’ 3’ 5’

19. . Polymerization completed R = Reporter Q = Quencher Taqman PCR Chemistry Q Q R R forward primer 5’ 5’ 3’ 5’ 3’ 5’

20. ß-Actin∆Rn vs Cycle Number 2.2 2 no template 1.8 20000 1.6 10000 5000 1.4 2000 1.2 1000 ∆Rn 1 500 0.8 100 0.6 0.4 0.2 0 0 10 20 30 40 cycle number

21. Why Real Time Detection allows Accurate Quantitation?

22. 96 replicates Fluor (delta Rn) Cycle Number Problems with End-Point Quantitation Ct 30

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

24. Accuracy How well a measured sample matches the true value = the measure of exactness. Important for absolute quantitation.

25. Precision The degree that multiple samplings of the same homogenous source give similar values = degree of agreement. Precision is critical for quatification.