Introduction to Real-Time PCR Joachim Hegstad Forsker Avdeling for Mikrobiologi og Smittevern Universitetssykehuset

1. Introduction to Real-Time PCR Joachim Hegstad Forsker Avdeling for Mikrobiologi og Smittevern Universitetssykehuset Nord-Norge

2. Main Topics What is PCR? PCR detection methods Real-Time PCR Instruments Software analyses Clinical value

3. Polymerase Chain Reaction (PCR) (1) PCR was invented by Dr. Kary Mullis i 1983 –Nobel price in chemistry ten years later. PCR is by far one of the most important and used molecular tool in diagnostic and science. By May 2009, 380 990 paper were published using PCR as a tool (PubMed) PCR imitate natures way of copying DNA by amplifying DNA sequence specified/limited by the primers.

4. Polymerase Chain Reaction (PCR) (2) PCR mix must contain: Template (DNA/RNA sample containing the target) DNA polymerase Co-factor such as Mg2+ Primers ( 20 – 30 bp) Deoxyribonucleotides (dATP, dCTP, dGTP, and dTTP)

5. Polymerase Chain Reaction (PCR) (3) The PCR program normally cycles bewteen 3 temperatures which are repeated Denaturation of DNA 94 – 96°C in = 20 sec. DNA is denatured to single stranded DNA Annealing, 50 – 65°C in = 20 sec. Primers binds to complementary sequence on both target sequences of single stranded DNA Extention, 72°C in = 20 sec. Polymerase binds to and incorporate complementary nucleotides from primers and downstream target sequence

6. Detection of PCR product by electrophoresis PCR product is loaded in wells on agarose gel (0,6-2,5%) Electrophoresis separates DNA by size Visualisation in UV-light after staining by EtBr

7. Fluorescence-detection methods

8. SYBER Green detection

9. Double Dye probes TaqMan probe Doube-Dye LNA probe MGB probe

10. Molecular Beacons

11. FRET probes or Hybridisation probes(fluorescence resonance energy transfer)

12. Fluorophores and quenchers (1)

13. Fluorophores and quenchers (2)

14. Real-Time PCR Instruments ABI Prism SDS: 7900 Fast 7500 Fast 7300 7000 Roche Light Cycler 1.0/2.0 LightCycler® 480 Cepheid Smart Cycler BioRad Icycler iQ5 Stratagene Mx3005P Mx3000P Mx4000 Corbett/Qiagen Roto-Gene 3000 Rotor-Gene 6000/Qiagen Gene Q NucliSens MB Analyzer

15. Real-Time PCR Instruments (2)ABI 7900 Fast 96 or 384 well format 40 cycles in 30 min in 96-Fast-format and 55 min in 384-format Closed well system Minimal hands-on Dynamic range of 6 orders of magnitude Multi-color detection Qualitative and quantitative testing

16. Real-Time PCR Instruments (3)Real-Time PCR Set-up Plastics Optical 96-well 384-well plates Tubes (only on 96 cycler) Reaction volume 96-well < 100 µl Reaction volume 384-well < 20 µl Optical adhesive covers or caps

17. Real-Time PCR Instruments (4) Applied Biosystems Line

18. Real-Time PCR Instruments (5) Roche Glass capillaries Air heating and cooling 30-40 cycles in 20-30 min 32 sample carousel 20 µl reactions

19. Real-Time PCR Instruments (6) Cepheid Smart Cycler

20. Real-Time PCR Instruments (7)

21. Real-Time PCR Instruments (8) Corbett Research/Qiagen Rotor-Gene 3000 Rotor-Gene 6000/Qiagen Gene Q BioMerieux NucliSens Easy Q Analyzer System

22. Recommended dyes combinations for multiplex assays on every thermocycler

24. compatibility

25. Data analyses Baseline: PCR cycle number in which signal is accumulating but beneath the limit of detection level of the instrument. Threshold: Purpose: find a level of fluorescence where samples can be compared. Sat in the area where the lines are in parallel and amplification is exponential. Fluorescent signal above threshold is used to calculate cycle threshold (Ct). Ct : (threshold cycle) is defined as the cycle number at which the fluorescence emission exceeds the fixed threshold. It gives the quantitative relationship between amount template in start reaction and amplified product in exponential phase. (quantitation: mRNA expression or DNA copy no). The fewer cycles it takes to reach detectable fluorescence, the greater initial copy number of target. Rn: Rn+ is the Rn value of a reaction containing all components (the sample of interest); Rn- is the Rn value detected in NTC (baseline value). ?Rn is the difference between Rn+ and Rn-. It is an indicator of the magnitude of the signal generated by the PCR. It is the ?Rn plotted against cycle numbers that produces the amplification curves and gives the CT value.

26. Software analysis of real time PCR

27. SYBR green-detection with dissociation curve

28. Exponential growth phase = linear part in logarithmic graphic

29. Clinical Value Qualitative (pos/neg) nucleic acid tests are especially valuable for the detection of infectious agents that are: Unculturable Present in extremely low quantities Fastidious or slow-growing

30. Clinical Value (2) Quantitative (viral load) methods are important for monitoring certain chronic infections. These tests allow us to: monitor therapy detect the development of drug resistance predict disease progression

31. Real time PCR vs PCR and detection by electrophoresis Real time PCR Software detection Fast – 30 min -2 hours 96 and 384 well formate Very suitable for quantification – no end point analysis Achieves no data on size of ampilfied DNA PCR machine is rather expensive PCR and electrophoresis Size determination of amplicon Time and labour intensive Few samples per gel Pour quantification – end point analysis