1. Introduction to Real-Time PCR Joachim Hegstad Forsker Avdeling for Mikrobiologi og Smittevern Universitetssykehuset Nord-Norge Dept. for microbiology and infection contol

2. Main Topics What is PCR? What is PCR? PCR detection methods PCR detection methods Real-Time PCR Real-Time PCR Instruments Software analyses Software analyses Clinical value 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 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. 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) 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. 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) Template (DNA/RNA sample containing the target) DNA polymerase DNA polymerase Co-factor such as Mg 2+ Co-factor such as Mg 2+ Primers ( 20 – 30 bp) Primers ( 20 – 30 bp) Deoxyribonucleotides (dATP, dCTP, dGTP, and dTTP) Deoxyribonucleotides (dATP, dCTP, dGTP, and dTTP)

5. Polymerase Chain Reaction (PCR) (3) The PCR program normally cycles bewteen 3 temperatures which are repeated The PCR program normally cycles bewteen 3 temperatures which are repeated 1. Denaturation of DNA 94 – 96°C in ≥ 20 sec.  DNA is denatured to single stranded DNA 2. Annealing, 50 – 65°C in ≥ 20 sec.  Primers binds to complementary sequence on both target sequences of single stranded DNA 3. 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%) PCR product is loaded in wells on agarose gel (0,6- 2,5%) Electrophoresis separates DNA by size Electrophoresis separates DNA by size Visualisation in UV-light after staining by EtBr Visualisation in UV-light after staining by EtBr

7. Fluorescence-detection methods

8. SYBER Green detection

9. Double Dye probes Eurogentec Real-Time qPCR High Quality probes for Real-Time qPCR TaqMan probe TaqMan probe Doube-Dye LNA probe Doube-Dye LNA probe MGB probe MGB probe

10. Molecular Beacons

11. FRET probes or Hybridisation probes (fluorescence resonance energy transfer) Eurogentec Real-Time qPCR High Quality probes for Real-Time qPCR Donor probe Acceptor

12. Fluorophores and quenchers (1) Eurogentec Real-Time qPCR High Quality probes for Real-Time qPCR

13. Eurogentec Real-Time qPCR High Quality probes for Real-Time qPCR 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. (2) ABI 7900 Fast Real-Time PCR Instruments (2) ABI 7900 Fast 96 or 384 well format 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 ABI 7900 HT ABI 7300 ABI 7500 ABI 7000

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 LightCycler LightCycler® 480 96 well and 384 well Peltier-based heating/cooling <40 minutes (384-well PCR plate) 96-well PCR plate:10 - 100 μl 384-well PCR plate:5 - 20 μl Excitation source: Xenon lamp, 420 nm - 670 nm

19. Real-Time PCR Instruments (6) Cepheid Smart Cycler 16 individual Real-Time cyclers in one box Fan Optic Blocks Heater Tube I-CORE Board

20. Real-Time PCR Instruments (7) BioRad iCycler Stratagene Mx4000 and Mx3005P

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 Eurogentec Real-Time qPCR High Quality probes for Real-Time qPCR

23. Eurogentec Real-Time qPCR High Quality probes for Real-Time qPCR

24. compatibility

25. Data analyses Baseline: PCR cycle number in which signal is accumulating but beneath the limit of detection level of the instrument. 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. 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 (C t ). Sat in the area where the lines are in parallel and amplification is exponential. Fluorescent signal above threshold is used to calculate cycle threshold (C t ). C t : (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. C t : (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). ΔRn is the difference between Rn+ and Rn-. It is an indicator of the magnitude of the signal generated by the PCR. It is the ΔRn plotted against cycle numbers that produces the amplification curves and gives the CT value. 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). ΔRn is the difference between Rn+ and Rn-. It is an indicator of the magnitude of the signal generated by the PCR. It is the ΔRn 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 Samples must be compared in the exponential phase, easier to find when converted to log scale Logarithmic graphExponential growth phase Threshold Ct=26Ct=22 Sample A>Sample B; why? Fluorescence signal is proportional to DNA amount in sample, fluorescence appears earlier from sample A than sample B=>higher initial copy number Sample ASample B

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