IntroductionIntroduction to Real-Time PCR to Real-Time PCR

Joachim HegstadJoachim HegstadForskerForsker

Avdeling for Mikrobiologi og SmittevernAvdeling for Mikrobiologi og SmittevernUniversitetssykehuset Nord-NorgeUniversitetssykehuset Nord-Norge

Dept. for microbiology and infection contol

Main Topics Main Topics • What is PCR?What is PCR?• PCR detection methods PCR detection methods • Real-Time PCR Real-Time PCR Instruments• Software analysesSoftware analyses• Clinical valueClinical value

Polymerase Chain Reaction (PCR) (1)Polymerase Chain Reaction (PCR) (1)

PCR was invented by Dr. Kary Mullis i 1983 PCR was invented by Dr. Kary Mullis i 1983 –Nobel price in –Nobel price in chemistry chemistry ten years later. ten years later.

PCR is by far one of the most important PCR is by far one of the most important and used molecular tool in diagnostic and and used molecular tool in diagnostic and science.science.

By May 2009, By May 2009, 380 990 380 990 paper were paper were published using PCR as a tool (PubMed) published using PCR as a tool (PubMed)

PCR imitate natures way of copying DNA PCR imitate natures way of copying DNA by amplifying DNA sequence by amplifying DNA sequence specified/limited by the primers.specified/limited by the primers.

Polymerase Chain Reaction (PCR) (2)Polymerase Chain Reaction (PCR) (2)

PCR mix must contain:PCR mix must contain: Template (DNA/RNA sample containing the Template (DNA/RNA sample containing the

target)target) DNA polymeraseDNA polymerase Co-factor such as MgCo-factor such as Mg2+2+

Primers ( 20 – 30 bp) Primers ( 20 – 30 bp) Deoxyribonucleotides (dATP, dCTP, dGTP, and Deoxyribonucleotides (dATP, dCTP, dGTP, and

dTTP)dTTP)

Polymerase Chain Reaction (PCR) (3)Polymerase Chain Reaction (PCR) (3)

The PCR program normally cycles bewteen 3 The PCR program normally cycles bewteen 3 temperatures which are repeatedtemperatures which are repeated

1.1. Denaturation of DNA 94 – 96Denaturation of DNA 94 – 96°°C in C in ≥≥ 20 20 sec.sec.

DNA is denatured to single stranded DNADNA is denatured to single stranded DNA2.2. Annealing, 50 – 65Annealing, 50 – 65°°C in C in ≥ 20≥ 20 sec. sec.

Primers binds to complementary sequence on Primers binds to complementary sequence on both target sequences of single stranded DNAboth target sequences of single stranded DNA

3.3. Extention, 72Extention, 72°°C in C in ≥ 20≥ 20 sec. sec.1.1. Polymerase binds to and incorporate Polymerase binds to and incorporate

complementary nucleotides from primers and complementary nucleotides from primers and downstream target sequencedownstream target sequence

Detection of PCR product by Detection of PCR product by electrophoresiselectrophoresis

PCR product is loaded PCR product is loaded in wells on agarose gel in wells on agarose gel (0,6-2,5%)(0,6-2,5%)

Electrophoresis Electrophoresis separates DNA by sizeseparates DNA by size

Visualisation in UV-Visualisation in UV-light after staining by light after staining by EtBrEtBr

Fluorescence-detection Fluorescence-detection methods methods

SYBER Green detectionSYBER Green detection

Double Dye probes Double Dye probes

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

TaqMan probeTaqMan probe Doube-Dye LNA Doube-Dye LNA

probeprobe MGB probeMGB probe

Molecular BeaconsMolecular Beacons

FRET probes or FRET probes or Hybridisation probesHybridisation probes

(fluorescence resonance energy transfer)(fluorescence resonance energy transfer)

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

Donor probe Acceptor

Fluorophores and quenchers Fluorophores and quenchers (1)(1)

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

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

Fluorophores and quenchers Fluorophores and quenchers (2)(2)

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

Real-Time PCR Instruments (2) (2)ABI 7900 FastABI 7900 Fast

96 or 384 well format96 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

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

Real-Time PCR Instruments (4) Applied Biosystems Line

ABI 7900 HT

ABI 7300

ABI 7500

ABI 7000

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

Real-Time PCR Instruments (6) Cepheid Smart Cycler

16 individual Real-Time cyclers in one box

Fan

Optic Blocks

Heater

Tube

I-CORE Board

Real-Time PCR Instruments (7)

BioRad iCycler Stratagene Mx4000 and Mx3005P

Real-Time PCR Instruments (8)

Corbett Research/Qiagen Rotor-Gene 3000 Rotor-Gene 6000/Qiagen

Gene Q

BioMerieux NucliSens Easy Q

Analyzer System

Recommended dyes combinations for Recommended dyes combinations for multiplex assays on every thermocyclermultiplex assays on every thermocycler

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

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

compatibilitycompatibility

Data analysesData analyses BaselineBaseline: PCR cycle number in which signal is : PCR cycle number in which signal is

accumulating but beneath the limit of detection accumulating but beneath the limit of detection level of the instrument.level of the instrument.

ThresholdThreshold: Purpose: find a level of fluorescence : Purpose: find a level of fluorescence where samples can be compared. where samples can be compared.

Sat in the area where the lines are in parallel and Sat in the area where the lines are in parallel and amplification is exponential. Fluorescent signal amplification is exponential. Fluorescent signal above threshold is used to calculate cycle above threshold is used to calculate cycle threshold (Cthreshold (Ctt).).

CCtt : ( : (threshold cycle) threshold cycle) is defined as the cycle is defined as the cycle number at which the fluorescence emission number at which the fluorescence emission exceeds the fixed threshold. It gives the exceeds the fixed threshold. It gives the quantitative relationship between amount quantitative relationship between amount template in start reaction and amplified product in template in start reaction and amplified product in exponential phase. (quantitation: mRNA exponential phase. (quantitation: mRNA expression or DNA copy no). The fewer cycles it expression or DNA copy no). The fewer cycles it takes to reach detectable fluorescence, the takes to reach detectable fluorescence, the greater initial copy number of target.greater initial copy number of target.

Rn:Rn: Rn+Rn+ is the Rn value of a reaction containing all is the Rn value of a reaction containing all components (the sample of interest); components (the sample of interest); Rn-Rn- is the Rn is the Rn value detected in NTC (baseline value). value detected in NTC (baseline value). ΔΔRn is the Rn is the difference between Rn+ and Rn-. difference between Rn+ and Rn-. It is an indicator It is an indicator of the magnitude of the signal generated by the of the magnitude of the signal generated by the PCRPCR. It is the . It is the ΔΔRn plotted against cycle numbers Rn plotted against cycle numbers that produces the amplification curves and gives that produces the amplification curves and gives the CT value.the CT value.

Software analysis of real time Software analysis of real time PCRPCR

SYBR green-detection with dissociation SYBR green-detection with dissociation curvecurve

Exponential growth phase = linear part in Exponential growth phase = linear part in logarithmic graphiclogarithmic graphic

Samples must be compared in the exponential phase, easier to

find when converted to log scale

Logarithmic graph

Exponential growth phase

Threshold

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 A Sample B

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

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

Real time PCR vs PCR and Real time PCR vs PCR and detection by electrophoresisdetection by electrophoresisReal time PCRReal time PCR+ Software detectionSoftware detection+ Fast – 30 min -2 hoursFast – 30 min -2 hours+ 96 and 384 well formate96 and 384 well formate+ Very suitable for Very suitable for

quantification – no end quantification – no end point analysis point analysis

- Achieves no data on Achieves no data on size of ampilfied DNAsize of ampilfied DNA

- PCR machine is rather PCR machine is rather expensiveexpensive

PCR and electrophoresisPCR and electrophoresis+ Size determination of Size determination of

ampliconamplicon- Time and labour Time and labour

intensiveintensive- Few samples per gelFew samples per gel- Pour quantification – Pour quantification –

end point analysisend point analysis