The basics of real-time PCR Introduction Dr BERRAZEG Meryem MCA à l’Université Oran1 Chercheur associée à l’Institut Pasteur d’Algérie

PCR Dreischrittreaktion Denaturation 95°C Annealing 50-60°C Extension 72°C Cycling Exponential amplification of PCR products Strand denaturation and primer annealing Primer extension Primers and DNA Polymerase Chain Reaction

Plateau phaseLinear phaseExponential phase End point analysis on agarose gels Cycles PCR Product Polymerase Chain Reaction

Variable plateau phase Time Point of PCR Analysis 96 Replicas Cycles High precision during exponential phase PCR Product

Signal Generation with a TaqMan ® Probe The 5-Nuclease Assay This method uses 2 principles: FRET Technology 5- Nuclease Activity of the Taq Polymerase

5’ 3’ 5’ 3’ 5’ Forward Primer Reverse Primer TaqMan ® Probe Q R 5’ Nuclease Assay using TaqMan ® probes FAM™, VIC ® TAMRA™ dyes PCR specificity (primer) Hybridization specificity (probe)

5’ 3’ 5’ 3’ 5’ R Q Fluorescence Resonance Energy Transfer (FRET) from high energy to low energy dye  No reporter signal with intact probe 5’ Nuclease Assay using TaqMan ® probes

3’ 5’ 3’ 5’ R Q p R Q Displacement of probe by 5‘ nuclease activity of polymerase 5’ Nuclease Assay using TaqMan ® probes

5’ 3’ 5’ 3’ 5’ R Q Cleavage of probe by 5‘ nuclease activity of Taq polymerase  FRET disabled, generation of reporter signal 5’ Nuclease Assay using TaqMan ® probes

5’ 3’ 5’ 3’ 5’ Polymerization completed One reporter signal for each new DNA copy Q R 5’ Nuclease Assay using TaqMan ® probes

TAMRA™ Dye Excitation Emission Quenching Process Reporter emission spectra and quencher excitation spectra overlap FRET Fluorescence Resonance Energy Transfer FAM™ Dye

MGB R TaqMan ® MGB probes R NFQ MGB: Minor Groove Binder  Stabilizes the last 5-6 bp on 3‘ end  Shorter and more specific probes for a given Tm MGB NFQ: Non-Fluorescent Quencher  Opens up another window for other reporter dyes

SYBR ® Green 1 dye binds to the minor groove of ds DNA Detects specific and unspecific products Signal Generation with SYBR ® Green 1 dye

When ?Post PCR Analysis How ? Gel or Real-Time PCR System Gels Real-Time PCR System Melting curve analysis Specificity check SYBR ® Green 1 dye

Chemistry Comparison TaqMan ® Probe SYBR ® Green 1 Dye Universal guidelines Check Primer dimer formation Spezifität Primer binding - PCR conditions Specificity Primer binding Probe hybridization PCR conditions Flexibility Multiplex SNP Detection +/- Application Optimization - Universal guidelines -

Sample 2 FAM™ dye Sample 1 FAM™ dye Passive Reference ROX™ Dye ROX™ dye normalizes for non-PCR related fluorescence variations ROX™ dye Fluorescence RnRn RnRn Rn =Rn = Reporter Passive Reference

Baseline ThresholdCt value Rn Cycles From Fluorescence to Results Step 1 Determination of Ct Value

Log copy number Ct values Ct‘s The well contains 400 target copies From Fluorescence to Results Step 2 „Comparison“ of Ct Values

Rapid Assay Development Guidelines Universal Master Mixes Universal Assay Conditions Primer and Probes (Assays) Universal Thermal Cycling Protocol  Follow design guidelines  Design with Primer Express ® Software  or use ready-to-use Genomic Assays

Universal Primer and Probe Concentrations Universal Assay Condition No optimization required  reduced assay development time Setup conditions Probe concentration: 200 nM (FAM™ / VIC ® dyes) Primer concentration:900 nM (each primer)

Universal Thermal Cycling Protocol Denaturation Activation of AmpliTaq Gold ® polymerase Annealing/ Extension

Universal Fast Thermal Cycling Protocol Applied Biosystems 7500 Fast Real-Time PCR System Applied Biosystems 7900 Fast Real-Time PCR System