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

Présentation au sujet: "The basics of real-time PCR Introduction Dr BERRAZEG Meryem MCA à l’Université Oran1 Chercheur associée à l’Institut Pasteur d’Algérie."— Transcription de la présentation:

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

2 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

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

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

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

6 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)

7 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

8 3’ 5’ 3’ 5’ R Q p R Q Displacement of probe by 5‘ nuclease activity of polymerase 5’ Nuclease Assay using TaqMan ® probes

9 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

10 5’ 3’ 5’ 3’ 5’ Polymerization completed One reporter signal for each new DNA copy Q R 5’ Nuclease Assay using TaqMan ® probes

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

12 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

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

14 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

15 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 -

16 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

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

18 Log copy number Ct values Ct‘s 01234567 The well contains 400 target copies From Fluorescence to Results Step 2 „Comparison“ of Ct Values

19 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

20 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)

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

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