Fundamentals of Real Time PCR
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1 Fundamentals of Real Time PCR Mohamed Abdel Fattah Senior Technical Specialist Scientific Support - Molecular Biology Dept. AnalysisAB Co. Mobile: analysis@analysis-ab.com
2 What is that DNA? Cytosine (C) Adenine (A) Thymine (T) Guanine (G) Guanine (G) Thymine (T) Adenine (A) Cytosine (C) Deoxyribose (Sugar molecule) Phosphoric Acid (Phosphate molecule)
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4 PCR As Exponential Amplification 25 cycles 2 25 copies (33,554,432)
5 PCR In Theoretical Aspect N = N 0 x 2 n N: number of amplified molecules N 0: initial number of molecules n: number of amplification cycles
6 PCR In Graphical Aspect
7 Limitations of PCR Efficiency, Plateau Effect The Plateau Effect is caused by: Enzyme instability. Reagents consumption. Product inhibition. e.g. pyrophosphate accumulation. Competition of re-annealing amplification product with primers.
8 Limitations of PCR Efficiency
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13 From PCR to Real-Time PCR Real-Time PCR turns the question HOW MUCH? Into the question WHEN?
14 From PCR to Real-Time PCR Plateau End Point Log-Linear Real-Time Baseline
15 Towards Real-Time PCR 1 st st Point Detection of Specific PCR Product by utilizing the to 3 Exonuclease Activity of Thermus Aquaticus DNA Polymerase
16 Towards Real-Time PCR 2nd Point Fluoresceins Rhodamines FRET What is FRET?, Flourochromes
17 Towards Real-Time PCR What is FRET?, Flourochromes Reporter (Fluoresceine) Energy Transfer Quencher (Rhodamine) Fam Vic Tet Phosphate Group 3 Excitation
18 Towards Real-Time PCR What is FRET?, Flourochromes Reporter (Fluoresceine) Quencher (Rhodamine) Fam Vic Tet Cleaved Probe Phosphate Group 3 Excitation
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20 Real-Time PCR Chemistries TaqMan Probe-Based Chemistery 3 Forward Primer TaqMan Probe R Q p Reverse Primer 3
21 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization 3 forward primer R Q 3 R----- Reporter Q----- Quencher reverse primer
22 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization 3 forward primer R Q 3 R----- Reporter Q----- Quencher reverse primer
23 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization 3 forward primer R Q 3 R----- Reporter Q----- Quencher reverse primer
24 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization 3 forward primer R Q 3 R----- Reporter Q----- Quencher reverse primer
25 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization 3 forward primer R Q 3 R----- Reporter Q----- Quencher reverse primer
26 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization 3 forward primer R Q 3 R----- Reporter Q----- Quencher reverse primer
27 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization 3 forward primer R Q 3 R----- Reporter Q----- Quencher reverse primer
28 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization 3 forward primer R Q 3 R----- Reporter Q----- Quencher reverse primer
29 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization. Strand Displacement R Q 3 forward primer 3 R----- Reporter Q----- Quencher reverse primer
30 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization. Cleavage 3 forward primer R Q 3 R----- Reporter Q----- Quencher
31 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization. Cleavage 3 forward primer R Q 3 R----- Reporter Q----- Quencher
32 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization. Cleavage R Q 3 forward primer 3 R----- Reporter Q----- Quencher
33 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization. Cleavage R Q 3 forward primer 3 R----- Reporter Q----- Quencher
34 TaqMan Probe-Based Chemistery Denaturation Annealing Polymerization. Polymerization Completed R Q 3 forward primer 3 R----- Reporter Q----- Quencher
35 TaqMan Probe-Based Chemistery Denaturation Annealing R Polymerization. Polymerization Completed Q 3 forward primer 3 R----- Reporter Q----- Quencher
36 Real-Time PCR Principles Real-Time Amplification Plots Rn: Reporter Signal Cycle
37 SYBR green chemistry Double stranded DNA Binding Dye
38 SYBR Green detection Primer SYBR Green template Taq Polymerase
39 SYBR Green detection
40 SYBR Green detection
41 SYBR Green detection
42 SYBR Green detection
43 SYBR Green detection
44 CARRY-OVER PREVENTION WITH UNG
45 CARRY-OVER PREVENTION WITH UNG
46 CARRY-OVER PREVENTION WITH UNG
47 CARRY-OVER PREVENTION WITH UNG
48 CARRY-OVER PREVENTION WITH UNG
49 CARRY-OVER PREVENTION WITH UNG / AmpliTaq Gold TM ACTIVATION
50 CARRY-OVER PREVENTION WITH UNG / AmpliTaq Gold TM CYCLING
51 CARRY-OVER PREVENTION WITH UNG / AmpliTaq Gold TM CYCLING
52 CARRY-OVER PREVENTION WITH UNG / AmpliTaq Gold TM CYCLING
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73 please wait for Q&A
74 Mohamed Abdel Fattah Senior Technical Specialist Scientific Support - Molecular Biology Dept. AnalysisAB Co. Mobile: analysis@analysis-ab.com
Module1TheBasicsofRealTimePCR Monday, March 19, 2007
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