Real Time PCR and the iCycler iQ™
Real Time PCR Detection System
for Quantitative PCR
PCR FORMULA
Correlazione tra la quantità di amplificato e la quantita iniziale
Y = X (1 + E) n -1
Y = PCR amplificato DNA quantità X = Quantità diTarget DNA prima
della PCR
E = Efficienza di Amplificazione
n = numero di cicli
PCR Numbers
Cycle Relative Amount
1 2
2 4
3 8
4 16
5 32
6 64
7 128
8 256
9 512
10 1024
11 2048
12 4096
13 8192
14 16384
15 32768
16 65536
17 131072
18 262144
19 524288
20 1048576
21 2097152
22 4194304
23 8388608
24 16777216
25 33554432
26 67108864
27 134217728
28 268435456
29 536870912
30 1073741824 ….……..
La reazione
5’
5’
3’
3’
d.NTPs Thermal Stable DNA Polymerase Primers
5’
5’ 3’
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5’
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5’
5’ 3’
3’
Master Mix contenuto
Denaturare 95°C
5’
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5’ 3’
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Addizionare al tubo di reazione
Tecniche strumentali in Biochimica REAL TIME PCR
Estensione
5’ 3’
3’ 5’
5’ 3’
5’
3’
Estensione continua
5’ 3’
3’ 5’
5’
5’
Taq
Taq
3’
3’ 5’
Taq
Taq
5’
5’
Ripetere per n cicli
La reazione
La reazione
5’
3’
5’ 3’
5’
5’ 3’ 3’
5’ 5’
3’ 3’
5’
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5’ 3’
Cycle 2
4 Copie
Cycle 3 8 Copie
3’ 5’
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5’ 3’
3’ 5’
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5’ 3’
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5’ 3’
Tecniche strumentali in Biochimica REAL TIME PCR
n
La crescita
esponenziale è limitata
n
Effetto plateaus
Cycle #
Teorica
Reale
Log Target DNA
PCR Amplification Curve
Threshould
Lockey etal. (1998) Biotechniques 24:744-6
Comparazione dei risultati ad end point.
Con gel e Real Time
Tecniche strumentali in Biochimica REAL TIME PCR
Real Time Vs End Point
9,048 9,498 10,180 9,238 9,111 12,885 10,539
Relative Fluorescence Relative Fluorescence
Misurazione
Misurazione End Point? End Point?
Tecniche strumentali in Biochimica REAL TIME PCR
96 96 Repliche Repliche di di una una identica
identica reazione reazione , , mostrano mostrano differenti
differenti efficienze efficienze al al termine termine della
della reazione reazione
Threshold Cycle, C t , dei 96 replicati mostra un
identico valore
Tecniche strumentali in Biochimica REAL TIME PCR
0 0.4 0.8 1.2 1.6 2
0 10 20 21 30 40
Threshold Baseline Sample
C C T T
Threshold Cycle (C T )?
Threshold cycle, C t
Detection of 125 genomic equivalents from 250. Two-fold serial dilutions of human genomic DNA (gDNA) from 125 to 16,000 genomic equivalents were assayed for β-actin.
Tecniche strumentali in Biochimica REAL TIME PCR
C o p y N u m b e r v s. Ct - S t a n d a r d C u r v e
y = - 3 . 3 1 9 2 x + 3 9 . 7 7 2
R2 = 0 . 9 9 6 7
0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0
0 1 2 3 4 5 6 7 8 9 1 0 1 1
L o g o f c o p y n u m b e r ( 1 0n)
Ct
Threshold Cycle, C t , è un indicatore del numero di
copie iniziali
Intercalating Dyes
n
Intercalating Dyes are inexpensive compared to hybridization probes (depending on the reactions performed).
n
A dye based strategy allows one to take a “big picture”
- that is - get a general confirmation of amplification.
n
Russ Higuchi demonstrated the key principle of Real Time PCR using Ethidium Bromide -
u u EtBr EtBr fluoresces 25 times more brightly when bound fluoresces 25 times more brightly when bound to to dsDNA dsDNA
n
SYBR Green, a more sensitive intercalating dye is an even more attractive approach
u u SYBR Green fluoresces 200 times more brightly SYBR Green fluoresces 200 times more brightly
Tecniche strumentali in Biochimica REAL TIME PCR
5’
5’
3’
3’
d.NTPs Thermal Stable DNA Polymerase Primers
5’
5’ 3’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’ 3’
3’
Add Master Mix and Sample
Denaturation
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5’ 3’
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Annealing Reaction Tube
Intercalating Dyes
Intercalation Dyes
Taq ID
λ
Extension
5’ 3’
3’ 5’
5’ 3’
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Extension Continued Apply Excitation
Wavelength
5’ 3’
3’ 5’
5’
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Taq
Taq
3’
3’ 5’
Taq
Taq
5’
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Intercalating Dyes
ID ID
ID ID ID
ID ID ID
ID ID
λ λ λ
λ λ
Tecniche strumentali in Biochimica REAL TIME PCR
Hybridization Probes
n
Cleavage Based Assay - TaqMan Assays
n
Displaceable Probe Assays
u Molecular Beacons
u Dual oligo FRET probes
n
Probes incorporated directly into the primers
u Amplifluor
u Scorpions
Today Hybridization Probe Strategies
fall into three main categories:
Taqman Probe
R Q
R
Q After enzyme cleavage
Before enzyme cleavage
Tecniche strumentali in Biochimica REAL TIME PCR
TaqMan TM
5’
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3’
d.NTPs Thermal Stable DNA Polymerase Primers
5’
5’ 3’
3’
5’
3’
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3’
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3’
5’
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5’
5’ 3’
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Add Master Mix and Sample
Denaturation
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5’ 3’
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Annealing Reaction Tube
Taq
λ
5’ 3’
R Q
Probe
5’ 3’
R Q
5’ 3’
3’ 5’
TaqMan TM
5’
3’
Q R
Extension Step
5’ 3’Q Taq 3’
1. Strand Displacement
R
5’
5’ 3’
3’
Q Taq
R
5’
2. Cleavage
3. Polymerization Complete
5’ 3’
Taq Q R
3’ 5’
4. Detection
5’ 3’
3’
Taq Q R
5’
λ
R
Tecniche strumentali in Biochimica REAL TIME PCR
TaqMan TM
Eight log orders of dynamic range. Ten-fold serial dilutions from 101to 109copies of a beta- Actin containing plasmid were prepared and amplified in Real-Time using a Taqman designed probe.
Cleavable Hydrolysis Probe - Multiplex
Texas Red
IL-1 beta
Single
Multiplex Ct = 23.254
FAM
Factor 8
Single
Multiplex Ct = 23.672
Tecniche strumentali in Biochimica REAL TIME PCR
10 100 1000
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49
Tubulin GAPDH beta Actin Cyclophilin
Cleavable Hydrolysis Probe - Multiplex
4 Color/4 Probe
Hex FAM
Texas Red
Cy5
Log Relative Fluorescence
Target GAPDH Cyclophilin
Tubulin β-Actin
Reporter HEX FAMCy5 Texas Red
Quencher DABCYL Black Hole
DABCYL Black Hole Concentration
Threshold Cycle
Tubulin r2= 0.988 y = -3.50x + 39.67 GAPDH r2= 0.997 y = -3.28x + 37.30 β-Actin r2= 0.970 y = -2.88x + 33.89
Cyclophilin r2= 0.964 y = -4.68x + 49.74
10 15 20 25 30 35 40 45
1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 Concentration
Cycle
Tubulin GAPDH beta Actin Cyclophilin
Cleavable Hydrolysis
Probe - Multiplex
Tecniche strumentali in Biochimica REAL TIME PCR
Cleavable Hydrolysis Probe - Multiplex
Cycle
Log Relative Fluorescence
Texas Red FAM
Hex Cy5
10 100 1000
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49
Tubulin Cyclophilin beta Actin GAPDH
Cleavable Hydrolysis Probe - Multiplex
Concentration
Threshold Cycle
Tubulin r2= 0.994 y = -3.36x + 39.42
GAPDH r2= 0.995 y = -3.93x + 37.64 β-Actin r2= 0.995 y = -3.82x + 38.89 Cyclophilin r2 = 0.996 y = -3.65x + 38.43
10 15 20 25 30 35 40
1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06
Tubulin Cyclophilin beta Actin GAPDH
Tecniche strumentali in Biochimica REAL TIME PCR
Cleavable Hydrolysis Probe - Multiplex
FAM/Tubulin
Cleavable Hydrolysis Probe - Multiplex
HEX/ Cyclophilin
Tecniche strumentali in Biochimica REAL TIME PCR
Cleavable Hydrolysis Probe - Multiplex
Tex Red/ β-Actin
Cleavable Hydrolysis Probe - Multiplex
Cy5/GAPDH
Tecniche strumentali in Biochimica REAL TIME PCR
5’
5’
3’
3’
d.NTPs Thermal Stable DNA Polymerase Primers
5’
5’ 3’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’ 3’
3’
Add Master Mix and Sample
Annealing Reaction Tube
Molecular Beacons
Denaturation
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5’ 3’
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Taq
5’ 3’
R Q
R Q Molecular
Beacon
5’ 3’
5’
3’
Extension Step
5’ Taq 5’ 3’
1. Strand Displacement
2. Polymerization Complete
Probe Silent
λ
Molecular Beacons
3’ 5’
R Q
Detection
3’ 5’
R Q
5’ 3’
3’ 5’
Taq
R Q Molecular
Beacon
Tecniche strumentali in Biochimica REAL TIME PCR
Molecular Beacons
A serial dilution from 1x103 to 1x108plasmids copies of IL1-b (ATCC#581768) and beta-Actin were assayed with hIL1-b Taqman PDAR kit (Perkin-Elmer) and hb-Actin PDAR (Perkin-Elmer).
VIC Labeled
FAM Labeled
FRET Probes
5’ 3’
5’
3’
λ
3’
D R
5’
Detection
Extension Step
5’ 3’
1. Strand Displacement System Silent
2. Polymerization Complete
System Silent
5’ 3’
3’ 5’
Taq
3’
D
1-5 bases R
D R
Taq 5’
R
5’
Tecniche strumentali in Biochimica REAL TIME PCR
Primer Based FRET
λ
3’ 5’
R Q
Heat
Incorporation
R Q
λ
Scorpions Primer
λ
3’ 5’
R Q
3
This sequence is complementary to a sequence of the amplified
fragment
Taq
Taq
Tecniche strumentali in Biochimica REAL TIME PCR
Scorpions Primer after the Extention
λ
3’ 5’
R Q
3
The tail of the Scorpion is complementary to a sequence of the amplified
fragment
Amplifluor Primer
5’
Extension 2
3’ R 5’
Q
5’ 3’ R Q 5’
Annealing/Extension 1
3’
R Q 3’
5’ 3’
5’
λ
Detection
Tecniche strumentali in Biochimica REAL TIME PCR
