multiplexing chem is tries
TRANSCRIPT
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Real-Time PCR
Optimization of Single and MultiplexReactions on the iCycler iQ System
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Real-Time PCR
Through the use of fluorescentmolecules, real-time PCR has the
ability to directly measure thereaction while amplification istaking place.
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How fluorescence works
Fluorescence is the emission of light at longerwavelengths in response to excitation of light atshorter wavelengths
Every fluorophore has a particular wavelength that is mostefficient at exciting it.
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Real-Time PCR
These fluorescent molecules canbe
Non-specific DNA binding dyes
SYBR® Green IEthidium Bromide
Specific Hybridization Probes
TaqManTM
molecular beacons
ScorpionsTM /AmplifluorTM
dual-oligo FRET pairs
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Intercalating dyes
d.NTPs
Thermal StableDNA Polymerase
Primers Add Master Mixand Sample
Denaturation
Annealing
Reaction Tube
Intercalation Dyes
Taq IDl
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Intercalating dyes
Extension
5’ 3’
Extension Continued Apply Excitation
Wavelength
5’ 3’
5’ 3’
Taq
Taq
3’
5’ 3’
Taq
Taq
Repeat
ID ID
ID IDID
ID ID ID
ID ID
l l l
ll
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Taqman
Once freed from the quencher the reporter fluorescence can be detected
indicatingamplification has occurred
•A hybridization probe is constructed with a
fluorescent reporter at one end to a nearby quencher.
•The reporter is excited but its emitted fluorescence
is captured by the nearby quencher.
No reporter fluorescence is detected.
Reporter
During theextension step, thepolymerase
encounters theTaqMan probe andchews off the end.
Fluorescent Tag
Target Sequence
Reporter
Quencher
TaqPolymerase
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Molecular Beacons
5’
5’
3’
3’
d.NTPs
Thermal StableDNA Polymerase
Primers Add Master Mixand Sample
Annealing
Reaction Tube
Denaturation
R Q
R Q
MolecularBeacon
Taq
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Molecular Beacons
5’ 3’
Extension Step5’ 3’
1. Strand DisplacementTaq
2. Polymerization
CompleteProbe Silent
l
R Q
Detection
R Q
5’ 3’
5’
Taq R Q
MolecularBeacon
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Primer based
l
3’
R Q
HeatIncorporation
R Ql
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FRET Probes
5’ 3’
l
D R
5’
Detection
Extension Step
5’ 3’
1. Strand DisplacementSystem Silent
2. PolymerizationComplete
System Silent
5’ 3’
5’
Taq
D
1-5 bases
Taq R
5’
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Reality vs.Theory
Cycle #
L o g
T a r g e
t D N A
Theoretical
Amplification is exponential, but theexponential increase is limited:
Real-Time PCR allows usto „see‟ the exponential
phase so we can
calculate how much we
A linear increasefollows exponential
Real Life
• Eventually plateaus
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End Point Measurements
96 replicates of the identical reaction canhave very different final amounts offluorescence
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Threshold Cycle, CT
The point at which the fluorescence risesappreciably above background
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Threshold Cycle, CT
of the same 96 replicates showsnearly identical values
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Threshold Cycle, CT
The least? Which one
has themost?
Correlates strongly with the starting copy
number
Is linear with the log of starting copy number
over at least 6 orders
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Threshold Cycle, CT, is areliable indicator of initial copy
number
C o p y N u m b e r v s. C t - St a n d a r d C u r v e
y = -3 .3 1 92 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 0 n )
C t
r = is a measure of how well the actual datafit to the standard curve.
= (explained variation/total variation)
The slope of the standard curve can bedirectly correlated to the efficiency of thereactions:
Efficiency () = [10(-1/slope) ] - 1
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Threshold Cycle, CT
Two-fold serial dilutions of human genomic DNA(gDNA) from 125 to 16,000 genomic equivalentswere assayed for b-actin.
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Good Lab Technique
Do not underestimate theimportance of using:
screwcap tubes
aerosol-barrier tips
hot-start polymerase
replicates
master mixes
And the golden rule:
Pipet only once into each well
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MultiplexingReactionsObjectives
Maximize Efficiency
Equalize Efficiency
Eliminate Cross-Reactivity
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Individual Reactions
1. Choose amplicon of 75-150 bp
Design and order primers
2. PCR dilution series with SYBRGreen I
3. Check for specificity
Use Melt Curve then confirm with gel
4. Design and order specific probe
5. Confirm probe behavior over wide
dynamic range (5 or 6 orders)
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Testing with SYBR
IL-1b plasmid with SYBR detection
5-fold dilution series: 10,000 to 16copies
No resolution below 2000copies
r = 0.957
= 153%
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Melt Curve: what is it?
Programming a slow change in temperatureto determine the temperature at which the
DNA separates into two strands - or “melts”apart
This temperature is referred to as the “Tm” or
melting temperature for that piece of DNA
Tm is a characteristic, not unlike the isoelectricpoint of proteins, and can be used to makeobservations about the sequence of DNAwithout directly sequencing it
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Melt CurveFunctionality
Melt Curve can be used for:
Identification of non-specific products
Characterization of molecular beacons
Mutation detection/allelic discrimination
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Identify Primer-
Dimers by Melt Curve
10,000 copies
No template control 400 copies
2,000 copies
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Primer-Dimers
IL-1b plasmid with SYBR detection collected at 82
Poor resolution below2000 copies
Poor replicates
= 93%
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Primer-Dimers:
No template controlsIL-1b
collected at 60 ºCIL-1b
collected at 82 ºC
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Primer-Dimers
Same primers with a specific Texas Red pr
Poor resolution below 200
Poor replicates
= 71%
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Primer-Dimers
After primer re-design to eliminate primer-dim
r = 0.999
= 91.3%
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Before Multiplexing
Objectives
Maximize individual efficiencies
Equalize individual efficiencies
Demonstrate lack of cross
reactivity
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Maximizing Individual
Reaction Efficiencies Target:
If templates differ by < 1000-fold >80% efficiency per reaction
If templates differ by > 1000-fold
(or an unknown amount)
>90% efficiency per reaction
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Adjusting Efficiency
Model Secondary Structure of
Amplicon at Anneal/Extend
temperature Move Primers to avoid areas of
secondary structure
Model Secondary Structure ofPrimers at Anneal/Extend
temperature
Redesign Primers
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Optimizing Primerlocation
Template with secondary structure
Cyclophilin
Target Amplicon
Forward Primer Reverse Primer
1
110
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Optimizing Primer location
= 66.3 %
Reverse primer A
Forward Primer
1 110
Reverse PrimerA
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Optimizing Primer location
= 95.8 %
Reverse primer B
Forward
Primer1
110
Reverse Primer
B
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Equalizing Individual
EfficienciesTarget:
If templates differ by < 1000-fold
10% differences okay
If templates differ by > 1000-fold
(or an unknown amount) 5% differences OK
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Unequal Efficiencies
a-tubulin r= 0.988=93.1%
GAPDH r= 0.997=101.8%
b-Actin r = 0.970=122.4%
cyclophilin r = 0.964=63.6%
TargetGAPDH
Cyclophilin
Tubulinb-Actin
ReporterHEXCy5
FAMTexas Red
QuencherDABCYL
Black Hole
DABCYLBlack Hole
10
15
20
25
30 35
40
45
50
1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08
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~ Equal Efficiencies
a-tubulin r= 1.000
=95.3%
GAPDH r= 1.000
=96.8%
b-Actin r = 0.998
=91.3%
cyclophilin r = 0.996=99.3%
T h r e s h o l d
C y c l e
10
15
20
25
30
35
40
2 3 4 5 6 7 8
Copy number (10n)
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Cross-reactivity
How we screen for it:
Primer Recognition (BLAST)
Why it may occur:
Competition for Enzyme
Competition for dNTPs
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Multiplexing GenomicTargets
Cy5 - Factor VIII
single = 26.15 0.09multiplex = 25.96 0.10
FAM - a-Tubulin single = 24.13 0.08multiplex = 24.24 0.08
HEX - GAPDH single = 23.11 0.11multiplex = 22.98 0.14
Texas Red - IL1-b
single = 23.56 0.07multiplex = 23.62 0.11
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An Individual FAMReaction
= 101%
r = 0.997
103, 105 and 107 copies1.25 U Taq , 3 mM MgCl2, 0.8 mM dNTPs
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FAM in multiplex with TET
103, 105 and 107 copies1.25 U Taq , 3 mM MgCl2, 0.8 mM dNTPs
= 131%
r = 0.982
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Individual FAM Reaction
102 - 106 copies1.25 U Taq , 3 mM MgCl2, 0.8 mM dNTPs
= 97.6%
r = 0.998
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FAM in multiplex with TET
102 - 106 copies of each template3.5 U Taq , 5 mM MgCl2, 1.8 mM dNTPs
= 95.8%
r = 0.999
Eff f Addi i l
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Effect of AdditionalEnzyme, MgCl2 and
dNTPs
Single Reaction
Multiplex Reaction
Single Reaction
Multiplex Reaction
nothing extra added: additional reagents:
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Reaction Conditions
Taq enzyme1.25 U/50 ml rxn
dNTPs G
G
A
T
C
A
T
C
G
T
C
A
800 mM
3.0 mM MgCl2 Mg+2
3.5 U/50 ml rxn
1800 mM
5.0 mM
300 nM 150-400 nMprimers
Single Reaction Multiplex Reaction
200 -500 nM 200-500 nMprobes
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Our favorite probes
Are quenched with darkquenchers:
Black Hole Quenchers
DABCYL
This helps us obtain a high signal-to-noise ratio
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Choosing a Quencher
Quenchers:
BHQ-3
BHQ-2
BHQ-1
QSY-7
TAMRA
Eclipse
DABCYL
Fluorophores:
FAMTET, VIC
HEX
JOE
CY3TAMRA
CY3.5, Redmond RedTexas Red, ROX
CY5 LC640
CY5.5 LC705
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Texas Red, ROX BHQ-2BHQ-3DABCYLEclipse
CY3.5, Redmond Red BHQ-2DABCYLEclipse
Choosing a Quencher
Quenchers:Fluorophores:
TAMRA
BHQ-1
QSY-7Eclipse
DABCYLFAMTET, VIC
BHQ-3BHQ-2CY5
CY5.5, LC705
LC640 BHQ-3
HEX
JOE
BHQ-2
BHQ-1
QSY-7
TAMRAEclipse
DABCYL
CY3TAMRA
BHQ-2
QSY-7Eclipse
DABCYL
4 C l M l i l
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4-Color MultiplexReaction
Texas Red - b-actin
r = 0.999
Cy5 - a-tubulin
r = 0.999
4 C l M lti l
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4-Color MultiplexReaction
HEX - GAPDH
r = 1.000
FAM - cyclophilin
r = 0.999
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Final Multiplex ConditionsThe final concentration of each component in the
50 ml reaction (for genomic template) was asfollows:
FAM - a-tubulin: 300 nM probe, 400 nM primers
HEX - GAPDH: 200 nM probe, 300 nM primers Texas Red - IL1-b: 150 nM probe, 150 nM primers Cy5 - Factor VIII: 300 nM probe, 400 nM primers
3.25 Units Platinum
Taq Polymerase, 5 mM MgCl2and 1.8 mM dNTPs
PCR conditions were 3 minutes at 95 oC, followedby 50 cycles of 10 seconds at 95oC, 60 seconds at
55 oC.
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Large Concentration
Differences
105 - 102 copies
of GAPDHalone
105 - 102 copiesof GAPDH with109 copies of a-tubulin
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Large Concentration
Differences
109 - 106 copies
of GAPDHalone
109 - 106 copiesof GAPDH with109 copies of a-tubulin
Large
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LargeConcentration
DifferencesGAPDH
Starting quantityMean Ct S.D. Mean Ct S.D.
109 11.32 0.10 11.32 0.14108 14.51 0.06 14.41 0.14107 17.89 0.11 17.70 0.18
10
6
21.03
0.06 20.99
0.18105 24.21 0.10 24.25 0.19104 27.65 0.24 27.54 0.11103 30.62 0.10 30.46 0.15
102 32.99 0.14 32.91 0.23
alone In multiplex
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Buyer beware!
Different dNTP Vendors
FAM dye layer of 2-color experiment
Two different dNTP sources
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Buyer beware!
Different Probe Vendors
Identical HEX probes by two differentvendors
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Buyer beware!
Probe lot-to-lot Variation
4 different lots of the same probe from thesame vendor
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Buyer beware!
Effect of Different Quenchers
Identical HEX probes made by the samevendor with two different quenchers:
Black Hole 2 and TAMRA
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Remember...
Real-Time PCR is not‘cookbook chemistry’ -a real-time instrument
will not optimize yourexperiments for you However, once you do
optimize yourreactions, you will getreproducible, accurateresults