Download Real-Time PCR Probe Design

Real Time PCR Basic Theory &
Experiment Design
Summary
•Real Time Basic Theory
•Experiment Design & optimization
The PCR Process
Denaturation
Primer Annealing
Elongation
3’
5’
5’
3’
Taq
3’
5’
5’
3’
Taq
Repeat
In theory, product accumulation is proportional to 2n,
where n is the number of amplification cycle repeats
Theory vs. Reality
Log Target DNA
Theoretical


Real
Life

Exponential increase is
limited
Linear increase follows
exponential
Eventually plateaus
Real Time PCR utilizes fluorescence detection
technology to allow us to monitor
a reaction while it is occurring.
How is quantitative data collected?
Quantitative
information comes
from monitoring the
early stages of
amplification
Log Target DNA
Theoretical
Detector
Real Life
Cycle #
Real-Time PCR Process
In real-time PCR reactions, fluorescent
molecules are used to monitor the reaction
while amplification is taking place.
Detection Chemistries
(检测中的化学荧光染料)
Real-Time PCR Detection
•These fluorescent molecules can be
–Non-specific DNA binding dyes
• SYBR® Green I
–Specific Hybridization Probes/Primers
• TaqMan™
• Molecular Beacons
• Dual-oligo FRET pairs
• Scorpions™/Amplifluor™ /LUX™
DNA Binding Dyes



Intercalating Dyes are inexpensive
compared to hybridization probes.
A dye based strategy allows one to get a
general confirmation of amplification.
SYBR Green, is a more sensitive
intercalating dye

SYBR Green I fluoresces 1000 times more
brightly when bound to dsDNA
DNA Binding Dyes
5’
3’
5’
3’
d.NTPs
Primers
Add iQ SYBR™
Green Supermix,
Primers & Sample
SYBR Green I
Thermal Stable
DNA Polymerase
Denaturation
l
Taq
SG
Annealing
DNA Binding Dyes
3’
5’
5’
3’
Extension
3’
SG
Extension Continued
5’
SG
Taq
l
SG
3’
l
SG
SG
5’
SG
l
l
Apply Excitation
Wavelength
SG
Taq
3’
Taq
SG
SG
SG
Taq5’
3’
l
Repeat
DNA Binding Dyes
Advantages
Inexpensive compared to hybridization probes
No additional design work than the primer used for PCR
reaction
Disadvantages
Not template specific, will bind ALL double stranded DNA
inducing primer-dimer and unspecific amplicon formation
Multiplex assays not possible
DNA Binding Dyes
Typical “first step” experiment:
• Evaluate Primer Specificity
• Using Melt Curve Analysis
• Evaluate Primer Pair Efficiencies
By running serial dilutions of template as standards
• Identify Sub-Optimal aspects of assay
Optimize further with thermal gradient, etc.
TaqMan®
5’
3’
5’
3’
d.NTPs
Primers
Thermal Stable
DNA Polymerase
R
5’
Probe
Q
3’
Denaturation
Add iQ Supermix,
Hybridization Probe
and Sample
Taq
l
R
5’
Annealing
Q
3’
TaqMan®
Q
R
5’
3’
R
Q
Primer Extension
R
Taq
5’
3’
R
Q
Cleavage
Taq
5’
3’
5’
R
Taq
Polymerization
5’
3’
5’
l
R
Taq
Detection
5’
5’
3’
TaqMan Probes
Advantages
•Generates a robust cumulative fluorescence signal
•Simple to design and synthesize compared to other
hybridization probes (i.e. beacons)
•Ideal approach for multiplex assays
•SNP (Single Nucleotide Polymorphism) assay possible
Disadvantages
•More expensive than DNA binding dyes
Commonly Used Fluorescent Probes
78%
TaqMan probes
19%
Molecular Beacons
15%
FRET probes
LUX fluorogenic
primers
9%
9%
MGB Eclipse probes
Other
Scorpion probes
0%
3%
2%
10%
Detection Chemistries
20%
30%
40%
50%
60%
70%
80%
Which Chemistry To Use?
• Each method has advantages and disadvantages
•
Bio-Rad Real-Time Instrumentation is equipped to handle
all chemistries (CFX 96 is optimized for FRET )
•
One method may be more appropriate for an application
over another
Dye/Quencher selection
•Select dyes with excitation/emission maxima compatible with the
excitation/detection ranges of the instrument.
•Select the appropriate quencher for each dye
•Select non-fluorescent quenchers (e.g. BHQs, Dabcyl) instead of
TAMRA
•Preferentially select dyes with good performance (usually indicated
by high extinction coefficients and quantum yields)
•When multiplexing, strive for minimal spectral overlap between dyes
•Label your least abundant target with the best performing dye
(usually FAM)
What is Threshold Cycle (CT)?
The point at which the fluorescence rises
appreciably above background
Threshold Cycle, CT
•
Correlates strongly with the starting copy number
• Is linear with the log of starting copy number
Which one
The least?
has the
most?
Threshold Cycle, CT
Of the same 96 replicates shows nearly identical values
End-point vs. Real-Time
Lockey et al. (1998) Biotechniques 24:744-6
Threshold Cycle, CT, is a reliable
indicator of initial copy number
Copy Number vs. Ct - Standard Curve
40
y = -3.3192x + 39.772
R2 = 0.9967
35
30
Ct
25
r = is a measure of how well the actual data fit to the
standard curve.
20
= (explained variation/total variation)
15
The slope of the standard curve can be directly
correlated to the efficiency of the reactions:
10
5
Efficiency () = [10(-1/slope) ] - 1
0
0
1
2
3
4
5
6
7
Log of copy number (10 n)
8
9
10
11
Standard Curve
Real time PCR
108
T
106
104
102
Real time PCR
105 copies/well
Real-Time Assay Options
•As with all real-time qPCR, analysis options include
–Absolute Quantitation
–Relative Quantitation
• Using Standard Curve
• Using Algorithm (e.g. 2-DDCt )
•Most gene expression analyses are interested in relative expression
(i.e. comparing expression in one sample to another)
Relative Quantification Methods
•
Δ Ct method: (no reference gene)
•
Δ Δ Ct method: (reference gene,same efficiency)
•
Pfaffl modification: (reference gene and efficiency)
•
Vandesompele: (Multiple reference gene)
ΔCt
Tissue #1:
GOI
22
Tissue #2:
24
Δ Ct:
Fold induction = 22 = 4
24-22 = 2
(2-ΔΔCt)
Reference
Tissue #1:
21
GOI
22
Tissue #2:
24
20
Δ Ct #1:
Delta
Δ Ct #2:
2nd Delta Δ Δ Ct:
1st
Fold induction = 23 = 8
22-21 = 1
24-20 = 4
4-1 = 3
Problem with the Δ Δ CT
Ct
24
22
90%
Starting quantity
Ct
24
22
90%
100%
Starting quantity
Relative Quantification
(CT GOI (control) - CT GOI
(sample)
)
EGOI
Relative Expression =
(sample)
(CT REF (control) - CT REF (sample))
EREF
GOI = Gene of Interest
REF = Reference Gene
Pfaffl method
Primer set #1Reference
(From Standard curve)
Primer set #2 GOI
Tissue #1:
21
22
Tissue #2:
20
24
Efficiency:
90% = 1.9
Delta Ct:
20-21 = -1
100% = 2
24-22 = 2
deltaCt target (24-22 = 2)
2target
Fold induction =
deltaCt reference (20-21 = -1)
1.9reference
4
=
0.53
=
7.5
Δ Ct method: (no reference gene)
Fold induction : 4
Δ Δ Ct method: (reference gene,same efficiency)
Fold induction : 8
Pfaffl modification: (reference gene and efficiency)
Fold induction : 7.5
Use of reference
(normalizer) genes
•Used to control for differences between samples:
–Amount of starting material (RNA isolation)
–Efficiency of cDNA synthesis
–Overall transcriptional activity of tissues or cells
•Normalizes target gene so that it is expressed as number of copies per
copy of reference gene, rather than absolute copy number
An ideal reference gene
•Should not vary in expression in the tissues or cells under
investigation
•Should not vary in expression in response to experimental
treatment
•Must be validated for each assay
Commonly Used Reference Genes
Use of Multiple Reference Genes
Normalization Factor (NF)
About cDNA Synthesis
• Reverse transciption of mRNA to cDNA
• A number of priming options
–Random oligos (e.g. random hexamers)
• Primes all RNA (not just mRNA); non-specific
• Can overestimate mRNA copy numbers
• Creates a cDNA pool for multiple, subsequent experiments
• Allows analysis of multiple targets
–Oligo dTs
• Primes only mRNA; hybridizes to 3’ poly A tail
• Requires high quality, full length RNA
• More specificity than random oligos
• Creates a cDNA pool for multiple, subsequent experiments
• Allows analysis of multiple targets
–Gene-specific primers
• Most specific option; primes only RNA for the gene of interest
• Highest yield of specific product
• Requires separate priming reaction for each target
Reverse Transcriptases
MMLV (Moloney Murine Reverse Transcriptase):
• Lower activity temp; 37 C
• Lower intrinsic Rnase H activity
• Better for full length or longer cDNAs (making libraries)
AMV (Avian Myoblastosis Virus):
• More robust than MMLV
• Higher intrinsic Rnase H activity
• Higher activity temp 41 C
• Eliminates problems with RNA secondary structure
Tth (Thermus thermophilus):
• Both RT and DNA polymerase
• High activity temp, 68-74 C
• Significantly less efficient than either above
About HRM
Intercalation Chemistries
SYBR™ Green I is toxic to PCR,
so concentration used is very low
Unsaturated binding allows dye to
relocate as melting begins
Saturating dye technology for HRM -
SYBR® Green I
LCGreen™ I, EVA Green, Syto 9
Saturation dyes are less toxic,
so concentration used
can be high enough to allow all
sites to be saturated
Saturation eliminates potential
for dye relocation-ideal for HRM
LC Green™ I
HRM Profile
0.02deg
Data Acquisition
•Melting curves-normalized by selecting linear regions before and
after the melting transition
•Two regions defined-upper 100% double stranded and lower
single stranded baseline
Homoduplexes C or T
C
T
G
A
Homozygotes represented by a single base change
are differentiated by a difference in Tm melt.
Heteroduplex C>T
C
T
T
C
A
G
+
A
G
+
C
T
+
A
G
Heterozygotes form heteroduplexes, the heterozygote (blue)
trace is a mix of 4 duplexes
SOFTWARE: Normalised HRM data
Wild typ e s
(C a lle le )
Muta nts
(T a lle le )
He te ro zyg o te s
•ACTN3
(R577X) (C—T).
•10 replicates.
•40 cycle fast
(~34 min).
Real-Time PCR: Applications
Real-Time reaction monitoring provides information for
relative or quantitative
measurements of starting material.







Gene Expression Studies
Microarray Validation
Transgenic Analysis
GMO Testing
Viral/Bacterial Load Studies
Molecular Diagnostics
Allelic Discrimination
Experimental Design
How to Obtain Exceptional Real-Time PCR Results
What makes a good PCR reaction?
•Good Laboratory Practices
•Good Primer/Probe Design
•Good Amplicon Design
•High Quality Template
•Optimal Reagent Concentrations
•Good Instrument Performance
•Optimal Cycling Protocols
•Good Experimental Design – Controls, Replicates, Standards,
Testing Assumptions
What makes a good PCR reaction?
•Good Laboratory Practices
•Good Primer/Probe Design
•Good Amplicon Design
•High Quality Template
•Optimal Reagent Concentrations
•Good Instrument Performance
•Optimal Cycling Protocols
•Good Experimental Design – Controls, Replicates, Standards,
Testing Assumptions
General Laboratory
Practices
•Use clean bench (hood)
•Wear gloves
•Use screwcap tubes
•Use aerosol-resistant filter tips
•Use calibrated pipettes dedicated to PCR
•Use large volumes (>5ml)
•Use PCR-grade water
•Use a hot-start polymerase
•Use master mixes
•Pipette only once into each tube
Same Reagents, Different Hands
Cycle
Good Technique
Cycle
Poor Technique
Experiment Design
•Primer/probe design (Target/Reference)
•Validation by SYBR Green ,for primer/probe
efficiency / specificity/ reproducibility
•Standards preparation
What makes a good PCR reaction?
•Good Laboratory Practices
•Good Primer/Probe Design
•Good Amplicon Design
•High Quality Template
•Optimal Reagent Concentrations
•Good Instrument Performance
•Optimal Cycling Protocols
•Good Experimental Design – Controls, Replicates, Standards,
Testing Assumptions
Good Primer/Amplicon Design
•Maximizes reaction efficiency
•Maximizes specificity
•Maximizes yield/sensitivity
•Minimizes non-specific amplification
•Minimizes cross-reactivity in multiplex reactions
Accurate, Reproducible
Results
Real-Time PCR Primer Design
•Targets an amplicon length of 70 to 400bp
(70~150bp for probe-based assays,100~400bp for SYBR
Green)
•30 to 80% overall GC content(ideally 50-60%)
•Maintain a melting temperature (Tm) between 50 and 65ºC
•DTm (difference in Tm between FWD and REV primers)
should be less than 2oC
Real-Time PCR Primer Design
•Limit stretch of G ’s or C’s longer than 3 bases
•Limit secondary structure
•No stable interactions between primers (primer/dimer)
•Place C’s and G’s on ends of primers, but no more than
2 in the last 5 bases on 3’ end
•Always BLAST your primers
(http://www.ncbi.nlm.nih.gov/blast/)
Real-Time PCR Probe Design
•
•
•
•
•
Probe length should be between 18 and 30 bp (ideally 20 bp)
GC content of probe should be 30-80% (ideally 40-60%)
Tm of probe should be 10oC higher than primers
3’ end of primer and 5’ end of probe on same strand between
1-10 bp distance
Avoid secondary structure in the complementary region of the
probe
Real-Time PCR Probe Design
•
•
•
Avoid runs of > 3 identical nucleotides (especially G’s)
within the probe
Avoid G’s at the 5’ end of the probe sequence
Use oligo analysis tools to check probe for:
dimerization,secondary structure,cross reactivity with
primers
To verify good primer/amplicon design…
•Test primers with SYBR Green I
–Efficiency
By running serial dilutions of template as standards
–Reproducibility
By running replicates of some definite templates
–Specificity
Using Melt Curve Analysis
•Run agarose gel
–Specificity
To verify good primer/amplicon design…
•Run SYBR Green assay using validated positive control for template (e.g.
plasmid containing GOI)
•Include negative (no template) control
•Set up dilution series with 3-4 orders of magnitude
•Range of concentrations should include expected target concentration
•Run reactions in triplicate
•Run Melting Curve analysis
Internet resource
Check for existing qPCR primers
•Real Time PCR Primer Sets
•
http://www.realtimeprimers.org/
•PrimerBank
•
http://pga.mgh.harvard.edu/primerbank/index.html
•RT Primer DB
•
http://medgen.ugent.be/rtprimerdb/
•Quantitative PCR Primer Database (QPPD)
•
http://lpgws.nci.nih.gov/cgi-bin/PrimerViewer
Find Target Sequence
•Entrez Gene (NCBI)
•http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Gene
•Ensembl Genome Browser (Sanger Institute/European
Bioinformatics Institute)
•http://www.ensembl.org/
•Sequence Server (Dolan DNA Learning Center)
•http://www.dnalc.org/sequences/
• Design Primers/Probes
• Primer 3 (Whitehead Institute, MIT)
• http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
• GeneFisher (Bielefeld University)
• http://bibiserv.techfak.uni-bielefeld.de/cgibin/gf_submit?mode=STARTUP&qid=na&sample=dna
• Fast PCR (Biocenter, University of Helsinki)
• http://www.biocenter.helsinki.fi/bi/bare-1_html/oligos.htm
•
• PerlPrimer (Owen Marshall)
• http://perlprimer.sourceforge.net/
• Primer Design Assistant (Division of Biostatistics and Bioinformatics,
National Health Research Institutes)
• http://dbb.nhri.org.tw/primer/
How to Prepare Serial Dilutions
Stock Concentration of Plasmid = 108 copies/ml, 100ul
• Remove 10ul from 108 tube and add to the tube marked 107
• Vortex and remove 10ul from 107 tube and add to the tube
marked 106, etc.
Each tube
contains
90ml H20
108
107
106
105
104
103
102
101
How to Prepare Replicates
•Pipet enough template into one tube for the number of replicates you plan to
run
•Add enough master mix to the replicate tube
•Vortex and spin before aliquoting into the experimental plate or tubes
•Pipet only once into wells or tubes
Validation with SYBR Green
•Check reaction efficiency, E,
using standard curve
•Check reproducibility/linearity
using R value
•Check specificity using Melting Curve
•Check for contamination using NTC
Reaction Efficiency, E
•
If efficiency is perfect (i.e amount of PCR product doubles
each cycle), E=2
•
Calculate E using the slope of standard curve:
E=10-1/slope
•
To calculate efficiency as a percentage:
% Efficiency =(E - 1) x 100%
Reaction Efficiency, E
E=10-1/slope = 10-1/-3.394 = 1.971
% Efficiency =(1.971 - 1) x 100% = 97.1%
Causes of Efficiencies <90%
•Poor primer/amplicon design
–Non-specific amplification
–Primer dimers
–Secondary structure
•Non optimal cycling protocols
–Annealing temperature too high (gradient optimization)
–Extension time too short
•Non optimal reagent concentrations
–Primers, polymerase, dNTPs, MgCl2
•PCR Inhibitors
•Inaccurate pipettes
•Poor laboratory procedures
Maximizing Efficiency
Primer Location
Reverse primer A
Forward Primer
110
1
Reverse Primer
A
 = 66.3 %
Maximizing Efficiency
Reverse Primer
B
Reverse primer B
 = 95.8 %
2nd generation 85 bp amplicon
Forward
Primer
110
1
Causes of Efficiencies >110%
•Poor primer design
–Non-specific amplification
•Poor dilution series preparation
•Inaccurate pipettes
•Poor pipetting
•Dynamic range too large for limits of reaction
Assay Optimization by Real-Time
Gradient PCR
Gradient feature can be
used to optimize primer
and/or probe annealing
conditions!
Assay Optimization by Real-Time
Gradient PCR
Validation with SYBR Green
•Check reaction efficiency, E,
using standard curve
•Check reproducibility/linearity
using R value
•Check specificity using Melting Curve
•Check for contamination using NTC
Reproducibility/Linearity
The correlation coefficient, R (iQ software) are indicators of how well the
data points fit the standard curve
Reproducibility/Linearity
•R range from 0 to 1.
•If all data points lie perfectly on the line, value will be 1.
•R values are dependent on amount of variation and sample size.
•With 6 samples x 3 replicates:
–R should be 0.992 or higher
Validation with SYBR Green
•Check reaction efficiency, E,
using standard curve
•Check reproducibility/linearity
using R value
•Check specificity using Melting Curve
•Check for contamination using NTC
Specificity
•Single, well-defined peak in melting curve
•No amplification in negative control
•Verify specificity by running an
agarose gel
What makes a good PCR reaction?
•Good Laboratory Practices
•Good Primer/Probe Design
•Good Amplicon Design
•High Quality Template
•Optimal Reagent Concentrations
•Good Instrument Performance
•Optimal Cycling Protocols
•Good Experimental Design – Controls, Replicates, Standards,
Testing Assumptions
DNA Template
•The source of template affects the accessibility of the
target and must be considered during optimization
•It is important to optimize the reaction for the template
concentrations that will be used in your experiment
DNA Template
•Genomic DNA (Intact, high MW DNA)
–Cut with a restriction enzyme that does not cut region to be amplified
–Boil DNA stock for 10 minutes and place immediately on ice
•Plasmid DNA
–If there are problems with amplification, linearize the plasmid with a restriction
enzyme that does not cut within the target
•cDNA
–RNA must be free from genomic DNA contamination. Treat with RNAse-free
DNase prior to reverse transcription.
–Design primers at splice junctions to avoid genomic DNA amplification.
–Ensure optimal, reproducible efficiency of RT reaction
What makes a good PCR reaction?
•Good Laboratory Practices
•Good Primer/Probe Design
•Good Amplicon Design
•High Quality Template
•Optimal Reagent Concentrations
•Good Instrument Performance
•Optimal Cycling Protocols
•Good Experimental Design – Controls, Replicates, Standards,
Testing Assumptions
Optimal Reagent Concentrations
•Primer Concentrations:
–100~600nM(start with 300nM)
–50~300nM(start with 150nM) for SYBR Green
•Probe Concentrations:
–50~300nM(start with 200nM)
•Mg++ concentration:
–3.5~5.5mM(start with 5mM)
–1.5~3.5mM(start with 2.5mM) for SYBR Green
What makes a good PCR reaction?
•Good Laboratory Practices
•Good Primer/Probe Design
•Good Amplicon Design
•High Quality Template
•Optimal Reagent Concentrations
•Good Instrument Performance
•Optimal Cycling Protocols
•Good Experimental Design – Controls, Replicates, Standards,
Testing Assumptions
Controls
• Always include a positive control AND a negative control
• Controls, like samples, should be run in replicate
• Positive control
–sample with predictable results
–demonstrates that assay works
–prevents “false negatives” from PCR inhibition (especially in +/- pathogen detection
assays)
• Negative control
–usually no template control
–tests for contamination
–if doing RT real-time PCR, include no RT controls to check for contamination by
genomic DNAReplicates
• Real-time reactions should be run in replicate
• Ideally samples (and controls and standards) should be run in triplicate
• Acceptable variation between replicates depends on the mean and number of samples
• As a general rule, variation should be less than 0.5 Ct (ideally less than 0.25 Ct)
• To obtain good replicates:
–Prepare a master mix with all reaction components, including the sample.
–Use a hot start enzyme to prevent nonspecific amplification during preparation
–Pipette once per well
Standards
•Appropriate template for standards includes:
• Plasmid containing gene of interest
• PCR product
• Synthetic oligo
• Positive control sample
• For Gene Expression assays:
–cDNA from gene of interest
–Genomic cDNA
•Standards should be quantified independently (e.g. UV Spectrophotometry,
VersaFluor)
Standards
•A standard curve should include at least 5 different concentrations
•“Unknown” samples should fall within the limits of the standard curve; if they
are outside the minimum/maximum standard concentration, quantification may
not be accurate
•To quantify an unknown sample using a standard curve, the assumption is
that the reaction efficiency of the standards is the same as the sample.
•It is NOT appropriate to import a standard curve from a different assay
Multiplexing
Fluor selection
490/530
575/620 635/680
5000
Cy5
0
5000
FAM™
0
5000
Texas Red™
0
Multiplexing
Testing primers
dynamic thermal gradient
•Primers must amplify under the same
conditions.
•Test primers with gradient concurrently to
determine their actual annealing
temperatures.
•Adjust annealing temp of primers by
increasing and reducing their length.
Multiplexing
Gene A
Gene B
Multiplexing
Mix of two primer sets without template
Not an optimal condition for multiplexing!
Temperature Gradients
Gene A
Gene B
Concentration Differences
105 - 102 copies of
GAPDH alone
105 - 102 copies of
GAPDH with 109
copies of a-tubulin
Limiting Primers
105 - 102 copies
GAPDH alone
GAPDH primers = 250 nM
105 - 102 copies
GAPDH with 109
copies of a-tubulin
GAPDH primers = 250 nM
a-tubulin primers = 25 nM0
Additional Reagents
r = 0.979
slope = -4.542
h=66%
2X enzyme, 2X dNTP, & Mg+2
r = 0.999
slope = -3.361
Standard 1X conditions
h=98%
3X enzyme, 2X dNTP, & Mg +2
~ Equal Efficiencies
Multiplexing
Verifying Multiplex Reactions: Singleplex vs. Fourplex
β-actin
OAZ
17.0 ± 0.0
20.8 ± 0.1
17.3 ± 0.1
20.8 ± 0.2
Cycle
ODC
AZI
23.0 ± 0.2
22.2 ± 0.1
23.1 ± 0.2
22.2 ± 0.1