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A brief History of PCR
Dr. Richard Molenkamp Medical Molecular Microbiologist
Visualisation of DNA I
Direct Staining Hybridisation
Visualisation of DNA II
Small amounts of signal:
Need amplification
PCR: Polymerase Chain Reaction
Principle of PCR
Conventional thermocycle profile:
Exponential amplification
Number of cycli on an agarose gel: 10 15 20 25 30 35 40
Early PCR
Addition of polymerase in each cycle
Breakthrough:
Thermostable (Taq) polymerase
Integrated Thermocyclers
Conventional PCR = End point analysis
no quantitative data
Real-time PCR
Sybrgreen reactions - Intercalating
fluorescence
DNA Target Sequence
Denaturation
Drawback: a-specifc product also fluoresent!
Sample block
Lens
Lens
Spectrograph
CCD camera
LASER
Dichrome
Mirror
MUX
Sybrgreen reactions – melting curve
analysis
Detection Formats (I)
Probe-based
- Taqman technology: specific double-dyed fluorescent hydrolysis probes
- FRET (Fluorescence Resonance Energy Transfer): hybridisation probes
- Partially double stranded, single-dyed, hybridisation probes
Novel Probe Technology: Partially Double-Stranded Linear DNA Probes
EmissionEmissionExcitationExcitation
R
Q
R
Q
RR
– Long target-specific probe with fluor
– Short quencher probe
– Fluorescence quenched when probes
are hybridized
– Long probe preferentially binds target
– Short quencher probe is dissociated
– Fluorescence is detected
Excitation / emission of fluorophores
Fluorescence
TAMRA quenching
Deep Dark/Black hole quenchers
5’ Nuclease Assay / Taqman assay
F = reporter FAM, VIC, Hex…
Q = quencher Black hole quencher (BHQ)….
Novel Probe Technology: Partially Double-Stranded Linear DNA Probes
EmissionEmissionExcitationExcitation
R
Q
R
Q
RR
– Long target-specific probe with fluor
– Short quencher probe
– Fluorescence quenched when probes
are hybridized
– Long probe preferentially binds target
– Short quencher probe is dissociated
– Fluorescence is detected
Partially double stranded linear DNA
probes
Probe free: MultiCode technology (I)
MultiCode Base Pair
(isoC:isoG)
Scott C etal, Nucl. Ac. Res. 2004 Iso G
Iso C
MultiCode PCR (II)
MultiCode technology
Ct / Cp / Cq
Calling
PCR positivity measured at Cycle threshold (Ct)-level
Number of cycles
0 10 20 30
Flu
ore
scen
ce
10x SD background
Threshold Cycle
Background fluorescence
Ct calling I
PCR positivity measured at Cycle threshold (Ct)-level
Number of cycles
0 10 20
30
Flu
ore
scen
ce
10x SD background
Threshold Cycle
Background fluorescence
Ct calling II
PCR positivity measured 2nd derivative max.
Number of cycles
0 10 20 30
Flu
ore
scen
ce
Crossing point
Background fluorescence
Calculates 2nd derivative and
determines its maximum.
CP: Where the rate of
increase of
fluorescence is greatest
Cp calling I
PCR positivity measured 2nd derivative max.
Number of cycles
0 10 20 30
Flu
ore
scen
ce
Cp calling II
Quantification
Quantification with real-time PCR
Principles of quantification –
real time PCR
Calculation of efficiency
Reverse transcription PCR
Mix of RT and PCR
enzyme:
M-MLV / Taqgold
Enzyme with RT and
DNA pol activity
rTth
Sequence variation
Influences design of PCR
PCR can be used to detect variation
HIV-1 group M sequence variation in Gag and Pol genes
Influence of mismatches on
hybridization temperature
tgggaggttctctccagcactagcagg
Length 27 nt
GC content 60%
Tm 69 ºC
tgggaggttctctccagcactagcagg
a t
Tm 62.6 ºC
tgggaggttctctccagcactagcagg
a t a
Tm 57.8 ºC
How to deal with sequence variation
Degenerate oligos GGTAYCCATGRTCAG
Dual target assay
Dual probe assay
IUB codes
R = A or G
Y = C or T
Dual target real time PCR
LTR Integrase
Genome HIV
5’- -3’
If there is a mutation in either of the primer/probe sites
the other PCR will ‘take over‘
Realtime PCR for detection of single
mutations
Conventional Sanger sequencing: ~25%
Sensitive methods:
LIPA/DNA microarray (hybridisation) 5-10%
Allele-specific PCR ~5%
Next generation sequencing (0.5% ?)
Quantitative real-time techniques: 1-10%
LNA/MGB probes (short high affinity probes)
Digital PCR
Probes used for detection of single
mutations (I)
Minor groove binding probes Locked nucleic acid probes
• Due to higher affinity binding shorter probes can be defined
• Taqman probes are 22-30 nt long; LNA/MGB probes 8-20 nt long
Hybridization temperature:
effect in MGB and LNA probes
GGAGG(+T)T(+C)TCT(+C)CAG(+C)A Length 17 nt
Tm 69 ºC
GGAGG(+T)T(+C)TCT(+C)CAG(+C)A
A Tm 59 ºC
tgggaggttctctccagcactagcagg
Length 27 nt
Tm 69 ºC
tgggaggttctctccagcactagcagg
a t Tm 62.6 ºC
tgggaggttctctccagcactagcagg
a t a Tm 57.8 ºC
Detection of oseltamivir resistant
influenza A/H1N1 H274Y
by real-time discrimination PCR using
LNA probes
NTC
+ control
+ controlNTC
+ control
+ control
Wild-type
cluster
Mutant
cluster
NA: 5’atcgaaaagggaaaggttactaaatcaatagagttaaatgcacccaattttCattatgaggaatgttcctgttacccagacactggc 3’
N1274Yfpr1
(30bp)
N1274Yrpr1
(24bp)
LNA:H274Y
T (mut)
LNA:H274H
(16bp)
Detection of lamivudine resistance
in HBV
Pas et al., Journal of Clinical Virology 32 (2005) 166–172
Effect of (enzyme) mastermix
on mismatch tolerance
Influence of mastermix on primer
bindingsite mismatch tolerance
> 50 different mutants Stadhouders et al., J. Mol. Diag., 2010
Primer bindingsite mismatch tolerance
Influence of mastermix on primer
bindingsite mismatch tolerance
MMLV / Taqgold
combination (ABI):
RT @ 48°C
Influence of mastermix on primer
bindingsite mismatch tolerance
rTth based mastermix:
RT@60°C
Digital PCR
Quant studio
Raindance technologies
Biorad QX200 droplet PCR Fluidigm
Principle of digital PCR (dPCR)
Limited dilution
0 0.368
1 0.368
2 0.184
3 0.061
4 0.015
5 0.003
>5 0.001
Poisson distribution
1. Manual dilution
2. Droplet PCR (ddPCR, Biorad)
3. Lab on a chip (Lifetechnologies)
4. Microfluidics (Fluidigm)
1
2
3
4
Applications of dPCR
Rare sequence / mutation detection (oncology, virology drug resistance)
Copy number quantification (standardisation controls)
Low level pathogen detection (in difficult samples)
Gene-expression (absolute quant. of (un)stimulated) gene expression)
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