Diagn
ostics
Advances in Quantitative PCRfor Research and Diagnostic Applications
Thomas W. Myers, Ph.D.Program in Core ResearchRoche Molecular Systems
qPCR 20073rd International qPCR Symposium &
Industrial Exhibition & WorkshopTechnical University of Munich
Freising-Weihenstephan, GermanyMarch 26, 2007
PCR with Taq DNA Polymerase
Diverse Applications for PCR
Many Tools in the PCR Toolbox!
infectious diseases
blood bank screening
genetics and cancer
time
MedicalOpportunity
Nucleic Acid Testing in Diagnostics
technology development*
Program in Core Research
• Perform basic & applied research (and a little development!) to create technology and advance our knowledge and products in the area of PCR amplification.
• Facilitate the implementation of these new technologies throughout RMS for the IVD business, Roche Applied Science for our research reagent business, & the rest of Roche (Diagnostics, Pharma, RCMG, etc.).
PCR Research Areas
• New Proteins– Biological Diversity– Molecular Design
• Modeling• Cloning• Expression• Fermentation• Purification• Characterization
• New Technologies– Instrumentation
• KTC (2 X 384 well)– Chemistry
• Reaction Conditions• Oligonucleotides
– Primers– Probes– Aptamers
• Modified Enzymes– “Gold” Technology
– Processes• RT/PCR• Sequencing• TaqMan
PCR ComponentsBuffer Salt - specificity, sensitivity, and processivity
Metal Buffer - relaxation of [metal cation] toleranceMetal Cation - specificity, sensitivity, substrate
and template recognition
dNTPs Anti-contaminationDecreased stability of PCR productIncreased stability of PCR product
Primers & Probes Sensitivity and specificityAptamers Sensitivity, specificity, reagent stability
Enzymes ThermostabilityTemplatesProcessivityFidelityNucleotide analog
Thermal Parameters Sensitivity and specificity
or rATP
Screening for DNA Polymerases with Novel PropertiesScreening for DNA Polymerases with Novel Properties
in vivo screens
in vitro screens
DNA Polymerase Domains3'-Nucleolytic “Domain”(3'-exonuclease)•Fidelity•Primer degradation•Primer mismatch extension
Synthetic “Domain”•DNA polymerase•Reverse transcriptase•Modified base incorporation
5'-Nucleolytic “Domain”(5'-SDSSE)•RNase H•Template degradation•Product degradation (plateau)•Detection
Fifty Years of Molecular (DNA/RNA) Diagnostics Clin Chem. 2005 Mar;51(3):661-71 (C.Wittwer, ed.)
PCR tube in thermocycler
spectrofluormeterfiberoptic
First Real-time PCR (1991)
CCD Camera Approach (1991/92)
Raw data
20000
30000
40000
50000
60000
70000
80000
90000
100000
0 10 20 30 40 50 60
fluor
esce
nce
10^510^410^310^210NegNegNeg
target copynumber
Normalized data
0.81.01.21.41.61.82.02.22.42.62.83.03.23.43.6
0 10 20 30 40 50 60
rela
tive
fluor
esce
nce 10^5
10^410^310^210NegNegNeg
threshold
target copynumber
Normalized growth curves from CCD camera approach
Growth Curves From CCD Camera Approach (1991/92)
PE-ABI 5700 - 1997
Integrated CCD Camera Approach (1995)
The First Quantitative PCR-based Diagnostic Platform
Improvements in Quantitative PCR by an Evolution of Thermalcyclers
The First Quantitative 5’-Nuclease Based Quantitative PCR
Probe Intact: Reporter (R) Fluorescence Emission Suppressed by Quencher (Q)
hν
Primer Probe
RQ
Probe Cleavage by 5’-Nuclease Activity of Enzyme
QR
Probe Cleaved: Fluorescence Emission Detected
hνQ
R
R = Reporter Q = Quencher
Real-time PCR: 5’-Nuclease Technology with Z05 DNA Polymerase
Diagnostic 5’-Nuclease Based Quantitative Real-time PCR Platforms
Burnt Fingers - The First HotStartMethod!
Carryover Contamination Control and UNG-mediated HotStart
Hydrolysis of uracil-glycosidic bonds at U-DNA sites and cleavage with alkaline pH and heat.
Wax Barriers - The Second HotStartMethod!
Antibodies Directed Against DNA Polymerases – The Third HotStart Method!
Recombinant Taq DNA polymerase complexed with a proprietary antibody that blocks DNA polymerase activity at ambient temperatures. Activity of the DNA polymerase is restored after the denaturation step in PCR.
General Issues
• The use of an antibody increases the likely-hood of contaminating proteins (RNases, DNases, proteases, etc.) Antibodies are thermolabile and cannot typically be purified as extensively as the thermostable DNA polymerases.
• Stability, especially in a master mix formulation is unknown.
• Nonreversible – once the antibody is denatured, the DNA polymerase is active.
• Optimization issues – pH, two proteins for RT/PCR
• Cost? – especially for RT/PCR – additional RT, RNase inhibitor, etc.)
• Not suited for single enzyme RT/PCR due to activation requirements.
Various amounts of human genomic DNA were used for the amplification of a single 130-bp fragment from the tissue plasminogen activator (tPA) gene.
Chemically Modified Taq DNA Polymerase for HotStart – The Fourth HotStart Method!
Thermally Activated DNA Polymerases
Improves Specificity, Sensitivity, & Product Yield
Modified DNA Polymerases for PCR
KTC PCR with Modified Enzymes
0
20000
40000
60000
80000
100000
120000
140000
0 20 40 60 80 100
cycle
sign
al
TaqGoldDMMDPM
dimethylmaleic anhydride cis-aconitic anhydridecitraconic anhydride diphenylmaleic anhydride
2X 384 Kinetic Thermal Cycling System- 768 PCRs at the same time!
ΔZ05 Gold DNA Polymerase for SNP Detection PCR
Watson et. al., (2004) "Increased sample capacity for genotyping and expression profiling by kinetic polymerase chain reaction" Anal. Biochem., 329:58-67.
Allele-specific Primers for HFE SNP Detection
Modified Primers – The Fifth Method of HotStart!
• Specificity of amplification is fundamental to the success of any PCR system, and is of paramount importance in multiplex amplifications.
• Specificity of amplification drives sensitivity.
• Future RMD assays that require extreme sensitivity are being developed with modified primers.
• These primers are chemically modified at their 3’-ends, the business end of primers.
• These modifications suppress the amplification of poorly matchedtemplates, and the amplification of primer-dimer artifacts.
• This has the effect of improving assay sensitivity and robustness.
OOH
O
O
O
O
O
O
N
N N
N
NH
O
OH
O
O
N
NO
NH2
HN
NO
O
N
HN N
N
O
OHO
O
O
O
O
O
O
N
NN
N
HN
O
OH
O
O
N
N O
NH2
NH
N O
O
N
NHN
N
O
BENZYLATED PRIMERS
PO
O
PO
O
PO
O
NH2
5'
3'
5'
3'
Structure of Benzyl dA Terminated Primers
HCV RNA in serum @ 20,000 copies/mlummodified vs. 3' alkylated primers
0.9
1.4
1.9
2.4
2.9
3.4
3.9
4.4
4.9
0 10 20 30 40 50 60CYCLES
RELA
TIVE
FLU
ORE
SCEN
CE
unmodif ied 42.6
unmodif ied 49.4
unmodif ied 48.9
3' alkylated 37.1
3' alkylated 36.5
3' alkylated 37.2
Baseline cycles: 10-25
VPN9747.1-1.0-0100-3
Effect of Alkylated Primers on Real-time PCR
Progression of RT/PCR Assays
MMLV RT/Taq rTth/rTth rTth
25
27
29
31
33
35
37
39
41
43
45
E A C D F G H I K L M N P Q R S T V W Y
DNA Polymerase
CT V
alue
s
Mg2+-Activated RT/PCR
Mg2+-Activated RT/PCRInstead of Mn2+ only
ZO5 ES112 ES113 ZO5 ES112 ES113 ZO5 ES112 ES113 ZO5 ES112 ES113
Mg2+ Activation Mn2+ Activation Mg2+ Activation Mn2+ Activation
104 copies of HIV transcript per reaction Negative reactions
ES112 Mutation Frequency vs. Other Kits
Point Mutation Frequency
1.0
1.9
3.1
2.32.7
0.0
1.0
2.0
3.0
4.0
5.0
AccuRT, lowMg++
AccuRT, kitcond.
Tth 1-Step Tth 2-step C.Therm Q 1-Step
mut
atio
ns/ n
ucle
otid
e pe
r 35
cycl
es (x
10e-
3)
16.0
~~
15.5
Point Mutation Frequency
1.0
1.9
3.1
2.32.7
0.0
1.0
2.0
3.0
4.0
5.0
AccuRT, lowMg++
AccuRT, kitcond.
Tth 1-Step Tth 2-step C.Therm Q 1-Step
mut
atio
ns/ n
ucle
otid
e pe
r 35
cycl
es (x
10e-
3)
16.0
~~
15.5
ES112 ES112
Aptamer for Z05 DNA Polymerase and ES112 – The Sixth HotStart Method!
ES112
ES112
Inhibition of DNA Polymerases by Aptamer
0%
20%
40%
60%
80%
100%
NoAptamer
1µMAptamer
0.1µMAptamer
NoAptamer
1µMAptamer
0.1µMAptamer
Perc
ent E
nzym
e A
ctiv
ity
ES112 Enzyme Taq Enzyme
105 104 103 102 neg 105 104 103 102 neg
ES112 ± Aptamer Hot Start
•Reactions set up at room temperature
•Aptamer has been shown to improve sensitivity and specificity.
+ Aptamer - Aptamer
RT/PCR: Approaching 30 Min
RNA Templated Reactions
32 min run 10 ^5 c/rxn10 ^4 c/rxn
10 ^3 c/rxn
10 c/rxn10 ^2 c/rxn
No Template
*
RNA Templated Reactions
32 min run 10 ^5 c/rxn10 ^4 c/rxn
10 ^3 c/rxn
10 c/rxn10 ^2 c/rxn
No Template
*
• Five min RT incubation
• One out of two replicates detected at 10 c/reaction
70 °C Hold
95 °C, 1" —› 59 °C, 10", 50 cycles
95 °C 30“
65 °C 5‘
50 °C, 30“
RT/PCR Profile :
70 °C Hold
95 °C, 1" —› 59 °C, 10", 50 cycles
95 °C 30“
65 °C 5‘
50 °C, 30“
RT/PCR Profile :
or rATP
Screening for DNA Polymerases with Novel PropertiesScreening for DNA Polymerases with Novel Properties
in vivo screens
in vitro screens
rNTP/dNTP Incorporation Ratios for rNTP/dNTP Incorporation Ratios for TaqTaqand and TmaTma Mutant DNA PolymerasesMutant DNA Polymerases
0
0.5
1.0
1.5
2.0
rNT
P/dN
TP
G46DTaq
G46DE615G
Taq
F730YTma30
E678GTma30
N/D
N/D
E678GTma25
U/dTrG/dGrC/dCrA/dA
(DNA)n + dNTP (DNA)n+1 + PPiPOLYMERIZATION
PYROPHOSPHOROLYSIS
Under conditions of normal DNA synthesis polymerization is favored over pyrophosphorolysis
Pyrophosphorolysis* - The Seventh HotStart Method!
*Going One Step Backward Before Going Forward!
Pyrophosphorolysis-activated Polymerization (PAP)Background
• Non-extendable PCR primers are activated by pyrophosphorolysis (the reverse of polymerization).
• Pyrophosphorolysis is highly specific for perfectly matched primer-template, increasing the specificity of the amplification.
• The pyrophosphorolysis step must be repeated at every cycle, providing specificity at every cycle.
• Applications:– Hot Start– SNP genotyping– Rare allele detection
AC
AC
A
AT
AT
AT
AT
A
AT
AT
Once a mismatch is extended,it remains throughout PCR!
Traditional Mismatch Discrimination Assays (Terminal 3’-mismatch)
CX
G
CG
TA
TA
CX
GTA
Pyrophosphorolysis
PAP Primer Hot Start
TCXTCX
TCX
TCX blocked primer
• Blocked primers cannot be extended.
• Polymerases with “E678G” mutation can remove blocked nucleotide from the primer by pyrophosphorolysis.
PAP Mismatch Discrimination
AX
C
GC
G
GC
AX
C
AT
AT
A
AT
AT
GX
CAT
Pyrophosphorolysis
Must occur at each cycle!
• Template titration in the presence of 25 ng genomic DNA (background).
• Specificity and sensitivity of amplification greatly improved.
PAP Primer Hot Start
Amplification of Unblocked (-OH) or Blocked Primers Using K-ras Plasmid DNA
-0.10.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.8
0 5 10 15 20 25 30 35 40 45 50 55 60Cycle Number
Rel
ativ
e Fl
uore
scen
ce
RSYC32 AM W118 14.6 RSYC32 AM W118 14.9NTC AM W118 26.6 NTC AM W118 26.5RSYC32 ESS149 15.7 RSYC32 ESS149 15.5NTC ESS149 - NTC ESS149 -threshold
Unblocked
Blocked
Unblocked Negative
Blocked Negative!
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0C
T
Unblocked 5.6 7.1 7.8 7.9 7.8 7.8 7.6
Blocked 7.5 10.7 16.8 20.2 23.9 36.3 30.1
10e6c 10e5c 10e4c 10e3c 10e2c 10e1c NTC
• Amplifications of mutant K-Ras in a background of 106 copies of wild-type K-Ras plasmid template.
• Comparison of amplifications with blocked vs. unblocked primers.
• Four orders of discrimination achieved: 100 copies mutant target in 106 copies background.
Amplification of Mutant Template in Wild-type Background
1318
2328
36
54
0
10
20
30
40
50
60
3 x 10e4 +10e6 w t
3 x 10e3 +10e6 w t
3 x 10e2 +10e6 w t
3 x 10e1 +10e6 w t
3 x 10e0 +10e6 w t
NTC + 10e6w t
Ct
• Increased NaPPi concentration improved specificity.
• Amplifications of mutant K-Ras in a background of 106 copies of wild-type K-Ras template.
• Three copy sensitivity in a background of 1,000,000 copies wild-type.
Amplification of Mutant Template in Wild-type Background
In Conclusion
The Keys to Optimal Quantitative PCR are:
Good Hardware,
Good Software,
and Great Chemistry -
with High Specificity and Sensitivity!
Program in Core ResearchKeith BauerOlga BudkerEllen Fiss Alvin LiMonica Lin Chris Long Tomás MartinezJohn Niemiec
Fred Reichert Nancy Schönbrunner Rachel ShahinianEd Smith Susanne Stoffel Shawn SukoBob Watson Sherry Zhang