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