Diagnostics Advances in Quantitative PCR for Research and Diagnostic Applications Thomas W. Myers, Ph.D. Program in Core Research Roche Molecular Systems qPCR 2007 3 rd International qPCR Symposium & Industrial Exhibition & Workshop Technical University of Munich Freising-Weihenstephan, Germany March 26, 2007
49
Embed
Advances in Quantitative PCR - Gene-Quantification
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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
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!