1. Homogeneous DNA Analysis
A. Real-time PCR: Past and Future
Russell Higuchi
Roche Molecular Systems
High resolution melting analysis for mutation scanning and genotyping
Carl Wittwer
University of Utah

2. Real-time PCR: Past and Future
R. Higuchi
ABRF meeting 2008
February 11, Salt Lake City

3. Research and diagnostic applications of real-time PCR
Viral quantification
mRNA profiling
microRNA
Microarray result validation
Genotyping
Copy number variation
Loss of heterozygosity
Tumor biology
Epigenetics
Developmental biology
Diagnostics
Viral load measurement
Blood screening
Sepsis
Hospital acquired infections
Oncology
STDs

4. Will “talk-story” “…sane people have variety when they talk-story” - Amy Tan
Some personal real-time PCR history
Why thermocycling is good for quantitative PCR
Notes on PCR efficiency from a target dilution series
Diagnostics
Tidbits

5. First real-time PCR 1991 “Fifty Years of Molecular Diagnostics”
PCR tube in thermocycler
spectrofluorometer
fiberoptic
“Fifty Years of Molecular Diagnostics”
Clin Chem Mar;51(3): (C.Wittwer, ed.)

6. CCD camera approach 1991/92

7. Growth curves from CCD camera approach

8. Iconic?

9. The break-up of Cetus (1992) and real-time PCR
It was an interesting time, where ABI was willing to work on a research instrument even before it merged with PE (who had the instrument IP rights)
First instrument depended on development of Taqman probes; dye alone (etbr, SYBR) deemed not specfic enough
For diagnostics Roche worked on an endpoint instrument (Amplicor) that has been very successful (“Don’t even tell my engineers about this (real-time PCR); it will only distract them”)
By aquiring Boehringer Manheim, Roche ended up partnering w/ Idaho Tech for research instruments (LightCyclersTM)
Took until 2001 for Roche, IVD-qualified real-time PCR instrument to enter diagnositcs
For diagnostics, automated sample-prep was probably the greater improvement

10. First commercial real-time PCR instruments
ABI 7700 – laser/fiberoptic-based
ABI 5700 – CCD camera-based
Idaho Technology LightCycler – capillary tubes

11. Why thermocycling is good for quantitative PCR
Exponential processes like PCR are not supposed to be easily reproducible
Small efficiency differences early on (due, say, to temperature differences) can be magnified into large differences between reactions by late cycles
Common misperception
The “stop/start” of temperature cycling synchronizes all reactions
Advantage of PCR over isothermal amplifications

12. Stop/start of thermocycling allows every reaction to keep up

13. Start/stop of thermocycling allows every reaction to keep up

14. Start/stop of thermocycling allows every reaction to keep up

15. Lack of start/stop synchronization makes quantitation between isothermal amplification reactions difficult
16mM
Mg2+
10mM
6mM
7.5 15 21 28 35 42 49 56
500000
50000
5000
500 50
H2O 7 14 20 26 33 40 46 53 59
mins
Note faster reaction and shallower slope with increasing Mg++.
Relative slope might be same
Issue in quantitation: if StDEV of threshold cross time constant, then loose resolution with fast reactions.
No information presented on specificity with increased Mg++

16. PCR efficiency estimation by target dilution40 30 20 10 1 2 3 4 5 6 7 8 9
cycle number
starting copy number
(101/slope - 1) x 100% = per-cycle efficiency

17. Slope (spacing between growth curves) of standard dilution curve is set by initial reaction efficiency

18. Marching band making a left turn and maintaining spacing between columns

19. Changing amplification efficiency “all at once” vs. “in phase”

20. Keeping PCRs in phase
Initial PCR efficiencies can be measured because PCRs stay in phase up to and even past plateau
To stay in phase, growth curve profiles change as a function of present copy number, not cycle number
Getting “out of phase” in a dilution series is indicative of something else happening – primer artifact, competitive PCR
“In theory, qPCR allows accurate and precise quantification of product during the exponential phase, where the amplification rate is similar across samples regardless of target amount.”
-CHI meeting blurb

21. Achieving well-to-well uniformity - the Roche LC480
Illumination uniformity
Temperature uniformity

22. LC480
ABI 7900
Amplicon Melt Analysis – 384 wells each (probe-less genotyping assay; Biotechniques :885-93)

23. LC480
ABI 7900
Genotype Scatter Plots

24. Real-time PCR in IVD diagnostics

25. Expression profiling on microarrays vs. real-time PCR
Many interesting mRNAs are below the level of detection by microarrays
The dynamic range of quantification by microarray hybridization is limited
Kinetic RT-PCR has a much broader dynamic range and much greater sensitivity
The number of mRNAs testable with real-time PCR is limited

26. M. Holland, J. Biol. Chem. 277:14363-66 (2002)
Microarray hybridization measurement of mRNA level in yeast vs kinetic RT-PCR measurement
Alpha 1 transcript
Quantification not possible
M. Holland, J. Biol. Chem. 277: (2002)

27. Having your cake and eating it too
A dense microarray of PCRs
Microfluidic partitioning of sample and master mix
Fluidigm
BioTrove

28. Perhaps another solution – next gen. sequencers
400,000 reads per run & 300 nt per read
10 nt per SAGE tag
1.2 x 106 SAGE tags/run
Roche/454 GSFLX

29. Genome-wide SNP association Illumina platform >500K SNPs
From Willer et al. Nature Genetics, online Jan 2008
Genome-wide SNP association Illumina platform >500K SNPs

30. Influence of Bob Watson
Folded-optics
Roche LC480
ABI 5700 prototype
Touch-screen prototype
- Circa 2001
ABI StepOnePlus™ System

31. Real-time PCR on Mars! (

32. Acknowledgements
Thanks to the many scientists and engineers in industry and academia who over the last 15 years have made real-time PCR the widely used tool that it is