Homogeneous DNA Analysis A Realtime PCR Past and

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

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

Research and diagnostic applications of real-time PCR • Research – Viral quantification – m. RNA profiling – micro. RNA – 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

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

First real-time PCR 1991 spectrofluorometer fiberoptic PCR tube in thermocycler “Fifty Years of Molecular Diagnostics” Clin Chem. 2005 Mar; 51(3): 661 -71 (C. Wittwer, ed. )

CCD camera approach 1991/92

Growth curves from CCD camera approach

Iconic?

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 (Light. Cyclers. TM) • Took until 2001 for Roche, IVD-qualified real-time PCR instrument to enter diagnositcs • For diagnostics, automated sample-prep was probably the greater improvement

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

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

Stop/start of thermocycling allows every reaction to keep up

Start/stop of thermocycling allows every reaction to keep up

Start/stop of thermocycling allows every reaction to keep up

Lack of start/stop synchronization makes quantitation between isothermal amplification reactions difficult 500000 5000 50 H 2 O 16 m. M Mg 2+ 0 7. 5 15 21 28 35 42 49 mins 56 500000 5000 50 H 2 O 10 m. M Mg 2+ 0 7 14 20 26 33 40 46 53 59 mins 500000 50000 6 m. M Mg 2+ 5000 50 H 2 O 0 7 14 20 26 33 40 46 53 59 mins

PCR efficiency estimation by target dilution 109 (101/slope - 1) x 100% = per-cycle efficiency 108 107 starting copy number 106 105 104 103 102 101 20 30 cycle number 40

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

Marching band making a left turn and maintaining spacing between columns

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

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, q. PCR 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

Achieving well-to-well uniformity - the Roche LC 480 Illumination uniformity Temperature uniformity

LC 480 ABI 7900 Amplicon Melt Analysis – 384 wells each (probe-less genotyping assay; Biotechniques. 2005 39: 885 -93)

LC 480 ABI 7900 Genotype Scatter Plots

Real-time PCR in IVD diagnostics

Expression profiling on microarrays vs. real-time PCR • Many interesting m. RNAs 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 m. RNAs testable with real-time PCR is limited

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

Having your cake and eating it too • A dense microarray of PCRs • Microfluidic partitioning of sample and master mix – Fluidigm – Bio. Trove

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

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

Influence of Bob Watson Folded-optics ABI 5700 prototype Roche LC 480 Touch-screen prototype - Circa 2001 ABI Step. One. Plus™ System

Real-time PCR on Mars! (http: //genetics. mgh. harvard. edu/Ruvkun. Web/projects/Life. On. Mars. html)

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