Modeling DNA Amplification Technology for Process Optimization

1. Modeling DNA Amplification Technology for Process Optimization Emily Stone, Utah State University Faculty Presenter David Eyre, Idaho Technology Industry Representative Partially funded by a USU CURI grant, and Idaho Technology

2. What is PCR? • PCR: polymerase chain reaction. • Invitro method for the enzymatic synthesis of specific DNA sequences. • Invented in 1985 by Kary Mullis (who won a Nobel prize for it). • Has revolutionized molecular biology, in both basic research and applications in medicine (diagnosis), or pathogen detection.

3. Enter Taq Polymerase • Found in the hot pots in Yellowstone. • High temperature stable enzyme allows for more specific amplification of sequences, since sequences other than the target will not form “accidentally” by themselves in a high temperature environment. • Yellowstone borders on Idaho….

4. Birth of Idaho Technology- 1990 • Founded by Kirk Ririe, graduate of Utah State University and University of Utah, to utilize the facilities at his father’s agricultural parts manufacturing plant in Idaho Falls during seasonal lag times. • First product: Light Cycler, an automated PCR device that performed 10 times faster than equipment used at that time. Now have many products, including the Rapid Cycler:

5. Rapid Cycler • The RapidCycler is the first instrument engineered to match the speed of biochemical reactions. The instrument's rapid temperature cycling system is based on heat transfer by hot air to samples contained in microcapillary tubes or thin walled micorcentrifuge tubes. Heating and cooling the samples with blasts of high velocity air results in nearly instantaneous temperature transactions, ensures temperature uniformity and rapid heat exchange within the sample.

6. IT Products, cont.

7. More on the R.A.P.I.D. System • (Ruggedized Advanced Pathogen Identification Device) The R.A.P.I.D. System is lab proven, field-tested, and has been used in real world events since 1998. Designed through a collaboration with the US Air Force, it is the only real-time thermocycling system designed for portability and ruggedness. The R.A.P.I.D. System weighs under 50 lbs., can operate in extreme environmental conditions, and has the flexibility of 3-channel optics for whatever real-time dye chemistry you choose. Our freeze-dried BioReagents and Detector analysis software allow the R.A.P.I.D. to be run by any user.

8. a division of Idaho Technology that specializes in the design and synthesis of reagents, probes, primers and buffers to enhance your experience using the RapidCycler, IndyCycler, LightTyper and LightCycler Instruments and our field-hardened instrument, the R.A.P.I.D. System.

9. The Light Typer: SNP detection device SNP: single nucleotide polymorphisms • Perform faster, easier analysis with the only homogeneous SNP assay available today-the new LightTyper SNP Instrument. Avoid the extensive post-PCR cleanup, reagent addition, and extra handling steps required by other SNP-analysis systems. For more information, visit Roche Applied Science LightTyper System.

10. Steps in the PCR reaction • Heat to 94 degrees C to denature DNA strands. • Cool to 60 degrees C, primer molecules bind on to each end of a target sequence. • Heat again to 70 degrees C, Taq polymerase does its job and single nucleotides are added to the single strand sequence. • Repeat 1-3 30 to 40 times.

11. Schematic of DNA Amplification by PCR

12. Quantitative Real Time PCR Uses fluorescent probes to detect completed strands TaqMan TaqMan probes are labeled on the 5' end with a reporter dye and on the 3' end with a quencher. During PCR, the fluorogenic probe hybridizes between the forward and reverse primers. As Taq DNA polymerase extends, the TaqMan probe is cleaved, separating the reporter from the quencher generating a fluorescent signal proportional to the number of amplicons produced.

13. Light Cycler amplification curves Fluorescing probe molecules detect completed strands. Quantitative PCR Want to determine initial amount of DNA target present in sample

14. “Famous” Model for PCR:Simple Doubling C(n) = 2^n C(0) Copy number at cycle n is 2 to the n times the initial copy number.

15. Mapping back to initial copy number using the “Famous” Model C_T “standard curve” C_T is the time to cross a set threshold fluorescence value

16. Problems with Current PCR Reaction Modeling • Identifying the exponential region. • Locating a good fluorescence threshold for all curves. • Errors in initial copy number are amplified. • Saturation in reaction clearly occurs… • But is is logistic? Not exactly... (low copy number curves especially)

17. Improved Modeling to Optimize Reaction Design • Reaction conditions need to be finely tuned for any new sequence to be amplified, which can take weeks in the lab. • A good model could reduce this time by narrowing regions of parameter space to be explored in the lab, with the purpose of optimizing both yield and the specificity of the reaction.

18. Banff GIMMC Workshop, June 2003

19. Fitting the Logistic Model: Least Squares and Nonlinear Optimization Least Squares fit: Mapping initial fluorescence to initial copy number Using nonlinear optimization to fit the data to the logistic map

20. Fitting the Taq model (modified efficiency) Two trial efficiency functions, r(y) Logarithmic regression shows that the data is not truly logistic Fitting the data to the model with nonlinear optimization software (matlab)

21. More work on the Taq model (aka “Junk” model) By University of Waterloo student, Tor Myklebust

22. Can find the efficiency function directly from the data Normalized data Un-normalized data

23. Can the asymmetry in the amplification curves be explained directly by latency in the reactions?

24. Building an ODE Model for the Reaction Reactionphases dissociation priming extension primer resource 5 coupled ODEs, with temperature dependent reaction rates

25. The three phases of the PCR Reaction dissociation priming annealing

26. Assuming perfect denaturing, perfect priming, R used only during this step Work in the extension phase only 2 conserved quantities:

27. Composing a 3-D map: R D B’ n Still looks pretty symmetrical!

29. Will adding in Taq dynamics capture the asymmetry? (on transparencies)

30. All very well, but how will the rate constants be determined? Answer: insinuate yourself into a laboratory doing real-time PCR

31. Capture and real-time detection of food pathogensProf.Marie Walsh, Nutrition and Food Science, USUCenter for Microbe Detection and Physiology • Detection of food pathogens hampered by large sample size • New techniques capture and concentrate bacteria from large volumes in short times (~5min) • Link this with real-time PCR for rapid analysis of large samples with mixed populations of microbes

32. Looking over the shoulder of a real-time microbiologist….

33. Designing primers and probes for real-time amplification of E.coli 015787

34. Summary • Direct fitting of PCR amplification curves (grad modeling camp and undergrad summer students). • Developing and analyzing ODE models for the reactions (masters project). • Parameterizing the model(s) with data from food pathogen detection system. • Ultimately using model to optimize reaction conditions for yield and specificity (a sequence dependent result).