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

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.

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….

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:

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.

IT Products, cont.

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.

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.

The Light Typer: SNP detection device

• 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.

SNP: single nucleotide polymorphisms

Steps in the PCR reaction

1) Heat to 94 degrees C to denature DNA strands.

2) Cool to 60 degrees C, primer molecules bind on to each end of a target sequence.

3) Heat again to 70 degrees C, Taq polymerase does its job and single nucleotides are added to the single strand sequence.

4) Repeat 1-3 30 to 40 times.

Schematic of DNA Amplification by PCR

Quantitative Real Time PCR

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.

Uses fluorescent probes to detect completed strands

TaqMan

Light Cycler amplification curvesFluorescing probe molecules detect completed strands.

Want to determine initial amount of DNA target present in sample

Quantitative PCR

“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.

Mapping back to initial copy number using the “Famous” Model

C_T is the time to cross a set threshold fluorescence value

C_T “standard curve”

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)

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.

Banff GIMMC Workshop, June 2003

Fitting the Logistic Model: Least Squares and Nonlinear Optimization

Mapping initial fluorescence toinitial copy number

Using nonlinear optimization to fit the data to the logistic map

Least Squares fit:

Fitting the Taq model (modified efficiency)Two trial efficiency functions, r(y)

Fitting the data to themodel with nonlinearoptimization software(matlab)

Logarithmic regression showsthat the data is not truly logistic

More work on the Taq model (aka “Junk” model)

By University of Waterloo student,Tor Myklebust

Can find the efficiency function directly from the data

Un-normalized data Normalized data

Can the asymmetry in the amplification curves be

explained directly by latency in the reactions?

Building an ODE Model for the Reaction

dissociation

priming

extension

Reaction phases

primer

resource 5 coupled ODEs,with temperaturedependent reaction rates

The three phases of the PCR Reaction

dissociation priming annealing

Work in the extension phase onlyAssuming perfectdenaturing, perfectpriming, R used onlyduring this step

2 conserved quantities:

Composing a 3-D map:

n

R

D

B’

Still looks pretty symmetrical!

Will adding in Taq dynamics capture the asymmetry?

(on transparencies)

All very well, but how will the rate constants be determined?

Answer: insinuate yourself into a

laboratory doing real-time PCR

Capture and real-time detection of food pathogens

Prof. 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

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

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

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).