February 21, 2003
University of Utah College of Medicine
Center for Homogeneous DNA Analysis
Carl T. Wittwer, MD, PhD
Department of Pathology
50 N. Medical Drive
University of Utah
Salt Lake City, UT 84132
Phone: 581-4737
FAX: 581-4517
Email:
First year request: $200,000, five-year cumulative request $850,000
First year period: 7/1/03 – 6/30/04, five-year period 7/1/03 – 6/30/08
Principle Investigator: (Carl Wittwer):______
Office of Sponsored Projects (Amy Sikalis):______
Technology Transfer Office: (Jayne Carney):______
Executive Summary
Imagine analyzing your DNA in 15 minutes. Imagine finding out your risk for cancer or drug reactions while you wait in a doctor’s office. Imagine testing for microorganisms, and within an hour, knowing what strain of bacteria or virus is present so you can effectively treat your infection. The Center for Homogeneous DNA Analysis can make this happen.
The Human Genome Project has completely sequenced the genome, but it is difficult to use this knowledge in routine medical practice because the methods to screen DNA are expensive and complex. Only when costs are significantly lowered and the methods dramatically simplified will DNA screening be used in every day clinical practice for effective disease detection and better treatment.
We propose a new Center to address this challenge by using a new technology that makes DNA screening simple and cost effective. We will leverage the expertise of the our team who has modified the Nobel-prize winning technique, polymerase chain reaction (PCR), so that DNA can be amplified over a million–fold within 15 minutes. By adding a fluorescent dye, one can also “watch” the DNA as it is rapidly amplifying. This process called “real-time” PCR is able to tell if a target (for example, HIV) is present by increases in fluorescence signal. How much is present can also be determined automatically without any additional work.
In medical applications and many other uses, we need to amplify a certain segment of DNA and also know if the DNA is one of several different types (for instance, normal versus disease-causing mutant). Therefore, some form of final analysis for typing (“genotyping”) is required. The method pioneered by our team uses thermal melting of DNA as a simple and elegant way to genotype. Two strands of DNA fall apart or “melt” as the sample is gradually heated from 40°C to about 90°C. Exactly how they melt depends on the genotype. Within the past year, we have found that high-resolution melting of DNA is more powerful than previously imagined. We can easily tell the difference between genotypes that differ in only a single base (the basic unit of DNA sequence). High-resolution melting takes only 1-2 minutes and can be performed in the same tube as real-time PCR, without any additional cost.
High-resolution melting is similar to high-definition TV. The ability to collect high-density information allows us to magnify images and reveal greater detail. Interpretation of data is greatly aided by software algorithms. The “images” of DNA-melting take the form of fluorescence vs temperature plots, or “melting curves”. For example, the series of graphs shown here are melting curves of different genotypes contained in a piece of DNA that is 110 bases long taken from a gene called beta-globin which is responsible for sickle cell anemia. Panel A shows the original melting curve data from six different genotypes, each replicated four times, including normal (AA), sickle cell disease (SS) and sickle cell trait (AS). Only when the image is normalized and magnified do the data cluster by genotype (Panel B). By overlaying the plots at high temperature (Panel C) it is even easier to discern samples of individuals who have two copies of the chromosome that are different (heterozygous AS, AC, SC) versus those who have two copies that are the same (homozygous AA, CC, SS). Finally, clustering is most obvious when the plots are further magnified and each plot is subtracted from the plot of a normal sample (Panel D). Using these methods, we have detected single base changes in PCR products over 600 bases in length.
For the first year of Center funding, we will focus on commercializing a first generation high-resolution melting system to scan DNA for mutations. This will be a research model which will test one sample at a time with the highest resolution possible. Basic software and reagents will also be provided. The system is covered by four pending patents assigned to the University of Utah. The Center will attempt to prove the value of the system through product R&D, and alpha-site testing conducted at two of the nation’s leading clinical diagnostic laboratories, and domestic and foreign academic centers. Idaho Technology Inc (Salt Lake City, UT) and Roche Diagnostics (Alameda, CA and Penzberg, Germany) have expressed interest in the first generation system, and while neither have obtained licenses to date, one company has committed initial funds of $1.4 million to match Center funding. The Center will consider licensing product manufacturing to a Utah-based company such as Idaho Technology Inc, or the alternative option to set up a new manufacturing company in Utah. Proximity between the Center and the commercial manufacturing site will be beneficial, particularly during technology transfer and the early commercial phase. Product sales and distribution is best done through alliance partner(s) with existing presence and global reach to the R&D and diagnostic markets, Roche Diagnostics being one example.
After the first generation system, the Center will continue to develop and prototype advanced applications, software and hardware to increase the breadth of the technology, volume throughput, and compatibility with existing real-time PCR instrumentation. Specific assays will be developed for the clinical diagnostic markets. These steps will require further innovation, but if successful, the system will ultimately eliminate 95-99% of the current use of the high-cost procedure known as DNA sequencing. The potential global market for the Center’s mutation scanning technology is around $400 million today (instrumentation and reagents combined). This is projected to grow yearly at 9-10%. In the fifth year after launching the first generation technology, we estimate that a 4% share of the market can be obtained, generating an annual revenue of $24 million to our commercial partner(s). The Center anticipates receiving a share of royalties from this revenue through the University of Utah system as a source of further matching funds.
1. Background
1.1 Technology Definition. Our technology is based on fluorescent detection and analysis of nucleic acids, during and after PCR. We have developed methods and instruments for rapid real-time PCR and analyses that take only 10-20 minutes to complete. Because the fluorescent probes or dyes we use are added prior to PCR, no additional post-PCR processes such as membranes, arrays, or gels are necessary. Our DNA analysis method is fast, simple, and powerful. Homogeneous analysis with fluorescence allows one to rapidly detect, quantify and characterize DNA.
We introduced melting curve analysis to characterize PCR products in 1997. Two fluorescent probes were first used for genotyping, known as “adjacent hybridization probes” or “kissing probes”. In 2000, we developed a method using only a single labeled probe, greatly simplifying design considerations and cost. These formats have already been licensed locally and successfully commercialized nationally and internationally.
Recently, we discovered a method that does not require any probes for genotyping. A new dye is added before amplification and a high-resolution melting curve is obtained after PCR is complete. No labeled oligonucleotides are necessary, adding very little cost to the cost of PCR itself. The only addition is a generic fluorescent dye that stains all PCR products. This dye is added before PCR and the tube is never opened during amplification or analysis. Such a “closed-tube” method is important to avoid PCR product contamination of future reactions. Best of all, high resolution melting analysis can be performed in only 1-2 minutes. Instead of analyzing the sample by some other complex method like sequencing or denaturing gradient high-performance liquid chromatography (dHPLC), high-resolution melting analysis requires only the same parameters that are used in real-time PCR, temperature and fluorescence.
We have found that certain dyes allow detection of subtle DNA differences between the two copies of DNA present in diploid cells. This provides a method to screen or “scan” PCR products for unknown mutations, and is the focus of our first year funding request. It is clear to us that scanning for mutations with high-resolution melting curves is a great commercial opportunity.
1.2 Technology Rights. Our laboratory specializes in new techniques, instruments, and analysis methods up to the point of commercialization. Many aspects of homogeneous DNA analysis have already been licensed to local companies. Indeed, because of our track record, matching funds are coming from private industry with an option to license future inventions.
In 2002, we discovered that homogeneous mutation scanning was possible with labeled primers and a patent application was filed. Later that same year, we developed methods for using simple dyes that stain double stranded DNA instead of labeled primers that are more costly and limited to use on one target. Two additional patents were filed in 2002 and one in early 2003 on the methods and software. The following patents applications have been filed in the past year, all owned by the University of Utah Research Foundation. These patents protect our mutation scanning methods, and none are yet licensed. We have also kept the identity of the dyes that work best in mutation scanning a trade secret.
· Mutation Scanning & Genotyping by Amplicon Melting Curve Analysis (60/386,975)
· Real-time PCR Analysis Using Saturation Dyes (60/420,717)
· Real-time PCR Analysis Using Saturation Dyes (USSN not yet assigned)
· Amplicon Melting Analysis with Saturation Dyes (USSN not yet assigned)
We anticipate that, with Center funding, the current technology will be licensed within the next year. These patent applications are optioned to Idaho Technology (believe it or not, a Utah company), who has expressed interest, but not yet elected to take a license. The options will expire during fiscal year 04 (ending June of 2004). If Idaho Technology fails to take out a license, we will form a new Utah company, license the technology, and pursue commercialization. After licensing occurs, continued Center funding will depend on additional intellectual property. This means that for each year (assuming success in licensing), further funding will be dependent on new technology generated and our ability to defend the potential for commercial success of the new material.
1.3 Program History/Status. We have been working with homogeneous DNA analysis for the past 10 years. Developing techniques, building instruments, and writing software are best developed hand-in-hand. Our first funding was from a Technology Innovation Grant from the University of Utah:
Instrumentation for quantitative rapid cycle PCR, Technology Innovation Grant. University of Utah Research Foundation., Principle Investigator, 7/94-6/96, $90,000.
This was followed by a Small Technology Transfer Research award from the National Institute of General Medical Sciences. This is similar to a small business grant, except that an academic (like myself) is allowed to be the principle investigator. We collaborated with the small business, Idaho Technology, who licensed the technology:
Continuous monitoring of rapid cycle PCR. NIH STTR Phase I and Phase II Grants, Principle Investigator, 9/94-9/98, $600,000.
This funding allowed us to build the prototype LightCycler, a popular real-time PCR instrument now distributed worldwide. We were also able to attract funding from the Whitaker national biomedical engineering foundation for similar research:
Temperature cycling by adiabatic compression. Biomedical Engineering Grant. Whitaker Foundation, Principle Investigator, 12/95-11/98, $210,000.
Idaho Technology then became interested in direct funding of my laboratory at the University. We had created a market for rapid fluorescent analysis of DNA, but there were many obvious opportunities to develop additional techniques and applications:
Fluorescent PCR techniques. Idaho Technology, Principle Investigator, 7/97-12/02, $950,000.
Work during this time was also aided by funds from an endowed chair. I was the first (rotating) holder of the Watkins endowed chair of Pathology at the University of Utah:
Endowed Chair of Pathology. University of Utah, 1/99 – 12/01, $180,000.
In addition, we were successful in obtaining more NIH funds through another collaborative grant with Idaho Technology. This grant focused on using DNA melting temperature to characterize DNA and has just been successfully completed. Using a combination of probe color and melting temperature (Tm) to analyze DNA is a powerful technique that is now widely with the LightCycler system:
Homogeneous multiplex PCR by color and Tm. NIH STTR Phase I and II Grant, Principle Investigator, 4/1/99-2/03, $620,000.
Recently, we have obtained further seed money from the University of Utah to develop a new probe system that is simpler and less expensive than other probe systems. These same probes can be used as primers for multiplexing homogeneous scanning techniques.
Single-Labeled Probes for Real-Time PCR. Technology Commercialization Project. University of Utah Research Foundation, Principle Investigator, 7/02-6/04, $70,000.
The probes have just been licensed by Idaho Technology and introduced commercially along with a new instrument, the LightTyper. Idaho Technology has decided to continue to fund research in my laboratory at the University of Utah in the area of homogeneous DNA analysis. Idaho Technology has an option to license our mutation scanning technology, but has not yet decided to exercise it. These funds will be used as the required 2:1 matching funds for at least the first year of the Center of Excellence grant.
Fluorescent PCR techniques. Idaho Technology, Principle Investigator, 1/03-12/07, $1,650,000.
2. Program Rationale
2.1 Program Objectives. Our first year objective is to ready a new mutation scanning technology for commercialization by introducing novel techniques, instrumentation, software, and reagents. Mutation detection, especially when the precise location is not known, is usually a difficult and costly procedure. We have found a way that is less expensive, faster, and appears to have greater potential than current techniques. In order to transition this method successfully into the marketplace, we have the following specific aims:
A. Techniques. Define the limits of our high-resolution melting method for mutation scanning. This requires determining sensitivity and specificity according to PCR product size, GC content, mutation location and type. How does product size affect sensitivity and specificity? Can all single base mismatches be detected? Is there an effect of different melting domains? Under what conditions can the technique fail? Later years will focus on applying high-resolution melting techniques to complex genotyping applications where only a single copy is present (bacteria, viruses) and complex diploid loci (HLA typing and short tandem repeat analysis).