February 20, 2004
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:
Second year request: $160,000, five-year cumulative request $750,000
Second year period: 7/1/04 – 6/30/05, five-year period 7/1/03 – 6/30/08
Principalal 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 risk and disease detection and better treatment.
We propose a newOur Center proposes to address this challenge by use of new technology that makes DNA screening significantly simple and cost effective. We will leverage the expertise of the University of Utah team who, during the past decade, modified the Nobel-prize winning technique, polymerase chain reaction (PCR), and made the process ten times faster so that DNA can be amplified over a million–fold within 15 minutes. When a fluorescent dye is added, then one can also “watch” the DNA as it is amplifying at that speed. This process called “real-time” PCR is able to tell if the 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. We have recently 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 or satellite imaging. The ability to collect high-density information allows us to magnify images and reveal greater detail by using software algorithms that focus on important characteristics. The “images” of DNA-melting are simple fluorescence vs temperature plots, or “melting curves”. For example, genotyping of single base changes is shown in the melting curves of Fig. 1. The PCR amplicon is 544 bp long and it melts in two stages or “domains”. The domain that melts first (at a lower temperature) is variable at a single base. Two individuals each of three genotypes are shown. (??) Using these methods, we can (currently) detect single base changes in PCR products (of up to)at least 1000 bases in length. Another example of using high-resolution melting analysis for genotyping is shown in Fig. 2. Seventeen members of a family were analyzed by melting curve analysis. A highly variable region important for transplantation (HLA) was used. The melting curves group into clusters. Family members who are compatible for transplantation have melting curves in the same cluster. The same principle can be applied to forensic medicines or microbe identification. High-resolution melting is a powerful genetic analysis techniquemarker that is the cornerstone of our Ccenter’s technology.
DNA melting is a fundamental property of DNA that is in the public domain. However, our analysis techniques, software, instruments and dyes, and fields of application can be patented. Competitive advantages of homogeneous DNA analysis include: 1) everything is done in solution (no physical separations are required), 2) the system is closed tube (no contamination risk), 3) only PCR is required (no expensive probes), and 4) the method is simple (no need for automation, reagent additions, or intermediate purification).
In the first year of Center funding, we focused on commercializing high-resolution melting to scan DNA for mutations. Basic software, reagents and instruments were licensed to a Utah company in Research Park (Idaho Technology) whicho also provided the matching funds. The application is covered by pending patents assigned to the University of Utah. A first generation system (HR-1ä instrument and LCGreenä I reagent) was launched commercially in the fall of 2003 in the US and distributors in Japan and Italy are now in place. Successful licensing and commercialization of the technology at this early stage has created six jobs in Utah (two engineering, two production, one biochemist, one marketing) with an average salary of $56,000. We anticipate that the number of jobs will double in the next year and that this business will continue to grow along our original projections. In addition, the use of these instruments may create more jobs indirectly.
With successful licensing of mutation scanning, our Center is now focusing on other areas of commercialization. Specifically, these areas are: 1) methods for homogeneous repeat typing and sequencing, 2) software for DNA analysis with the objective of spinning off “DNAWizards” as a dotcom company in the next two years, and 3) developing a highly parallel hardware platform for real-time PCR and melting analysis in collaboration with a new Center of Excellence being proposed by Dr. Bruce Gale (U. of Utah Engineering).
Our Center will demonstrate the value of products through R&D, an academic web server for software analysis (DNAWizards.path.utah.edu), and alpha-site testing at leading clinical diagnostic laboratories as well as domestic and foreign academic centers. Idaho Technology Inc (Salt Lake City, UT) and Roche Diagnostics (Alameda, CA and Penzberg, Germany) closely follow our work, and Idaho Technology has committed initial funds of $1.65 million to match Center funding. The Center will consider both licensing to a Utah-based company such as Idaho Technology Inc, or setting up a new service/manufacturing company in Utah. The software company, DNAWizards.com we plan to spin off as a new company. Proximity between the Center and commercial sites will be beneficial, particularly during technology transfer and the early commercial phase. Product sales and distribution is best done through local distributors or alliance partner(s) with existing presence and global reach to the R&D and diagnostic markets, Roche Diagnostics being one example.
After rapid commercial success of one application in our first year, our Center will continue to develop advanced methods, software and hardware for homogeneous DNA analysis to increase the breadth and penetration of this simple and powerful technology. These steps will require further innovation, but if successful, the methods will ultimately eliminate 95-99% of high-cost conventional DNA sequencing. The potential global market for the Center’s technology is around $400 million today (instrumentation and reagents combined). This is projected to grow yearly at 9-10%. In the fifth year, we estimate that a 4% share of the market can be obtained, generating annual revenues 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 matching funds and eventual independence.
1. Background
1.1 Technology Definition. Our technology is based on fluorescent detection and analysis of nucleic acids, during and after PCR. Methods and instruments have been developed to achieve rapid real-time PCR followed by rapid automated real-time analyses that take 10-20 minutes in their entirety. 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 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. 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.
In the first year of funding, we applied high-resolution melting to detect subtle DNA differences between the two copies of DNA present in diploid cells. This provided a method to scan PCR products for unknown mutations, and has just been licensed to a Utah company. We arehave now focused uponidentified additional promising commercial opportunities for homogeneous DNA analysis and these new targets form the basis or our ongoing efforts andcurrent renewal application.application.
1.2 Technology Rights. Our Center specializes in new techniques, instruments, and software for homogeneous DNA analysis up to the point of commercialization. We have 13 issued US patents on various aspects of homogeneous DNA analysis in addition to foreign counterparts. About an equal number of additional patents are applied for, but not yet granted. Some of the technology rights for homogeneous DNA analysis have already been licensed to Utah companies. Listed below we consider only the patents and invention disclosures that have not yet been licensed. Idaho Technology has provided matching funds for our Center grant and has a limited-time option on the following two inventions disclosed during the first year of Center funding:
· Homogeneous sequencing and repeat typing (U-).
· Massively parallel primer synthesis, real-time PCR and melting analysis on a chip (U-).
We are still working on proof-of-principle for these disclosures and anticipate filing patent applications during the next year. The following six items are not limited by options (no company funds were used or the option period has expired). The last four were developed during the first year with Center funds.
· Homogeneous multiplex hybridization by color and Tm (US patent #6,772,156).
· Simultaneous screening and identification of sequence alterations from amplified target (published US patent pending 2002-0142300)
· SNPWizard – Design and optimization of primers for amplification and homogeneous analysis of DNA having mutations in one or few bases (U-)
· ExonWizard – Design and optimization of primers for amplification and homogeneous analysis of DNA exons and splicing regions (U-)
· Automatic clustering and classification of homozygotes and heterozygotes by high-resolution melting curve similarity (U-).
· Logistic quantification of initial copy number from the plateau height, linear growth rate, and maximum second derivative of PCR amplification curves (U-).
1.3 Program History/Status. We have been working on homogeneous DNA analysis for the past 10 years. 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, 7/94-6/96, $90,000.This was followed by a STTR award from the NIH. 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, 9/94-9/98, $600,000.
This funding allowed us to build the prototype LightCycler, a popular real-time PCR instrument now distributed worldwide by Roche. 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, 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, 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 STTR grant. This grant focused on using DNA melting temperature (Tm) to characterize DNA: Homogeneous multiplex PCR by color and Tm. NIH STTR Phase I and II Grant, 4/1/99-2/03, $620,000.
Recently, we obtained further seed money from the University of Utah to develop new methods for SNP typing. Single-labeled probes for real-time PCR and SNP typing without probes. Technology Commercialization Projects. University of Utah Research Foundation, 7/02-6/04, $70,000. These methods have just been licensed by a Utah company and introduced commercially along with a new instrument, the LightTyper, currently distributed by Roche.
We are now in our first year of Center of Excellence funding from the state of Utah. All of our goals for the first year have been met. Mutation scanning was licensed to a Utah company and commercially launched in the fall of 2003, resulting in six new jobs. Center for homogeneous mutation scanning, State of Utah, 7/03-6/04, $150,000.
Idaho Technology continues to fund research in my laboratory at the University of Utah in PCR and fluorescent techniques. These funds were used as the required 2:1 matching funds for the first year of the Center, and will also be used in the second year. When funding comes in part from Idaho Technology, they have an option to license the technology by research contract. In the first year of Center funding, Idaho Technology has an option to license two of the six invention disclosures. When the company contributes funds, they deserve a right of first refusal. This arrangement has worked well in the past, and we anticipate it will work well in the future. Fluorescent PCR techniques. Idaho Technology, 1/03-12/07, $1,650,000.