February 25, 2005

University of Utah College School 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:

Third year request: $176,000, five-year cumulative request $790,000

Third year period: 7/1/05 – 6/30/06, five-year period 7/1/03 – 6/30/08

Principal Investigator: (Carl Wittwer):______

Office of Sponsored Projects (Elliott C Kukakowski):______

Technology Transfer Office: (Brent Brown):______

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 and what antibiotics they are sensitive to. The Center for Homogeneous DNA Analysis is making this happen.

The Human Genome Project has completely sequenced the human DNA, 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 assessment, disease detection and better treatment.

Our Center proposes to address this challenge by use of new technology that makes DNA screening 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 to the reaction, 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 an increase 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 know if the DNA is one of several different types (e.g. 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 this method, we can currently detect single base changes in PCR products of up to 1000 bases in length. Another example of using high-resolution melting analysis for matching transplant recipients to donors is shown in Fig. 12. If highly polymorphic HLA regions are amplified by PCR, the melting curves group into “compatibility” clusters. Family members who are compatible for transplantation have melting curves in the same cluster. The same principle can be applied to forensic medicine or microbe identification. High-resolution melting is a powerful genetic analysis technique that is the cornerstone of our Center’s technology.

DNA melting is a fundamental property of DNA that is in the public domain. However, our methods, software, instruments, and fields of application are being 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 the development of high-resolution melting to scan DNA for mutations. Patent rights were assigned to the University of Utah. Basic sFirst generation software, reagents and instruments were licensed to a Utah company in Research Park (Idaho Technology Inc) which also provided the matching funds. In the fall of 2003, A the first generation commercial system (HR-1 instrument and LCGreen I reagent) was available launched commercially in the fall of 2003 in the US, and distributors in Japan and Italy were established. To date, 50 systems have been sold, generating gross revenues of $500,000. In this second year of funding, a 96/384-well high-resolution melting instrument, the LightScanner is being launched. Out-licensing and commercialization of the technology has created twelve new jobs in Utah with an average salary of $58,000. We anticipate that the number of jobs will continue to grow as projected.

With successful licensing of mutation scanning, our Center is focusing on additional areas of technology development. Specifically, these areas are: 1) methods for homogeneous repeat typing, sequencing and matching, 2) software for DNA analysis with the objective of spinning off “DNAWizards” as a dotcom company in the next year, and 3) developing a “digital PCR” chip for real-time PCR and melting analysis in collaboration with Bruce Gale’s Engineering Center of Excellence.

Our Center will demonstrate the value of the technologyproducts through R&Dresearch publications, providing access to analytical software through 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 and Roche Diagnostics closely follow our work, and Idaho Technology committed initial funds of $1.65 million to match Center funding. The Center will consider both the out-licensing of the newer technologies, and the formation of a new service/manufacturing company in Utah which may or may not be independent of the new software company, DNAWizards.com. Proximity between the Center and commercial sites will be beneficial, particularly during technology transfer and the early commercial phases. Product sales and distribution is best done through regional distributors or alliance partner(s) with existing presence and global reach to the R&D and diagnostic markets, such as Roche Diagnostics.

In the years to come, 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 require further innovation, but if successful, the methods will ultimately eliminate 95-99% of high-cost conventional DNA sequencing. The global market for the Center’s technology is around $400 million today (instrumentation and reagents combined) growing at 9-10% year. Annual revenue of $24 million (4% share) in 2008 is achievable for the technology suites generated by the Center. The Center anticipates receiving a share of royalties from this revenue through the University of Utah system and plans an NSF Center of Excellence application in the next year. Two Fast Track STTR grants have been funded since the Center’s initiation (1.7M). Development of new technologies in years 3 through 5 will further strengthen the competitive advantage of high resolution melting, and will provide opportunities for new software, service, and device companies in Utah.

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 DNA amplification and analyses that are monitored in real-time PCR followed by rapid automated real-time analyses thatand take 10-20 min in their entirety. Because theThe fluorescent indicators that monitor the process (probes or dyes)we use are added before PCR, and therefore, no additional post-PCR processes such as membranes, arrays, or gels are necessary. Our DNA analysis method “high-resolution melting” greatly simplifies the process, and provides significantly more accurate information which allows one to rapidly detect, quantify and characterize DNA sequences.

In 1997, wWe introduced the concept of characterizing PCR products by melting curve analysis to characterize PCR products in 1997. Two fluorescent probes were used for genotyping, known as “adjacent hybridization probes” or “kissing probes”, were used for genotyping. In 2000, we developed a method using only a single labeled probe (SimpleProbe), greatly simplifying design considerations and cost. RecentlyIn 2002, 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 expense 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 analyzing temperature and fluorescence, the same physical parameters that are used in real-time PCR.

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 and was licensed to a Utah company. During the second year, we focused on new technologies and perfected SNP typing techniques. We are now pursuing additional promising commercial opportunities for homogeneous DNA analysis, such as our “digital PCR” chip, and these new targets form the basis of our ongoing efforts and renewal 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 14 issued US patents on various aspects of rapid PCR and homogeneous DNA analysis in addition to foreign counterparts. About an equal number of additional patents are applied for, but not yet grantedpending. 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 four inventions:

  1. Homogeneous sequencing and repeat typing (U-3601).
  2. Use of saturating DNA dyes, asymmetric PCR and 3’-blocked oligonucleotides for multiplex amplicon and site specific genotyping (U-3715).
  3. Method for parallel amplification and mixing of multiple samples in a closed tube system. (U disclosed 2/11/05).
  4. Method for introducing melting domains through primer tailing: application to allele-specific PCR (U disclosed 2/11/05).

We are still working on reducing the first disclosure to practice. The second disclosure is enabled and a utility application has been filed. The last two are recent filings. The following ten items are not limited by options (no company funds were used or the option has expired). The last eight were developed during the first two years with Center funds.

  1. Homogeneous multiplex hybridization by color and Tm (US patent #6,772,156).
  2. Simultaneous screening and identification of sequence alterations from amplified target (published US patent pending 2002-0142300)
  3. Massively parallel primer synthesis, real-time PCR and melting analysis on a chip (U-3570).
  4. SNPWizard – Design and optimization of primers for amplification and homogeneous analysis of DNA having mutations in one or few bases (U-3701)
  5. ExonWizard – Design and optimization of primers for amplification and homogeneous analysis of DNA exons and splicing regions (U-3702)
  6. Automatic clustering and classification of homozygotes and heterozygotes by high-resolution melting curve similarity (U-3703).
  7. Logistic quantification of initial copy number from the plateau height, linear growth rate, and maximum second derivative of PCR amplification curves (U-3704).\
  8. Background removal for oligonucleotide fluorescence vs temperature melting and amplification curves. (U disclosed 10/29/04).
  9. Multiplex amplification and melting analysis for HLA matching (U disclosed 2/11/05).
  10. Nearest neighbor thermodynamic parameters under real world conditions – superior estimates by eliminating multiple correction factors. (U disclosed 2/15/05).

1.3Program History/Status. We have worked on homogeneous DNA analysis for the past 10 years. Our first substantial funding was from a STTR award from the NIH, collaborating 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.

The above funding allowed us to build the prototype LightCycler, now a popular real-time PCR instrument now distributed worldwide by Roche. We were also able to attract funding from the Whitaker bioengineering foundation: Temperature cycling by adiabatic compression. Biomedical Engineering Grant. Whitaker Foundation, 12/95-11/98, $210,000. Idaho Technology then became interested in partial funding of my laboratory at the University: Fluorescent PCR techniques. Idaho Technology, 7/97-12/02, $950,000. Work during this time was also aided by funds from an endowed chair: 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 focusing 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, 7/02-6/05, $105,000. One of these methods was introduced commercially along with a new instrument, the LightTyper, a second was licensed last year by a Utah company and another is under final negotiation (also by a Utah company). We are now in our second year of Center of Excellence funding from the state of Utah. All of our goals for the first year were met and our progress on second year goals is on target. Mutation scanning was licensed to a Utah company and commercially launched in the fall of 2003, resulting in twelve new jobs. Center for homogeneous mutation scanning, State of Utah, 7/03-6/54, $294,000.

Idaho Technology continues to fund research in my laboratory at the University of Utah for PCR and fluorescent techniques, providing matching funds for the first two years of the Center. When the company contributes funds, they have 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.

In August of 2004, a Fast-Track STTR to continue commercialization of mutation scanning through a Utah company: Homogeneous mutation scanning, NIH STTR, $850,000, 8/04-1/07 was funded.In February of 2005, another Fast-Track STTR to continue development of an integrated real—time PCR machine with high-resolution melting, A system for rapid PCR, mutation scanning and genotyping, NIH STTR, $850,000, 3/05-9/07 was funded. Allowed amounts will also be used as matching funds.

Although second and third generation instruments were described in the original Center of Excellence grant, no state funds will be used to develop further scanning instrumentation. Only new technology, not yet licensed will be included in future work funded by the state.

2. Program Rationale

2.1Program Objectives. Our overall objective is to develop DNA analysis techniques that are simple and homogeneous. In the past, gels have been used to discriminate DNA fragments by size. Agarose gels stained with ethidiuim bromide and sequencing separations in capillaries are two common examples of DNA analysis that require separation. These non-homogeneous methods include multiple steps that require either manual handling or automation to perform. Amplified products are also exposed to the environment with risk of contamination of future reactions. Instead of size discrimination, our Center technology is based on using melting temperature as a means to differentiate different DNA fragments. DNA melting is a basic physical property of DNA, and we have developed methods to melt DNA very precisely (high-resolution melting analysis). DNA melting is homogeneous, requires no separation steps, and can be performed in the same tube that is used for PCR.

Our first year objective was to commercialize a new mutation scanning technology. This was achieved during the fall of 2003 with licensing to a Utah Company. The company has now sold about 50 instruments ($10K each) and about $40K worth of reagents. Two Fast-Track STTR grants were funded in the second year for further commercialization support. With licensing of this application, no further state funds will be used to further commercialize scanning instuments. All of our initial objectives were achieved. Future state funds will be used to commercialize other aspects of high-resolution melting. Specifically, these areas are: 1) methods for repeat typing, sequencing, and matching 2) software for DNA analysis with the objective of spinning off “DNAWizards” as a dotcom company in the next year, and 3) developing a digital PCR chip for real-time PCR and melting analysis in collaboration with Dr. Bruce Gale’s Center of Excellence (U. of Utah Engineering). We have the following specific aims: