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CASE 6 NTNU INVENTION DISCLOSURE FORM APRIL 2013 Norwegian University of Science and Technology Trondheim, Norway 1. Title of Invention Miniaturized Amplification Device Inventors: Name 1. Professor Chin Home Address Citizenship China Chinese Norway Norwegian Korea Korean (on sabbatical leave at NTNU) 2. Dr. North (NTNU Professor) 3. Dr. Kim 2. Has the invention been publicly disclosed, publicly used, presented, published or offered for sale? When? No Circumstances: 3. Is the invention going to be publicly disclosed, publicly used, presented, published or offered for sale? When? May 25, 2013 Circumstances: Dr. Kim will discuss a working model of the invention at a biotechnology conference to be held in Osaka, Japan on May 25, 2013. He is expected to sell three devices at the conference to a US company. LET THE NTNU TECHNOLOGY TRANSFER OFFICE KNOW WELL BEFORE ANY PRESENTATION, DISCLOSURE OR OFFER FOR SALE OF THE INVENTION. 4. Background of the Invention: (Brief description of background, including problems solved. Use additional sheets if needed) DNA amplification technology has many applications. These include use in genetic engineering, the medical sciences (diagnostics, research and development of new drugs), forensic (crime scene investigations) and military uses (detection of pathogens in the field). Current DNA amplification techniques use the following components: (1) a double-stranded DNA template to be analyzed, CASE 8 NTNU Invention Disclosure Form Pg. 2 (2) a pair of DNA primers that bind each of two ends of the template, (3) a thermostable enzyme (“DNA polymerase”) that binds the primers and extends them along the length of the template in the presence of (4) DNA building blocks called “dNTPs”. When (1)-(4) are combined together under suitable temperature conditions, the result is a rapid “chain reaction” of DNA amplification by which the enzyme makes many, many copies of the template (called a “Polymerase Chain Reaction” or “PCR”). So many copies of the template are made that they are readily detectable using standard laboratory procedures (e.g., gel electrophoresis). The PCR technique is so sensitive that only one copy of the original template is needed for amplification and detection purposes. The PCR technique is often the subject of an American made-for-TV crime series called CSI: Crime Scene Investigations-Las Vegas. Bad guys/gals in the series usually get arrested when they inadvertently leave a sample of their DNA at the crime scene. When brought back to the CSI lab, the PCR technique is used to amplify the DNA taken from the crime scene. This provides enough sample for analysis such as comparison of the crime scene DNA sample to a national/international DNA registry. Any matches are used to confirm the identity of the suspect and place him or her at the crime scene. Fans of the CSI crime series will remember that the PCR machine used by the team is very large and complex. To use the machine, the researcher needs to control the temperature of the process very carefully. The machine begins PCR begins by subjecting (1)-(4) to a high temperature. This denatures the template (Step 1-Denaturation). Next, the reaction is cooled to allow the DNA primers to bind the template. This is called annealing (Step 2-annealing). In the last step, called polymerization, the reaction is maintained at an intermediate temperature dictated by the enzyme (Step 3-polymerization). The enzyme extends the DNA primers along the length of the template using the DNA building blocks. The machine repeats steps 1, 2, and 3, many times to create the “chain reaction” of DNA amplification. An example of a commercially available PCR machine is the GENE AMP® PCR SYSTEM 9700 sold by Applied Biosystems, Inc. (Foster City, CA (USA). Despite widespread use of PCR devices, these machines have problems. First, they are large and complicated machines. Often, they are quite expensive. Much of the complexity, size and expense is due to need to change temperature in the samples. That is, a solid block or fluid as a heat source requires a means for controlling and changing the temperature of the heat source rapidly and uniformly. Also required, is a means for controlling the time interval of the temperature change. Typically, means for mechanically moving samples around the device are required, especially when many samples are to be analyzed. These requirements do not lend themselves to miniaturization. Despite these drawbacks, it is estimated that the worldwide sales of PCR machines, consumbles (eg., reagents, tubes, etc.), services (diagnostic, clinical, research, forensic) and related licensing revenues amounts to about $5 billion USD annually. CASE 8 NTNU Invention Disclosure Form Pg. 3 5. Describe the Invention: (Describe the structure, function and all uses of the invention. Use sketches and/or additional attached sheets as necessary.) The present invention addresses the shortcomings of prior PCR devices such as those shown on CSI:Crime Scene Investigations-Las Vegas. In particular, the PCR apparatus of the invention uses thermal convection to amplify DNA. That is, the invention achieves PCR amplification by forming many regions of different temperatures inside a sample to be analyzed. Formation of a temperature gradient in the sample drives the thermal convection. Fig. 1 Fig. 1 shows an example of a PCR amplification according to the invention in which (1) is a high temperature region, (2) is a low temperature region, (3) and (4) are heat sources, (5) is the convection region and (6) is a reaction vessel that holds the sample that is being amplified. As Fig. 1 also shows that spatial regions of different temperatures (2), (5), (1) are generated inside the reaction vessel (6). Inside the reaction vessel, there are regions for template denaturation, annealing of primers to template, and polymerization (amplification of template). Sample is circulated throughout the reaction vessel (6) through the different temperature regions. Denaturation, annealing and polymerization can occur sequentially and repeatedly, thereby resulting in massive PCR amplification. The process is relatively, simple, lends itself to miniaturization, is less expensive and avoids the need for complicated means for controlling temperature required by prior PCR machines. CASE 8 NTNU Invention Disclosure Form Pg. 4 Fig. 2 Fig. 2 shows a cross-sectional view of a PCR amplification apparatus of the present invention in which: (101) is a first heating block, (102) is a cooling block, (103) is a reaction vessel, (104) is a heating device (105) is an inlet for a cooling fluid (106) is an outlet for the cooling fluid (107) is an insulator (112, 117 and 111) are cavities for holding the reaction vessel In operation, the heating block (101) receives heat from the heating device (104) and transfers it to the bottom of the reaction vessel (103), thereby forming a high temperature region at the bottom of the sample. The cooling block (102) consisting of an inlet (105) and outlet (106) for a cooling liquid (eg., water) removes heat from the upper portion of the sample to create a low temperature region. In one application, the high temperature region is maintained at 94°C and the low temperature region is maintained at 45°C. PCR amplification occurs throughout the convection region, roughly found within the middle portion of the reaction vessel. Methods for immobilizing DNA polymerases to solid surfaces are known. The present inventors have found that heat sensitive DNA polymerases can be used in the PCR amplification apparatus by binding those enzymes in the low temperature region and/or in the upper portion of the convection region. Prior PCR devices typically use only thermostable (and relatively expensive) DNA polymerases because the entire sample is subjected to high temperature during cycling. The high temperature will destroy heat sensitive enzymes. 6. How is the invention new and non-obvious? Please list any prior references which may be relevant to your invention: CASE 8 NTNU Invention Disclosure Form Pg. 5 One of Dr. North’s graduate students (Dr. Kathryn Willows) published, as part of her Ph.D. research, a thesis entitled “ Use of Thermal Convection To Assist Microfluidic Reactions”. The thesis was duly filed with the NTNU library and made available to the public on July 25, 2011. Her thesis states that thermal convection has many important applications in microfluidics and that it “may be helpful in many synthetic biological reactions”. No specific mention of any particular biological reaction (including PCR) was made. Since graduating from NTNU, Dr. Willows moved to Las Vegas, Nevada where she has continued a long collaboration with Drs. North and Chin. She now runs her own forensic laboratory in Las Vegas. She recently told Dr. North that on July 1, 2013, her lab will begin using and selling a new PCR device based on thermal convection. Prior to starting her own forensics laboratory, Dr. Willows was briefly employed by PCR Inc., a large US firm devoted to the design and manufacture of PCR machines worldwide. Note: Where are the potential markets for the invention? Consider what regions/countries (other then Europe) where NTNU should seek to have patent protection. 7. Sponsored research involved? NO_X_________ YES (Identify Sponsor) Submitted under their Employment or Consulting Agreement by the following inventors: Signature Date Signature 1. 3. 2. 4. Date