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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,
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NTNU Invention Disclosure Form
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(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.
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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.
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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:
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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
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