News Column

Researchers Submit Patent Application, "Thermal Cycling System and Method of Use", for Approval

February 25, 2014

By a News Reporter-Staff News Editor at Life Science Weekly -- From Washington, D.C., NewsRx journalists report that a patent application by the inventor Ririe, Kirk Max (Salt Lake City, UT), filed on July 5, 2013, was made available online on February 13, 2014 (see also BioFire Diagnostics, Inc.).

The patent's assignee is BioFire Diagnostics, Inc.

News editors obtained the following quote from the background information supplied by the inventors: "Amplification of DNA by polymerase chain reaction (PCR) requires reaction mixtures be subjected to repeated rounds of heating and cooling. All commercially available instruments for PCR operate by changing the temperature of the environment of a reaction vessel, either by heating and cooling the environment, or by robotically moving the samples between environments. The most common instruments for temperature cycling use a metal block to heat and cool reaction mixtures. Thermal mass of the metal block is typically large, meaning temperature transitions are relatively slow and require a large amount of energy to cycle the temperature. The reaction mixture is typically held in microcentrifuge tubes or microtiter plates consisting of rigid injection molded plastic vessels. These vessels need to be in uniform contact with the metal block for efficient heat transfer to occur. Maintaining temperature uniformity across a large heat block has also been a challenge.

"Novel techniques have been devised to overcome the challenges of using instruments with metal blocks for heating and cooling samples. Airflow can be used to thermocycle samples in plastic reaction tubes (U.S. Pat. No. 5,187,084), as well as in capillary reaction tubes (Wittwer, et al, 'Minimizing the time required for DNA amplification by efficient heat transfer to small samples', Anal Biochem 1990, 186:328-331 and U.S. Pat. No. 5,455,175). Capillary tubes provide a higher surface area to volume ratio than other vessels. Using air as the thermal medium allows rapid and uniform temperature transitions when small sample volumes are used.

"Further, the capillary tubes themselves can be physically moved back and forth across different temperature zones (Corbett, et al., U.S. Pat. No. 5,270,183, Kopp et al., 1998, and Haff et al., U.S. Pat. No. 5,827,480), or the sample can be moved within a stationary capillary (Hunicke-Smith, U.S. Pat. No. 5,985,651 and Haff, et al., U.S. Pat. No. 6,033,880). With the latter technique, contamination from sample to sample is a potential problem because different samples are sequentially passed through the winding capillary tube. Additionally, tracking the physical position of the sample is technically challenging.

"The use of sample vessels formed in thin plastic sheets has also been described. Schober et al. describe methods for forming shallow concave wells on plastic sheets in an array format similar to a microtiter plate (Schober et al, 'Multichannel PCR and serial transfer machine as a future tool in evolutionary biotechnology', Biotechniques 1995, 18:652-661). After samples are placed in the pre-formed well, a second sheet is placed over the top, and the vessel is heat-sealed. The accompanying thermal cycling apparatus physically moves a tray of samples between different temperature zones (Schober et al. and Bigen et al., U.S. Pat. No. 5,430,957). The use of multiple heating blocks for the temperature zones makes this machine large and cumbersome.

"Another system using reaction chambers formed between two thin sheets of plastic has been described where the vessel has multiple individual compartments containing various reaction reagents (Findlay et al, 'Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction', Clin Chem 1993, 39:1927-1933, and Schnipelsky, et al., U.S. Pat. No. 5,229,297). The compartments are connected through small channels that are sealed at the beginning of the process. One apparatus has a moving roller that squeezes the vessel while traveling from one end of the vessel to another. The pressure from the roller breaks the seal of the channels and brings the sample into contact with reagents. Temperature is controlled by a heater attached to the roller mechanism (DeVaney, Jr., et al., U.S. Pat. No. 5,089,233). A second apparatus uses pistons to apply pressure to the compartments and move the fluid (DeVaney, Jr., U.S. Pat. No. 5,098,660). The temperature of one of the pistons can be altered while in contact with the vessel to accomplish thermal cycling. In both of these examples, the temperature of a single heating element is being cycled. Changing the temperature of the heating element is a relatively slow process.

"Another system uses a planar plastic envelope (Corless et al. WO9809728A1). The sample remains stationary and heating is provided by an infrared source, a gas laser.

"Real-time monitoring of PCR is enabled using reaction chemistries that produce fluorescence as product accumulates in combination with instruments capable of monitoring the fluorescence. Real-time systems greatly reduce the amount of sample transfer required between amplification reaction and observation of results. Additionally, in some systems, quantitative data can also be collected.

"A number of commercially available real-time PCR instruments exist that couple a thermal cycling device with a fluorescence monitoring system. Of these real-time instruments, thermal cycling in the Perkin-Elmer 5700 and 7700 and the Bio-Rad iCycler instruments are based on metal heat blocks. The Roche LightCycler, the Idaho Technology Ruggedized Advanced Pathogen Identification system (or R.A.P.I.D.) and the Corbett RotoGene all use air to thermocycle the reactions. The Cepheid SmartCycler uses ceramic heater plates that directly contact the sample vessel."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventor's summary information for this patent application: "The present invention provides a cycling system for use in various temperature-controlled processes, including but not limited to the polymerase chain reaction. The present invention also provides a new thermal cycling system capable of generally automatically and simultaneously varying the temperature of one or more samples. The present invention further provides a new thermal cycling system that allows a rapid and almost instantaneous change of temperatures between a plurality of temperatures by moving samples between temperature zones within each reaction vessel. Additionally the present invention provides a thermal cycling system for the detection and analysis of a reaction in real-time by monitoring cycle-dependent and/or temperature-dependent fluorescence.

"In an illustrated embodiment, a reaction mixture is placed in a soft-sided flexible vessel that is in thermal contact with a plurality of temperature zones comprising a plurality of movable heating or heater elements. When pressure is applied to the vessel by closing all except one set of the heater elements, the reaction mixture inside the vessel moves to the heater element that is left open. The reaction mixture can be moved between different portions of the vessel and can be exposed to different temperature zones by selective opening and closing of the heater elements. Temperature change of the reaction mixture occurs rapidly and almost instantaneously. The vessel can be of any shape, illustratively elongated, and made of a flexible material, such as thin plastic film, foil, or soft composite material, provided that the material can hold the reaction mixture and can withstand temperature cycling. Exemplary plastic films include, but are not limited to, polyester, polyethylene terephthalate (PET), polycarbonate, polypropylene, polymethylmethacrylate, and alloys thereof and can be made by any process as known in the art including coextrusion, plasma deposition, and lamination. Plastics with aluminum lamination, or the like, may also be used.

"A single vessel can be used for temperature cycling. Alternatively, for simultaneous temperature cycling, multiple vessels may be used simultaneously. The multiple vessels can be stacked together, as parallel channels in sheet format, or adjacent each other in a circle to form a disk. The heater elements can be made of, for example, thin-film metal heaters, ceramic semiconductor elements, peltier devices, or circuit boards etched with metallic (e.g. copper) wires, or a combination of the above, with optional metal plates for uniform heat dispersion. Thick metal heaters are also an option if the device need not be small. Other heaters known in the art may be used.

"The heater elements are held at, or around, a set of characteristic temperatures for a particular chemical process, such as PCR. When the chemical process is PCR, at least two temperature zones are required: one at a temperature that is effective for denaturation of the nucleic acid sample, the other at a temperature that allows primer annealing and extension. As illustrated, reaction vessels are inserted in the apparatus when the heater elements for both temperature zones are in an open position. To temperature cycle for PCR, the heater element of one temperature zone is brought to the closed position, pushing the reaction mixture toward the open temperature zone at the other end of the vessel. In the open temperature zone, the heater element is in thermal contact with the vessel wall. Following an appropriate incubation time, the element of the zone heater is brought to the closed position, while the element of the other zone is opened. This action forces the reaction mixture to move to the other temperature zone. This process of opening and closing temperature zones is repeated as many times as required for nucleic acid amplification. It is understood that additional heater elements may be used for processes requiring more than two temperatures. For example, PCR reactions often use a denaturation temperature, an annealing temperature, and an extension temperature.

"The foregoing and many other aspects of the present invention will become more apparent when the following detailed description of the preferred embodiments is read in conjunction with the various figures.


"FIGS. 1A to 1E are cross-sectional diagrammatic views of a reaction vessel containing a reaction mixture positioned between heater elements of the present disclosure.

"FIG. 1A is a diagrammatic view of the vessel positioned between at least three pairs of heater elements showing each element spaced-apart from the vessel.

"FIG. 1B is a diagrammatic view similar to FIG. 1A of the vessel positioned between two pairs of heater elements and showing a top pair of the elements in a closed position and a bottom pair of the elements in an opened position so that generally all of the reaction mixture is positioned between and heated by the bottom pair of elements.

"FIG. 1C is a diagrammatic view similar to FIGS. 1A and 1B showing the bottom pair of elements in the closed position and the top pair of elements in the opened position so that generally all of the reaction mixture is positioned between and heated by the top pair of elements at a different temperature than the bottom pair of elements.

"FIG. 1D is a diagrammatic view similar to FIG. 1B showing the bottom pair of elements in the opened position and the top pair of elements in the closed position and further showing a heat sink adjacent but spaced-apart from each of the bottom pair of elements.

"FIG. 1E is a diagrammatic view similar to FIG. 1D showing the bottom pair of elements in the closed position and the top pair of elements in the opened position and further showing each of the heat sinks having engaged the respective element to cool the bottom pair of elements.

"FIG. 2A is a perspective view of the reaction vessel showing a receptacle coupled to a flexible body of the vessel.

"FIG. 2B is a perspective view of an array of reaction vessels coupled to each other to form a single row.

"FIG. 3 is a perspective view of an illustrative thermocycling subassembly for use with a real-time PCR apparatus of the present disclosure (shown in FIGS. 5 and 6) showing a first and a second stepper motor of the subassembly, top and bottom pairs of heater elements, and the row of reaction vessels positioned between the pairs of elements.

"FIGS. 4A to 4C are side views of the thermocycling subassembly shown in FIG. 3 showing thermocycling of the reaction mixture contained within the vessels.

"FIG. 4A is a side view thermocycling subassembly showing the top and bottom pairs of elements in the opened position prior to heating the reaction mixture within the vessels.

"FIG. 4B is a side view of the thermocycling subassembly showing the bottom pair of elements in the closed position so that the reaction mixture is in thermal contact with the top pair of elements.

"FIG. 4C is a side view of the thermocycling subassembly showing the top pair of elements in the closed position and the bottom pair of elements in the opened position so that so that the reaction mixture is in thermal contact with the bottom of elements.

"FIG. 5 is a perspective view of the thermocycling subassembly integrated into the real-time PCR apparatus including the thermocycling subassembly and a fluorimeter subassembly.

"FIG. 6 is a side view of the real-time PCR apparatus shown in FIG. 5.

"FIG. 7 is a graph showing the results of real-time monitoring of PCR in which DNA amplification is detected by the increase in relative fluorescence in the annealing temperature zone. (.DELTA., .largecircle., , are negative controls; .gradient., , .diamond-solid., + are positive samples)

"FIG. 8 is a perspective view of an alternative real-time PCR apparatus showing a body of the apparatus including a slot for placing sample vessels therein and a pressurized gas chamber adjacent the slot, and showing the apparatus further including a lid hinged to the body and including a computer having a PC interface and display monitor.

"FIG. 9 is a part schematic, part diagrammatic sectional view of the components located within the body of the PCR apparatus shown in FIG. 8 showing an alternative thermocycling subassembly having pneumatic bladders and the fluorimeter subassembly positioned below the thermocycling subassembly.

"FIG. 5A is a perspective view of a twelve-compartment pouch assembly with a comb holding the plunger in position to adjust cavities to predetermined volumes.

"FIG. 5B is a back plan view of the fitment of the twelve-compartment pouch assembly at initial position prior to evacuation without the comb."

For additional information on this patent application, see: Ririe, Kirk Max. Thermal Cycling System and Method of Use. Filed July 5, 2013 and posted February 13, 2014. Patent URL:

Keywords for this news article include: Polymerase, Enzymes and Coenzymes, BioFire Diagnostics Inc..

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Source: Life Science Weekly

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