(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I lllll llllllll II llllll lllll llll I II Ill lllll lllll lllll 111111111111111111111111111111111
`
`(43) International Publication Date
`30 January 2003 (30.01.2003)
`
`PCT
`
`(10) International Publication Number
`WO 03/007677 A2
`
`(51) International Patent Classification:
`
`Not classified
`
`(21) International Application Number: PCT/US02/22543
`
`(74) Agents: LAMMERT, Steven, R. et al.; Barnes & Thorn-
`burg, 11 South Meridian Street, Indianapolis, IN 46204
`(US).
`
`(22) International Filing Date:
`
`16 July 2002 (16.07.2002)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`60/305,632
`
`English
`
`English
`
`16 July 2001 (16.07.2001) US
`
`(71) Applicant (jor all designated States except US): IDAHO
`TECHNOLOGY, INC. [US/US]; 390 Wakura Way, Salt
`Lake City, UT 84108 (US).
`
`(72) Inventor; and
`(75) Inventor/Applicant (jor US only): RIRIE, Kirk [US/US];
`1223 S 2000 E, Salt Lake City, UT 84108 (US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, Fl, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
`VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, Fl, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, SK,
`
`[Continued on next page]
`
`= (54) Title: THERMAL CYCLING SYSTEM AND METHOD OF USE
`iiiiiiii -!!!!!!!!
`iiiiiiii -iiiiiiii
`
`(57) Abstract: A temperature cycling system (10, 110) is pro(cid:173)
`vided for repeatedly heating and cooling a reaction mixture (16).
`The system (10, 110) includes a first heater (27) and a second
`heater (28) each movable between a first orientation in which the
`first or second heater (27, 28) affects the temperature of the re(cid:173)
`action mixture (16) and a second orientation in which the first or
`second heater (27, 28) does not substantially affect the temper(cid:173)
`ature of the reaction mixture (16). During temperature cycling,
`the second heater (28) is in the second orientation when the first
`heater (27) is in the first orientation, and the second heater (28)
`is in the first orientation when the first heater (27) is in the sec(cid:173)
`ond orientation.
`
`iiiiiiii
`!!!!!!!!
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`!!!!!!!!
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`THERMO FISHER EX. 1012
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`WO 03/00 7 6 7 7 A2
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`I lllll llllllll II llllll lllll llll I II Ill lllll lllll lllll 111111111111111111111111111111111
`
`TR), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`GW, ML, MR, NE, SN, TD, TG).
`ning of each regular issue of the PCT Gazette.
`
`Published:
`without international search report and to be republished
`upon receipt of that report
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`THERMO FISHER EX. 1012
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`-1-
`
`THERMAL CYCLING SYSTEM AND METHOD OF USE
`
`Field of the Invention
`
`The present invention relates to a thermal cycling device and method that
`
`5
`
`facilitates rapid, uniform temperature cycling of samples. Illustratively, the invention is
`
`designed to perform DNA amplification and detection of amplified products within a
`
`reaction vessel.
`
`Background
`
`Amplification of DNA by polymerase chain reaction (PCR) requires reaction
`
`10 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
`
`15
`
`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
`
`20
`
`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. 5,187,084), as well as in capillary
`
`reaction tubes (Wittwer, et al, "Minimizing the time required for DNA amplification by
`
`25
`
`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.
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`
`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.
`
`5
`
`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
`
`10
`
`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.
`
`15
`
`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
`
`20
`
`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
`
`25
`
`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
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`-3-
`
`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. W09809728Al). The
`
`sample remains stationary and heating is provided by an infrared source, a gas laser.
`
`5
`
`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.
`
`10
`
`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
`
`15
`
`RotoGene all use air to thermocycle the reactions. The Cepheid SmartCycler uses ceramic
`
`heater plates that directly contact the sample vessel.
`
`Summary
`
`The present invention provides a cycling system for use in various temperature(cid:173)
`
`controlled processes, including but not limited to the polymerase chain reaction. The
`
`20
`
`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
`
`25
`
`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.
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`-4-
`
`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
`
`5 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
`
`10
`
`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
`
`15
`
`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,
`
`20
`
`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 ifthe
`
`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
`
`25
`
`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
`
`30
`
`PCR, the heater element of one temperature zone is brought to the closed position, pushing
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`
`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
`
`5
`
`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.
`
`10
`
`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.
`
`Brief Description of Drawings
`
`• FIGS. IA to IE are cross-sectional diagrammatic views of a reaction vessel
`
`15
`
`containing a reaction mixture positioned between heater elements of the present
`
`disclosure.
`
`• FIG. lA 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. lB is a diagrammatic view similar to FIG. lA of the vessel positioned
`
`20
`
`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. lC is a diagrammatic view similar to FIGS. lA and lB showing the bottom
`
`25
`
`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.
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`
`• FIG. lD is a diagrammatic view similar to FIG. lB 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.
`
`5
`
`• FIG. lE is a diagrammatic view similar to FIG. 1 D 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
`
`10
`
`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)
`
`15
`
`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.
`
`20
`
`• 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
`
`25
`
`with the top pair of elements.
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`-7-
`
`• 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.
`
`5
`
`• 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 ofreal-time monitoring of PCR in which
`
`10
`
`DNA amplification is detected by the increase in relative fluorescence in the
`annealing temperature zone. (~. 0, •, D are negative controls; \l .~ , +, + 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
`
`15
`
`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
`
`20
`
`alternative thermocycling subassembly having pneumatic bladders and the
`
`fluorimeter subassembly positioned below the thermocycling subassembly.
`
`Detailed Description of the Drawings
`
`25
`
`A real-time thermal cycling apparatus or system 10 is provided, as shown in FIGS.
`
`5 and 6, for use in temperature controlled processes such as amplification of DNA by PCR
`
`or cycle-sequencing, for example, and optionally for use in detecting and analyzing a
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`-8-
`
`reaction by monitoring fluorescence. Illustratively, system 10 is used as a biological agent
`
`identification system for specifically identifying organisms by their unique genetic
`
`makeup. System 10 includes a thermocycling subassembly 12 and a fluorimeter
`
`subassembly 38. In general, thermocycling subassembly 12 subjects a reaction mixture or
`
`5
`
`sample 16 (shown in FIGS. lA-lE) including a nucleic acid sample to temperature
`
`cycling, or repeated rounds of heating and cooling, illustratively, for denaturation of the
`
`nucleic acid sample and for primer annealing and elongation. The samples 16 are sealed
`
`inside flexible plastic film vessels 18 and actuators of subassembly 12 squeeze the vessels
`
`18 back and forth so that samples 16 are moved between two or more temperature zones.
`
`10
`
`Subassembly 38 detects and analyzes the reaction in real-time by monitoring cycle
`
`dependent and/or temperature-dependent fluorescence. System 10 further includes a
`
`vertical support structure shown as mechanical breadboard 36 and a base 46 coupled to
`
`support structure 36. As is shown in FIG. 5, each subassembly 12, 38 is mounted on
`
`structure 36. Each of the subassemblies 12, 38 is discussed in greater detail below.
`
`15
`
`As mentioned above, reaction mixture 16 includes a nucleic acid sample, for
`
`example, and is contained within a single, soft-sided reaction vessel 18, as shown in FIG.
`
`2A. Vessel 18 is used in system 10 and includes a reaction vessel body 19 and a
`
`receptacle 24 for receiving the nucleic acid sample 16. The reaction vessel body 19 is
`
`formed between two planar faces of plastic sheeting sealed together to form sealed sides
`
`20
`
`22. Individual vessels 18, therefore are separated from neighboring vessels 18 by sealed
`
`sides 22. Liquid can be loaded into the receptacle 24 and moved to the reaction vessel
`
`body 19, that is initially squeezed flat, by means such as gravity or a vacuum applied to
`
`the outer walls of the vessel 18. Once samples 16 are loaded into the reaction vessel body
`
`19, the vessel 18 can be sealed to create seal 21 by heat-sealing, clamping, or through the
`
`25
`
`use of adhesives, for example.
`
`Alternatively, the receptacle 24 can be fitted with a plastic fitment (not shown)
`
`manufactured from polypropylene, for example. Each vessel body 19 may be tapered at
`
`the top to a point (not shown) with the plastic fitment coupled thereto so that through use
`
`of either a pipette or a syringe-like plug, samples 16 could be forced into the reaction
`
`30
`
`vessel body 19. Each plastic fitment attaches to the top of a respective vessel body 19 and
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`-9-
`
`includes an injection port into the respective vessel body. Liquid reagents, therefore, may
`
`be injected into body 19 using a pipette, for example. Excess air may then be squeezed
`
`out of body 19 prior to loading the vessels 18 into the thermocycling subassembly 12 for
`
`heat-sealing and thermal cycling, as is described in greater detail below.
`
`5
`
`Additionally, an illustrative polypropylene fitting or plastic cap 23 (shown in
`
`FIGS. 2A and 2B) may be used to provide a secondary closure of the vessels 18. The
`
`plastic caps 23 may also be used to seal the vessel body 19 at the fitment, thereby
`
`obviating the need to seal body 19 at sealing area 21. It is within the scope of this
`
`disclosure for such a plastic cap 23 to be threadably attached to vessel body 19, snap-fit
`
`10
`
`onto or within vessel body 19, and/or melted into body 19, etc.
`
`In yet another alternative embodiment, a larger plastic fitting or fitment (not
`
`shown) may also be used to allow sample 16 to be freeze-dried inside a plurality of
`
`openings in the fitting, for example, twelve openings. A single injection port is connected
`
`to several of the reaction vessels 18, and when a prepared DNA sample is inserted into the
`
`15
`
`port, the sample 16 is drawn into the body 19 of each vessel 18 automatically by the force
`
`of the vacuum. This automatic distribution of samples 16 may be used for testing sample
`
`16 for multiple pathogens or multiple genes from a single source. A syringe plunger is
`
`inserted into the top of the fitting and is pressed down automatically at the end of the
`
`freeze-drying process, thus sealing the reagent pellet in vacuum. The body 19 is then
`
`20
`
`vacuum sealed inside a protective bag for long-term stability. See U.S. Provisional
`
`Application No. 60/374,730, filed April 23, 2002, herein incorporated by reference.
`
`Vessel body 19 is made of a flexible material. Such flexible materials include but
`
`are not limited to thin plastic films, foil, or soft composite materials, provided that they
`
`can hold the reaction mixture 16, and can withstand repeated exposure to temperatures
`
`25
`
`used in the reaction without deformation, crazing, cracking, or melting. Plastic films of
`
`polyester, (PET), polycarbonate, polypropylene, polymethylmethacrylate, and alloys
`
`thereof made by coextrusion, plasma deposition, lamination or the like are preferred.
`
`Plastics with aluminum lamination, or the like, are also preferred. Further, vessel body 19
`
`has a coefficient of heat transfer approximately in the range of0.02-20 W/m*degK.
`
`30 Because vessel body 19 is thin, it does not effectively transfer heat between portions of
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`
`vessel body 19 in contact with different heaters at different temperatures, as is discussed
`
`below.
`
`If fluorescence monitoring of the reaction is desired, through the use of
`
`subassembly 38, for example, plastic films that are adequately low in absorbance and auto-
`
`5
`
`fluorescence at the operative wavelengths are preferred. Such material could be found by
`
`trying different plastics, different plastisizers and composite ratios, as well as different
`
`thickness of the film. For plastics with aluminum or other foil lamination, the portion of
`
`the vessel 18 that is to be read by the fluorescence detection device 3 8 can be left without
`
`the foil. In the example of PCR, film laminates composed of polyester (Mylar, Dupont,
`
`10 Wilmington DE) of about 0.0048 inch (0.1219 mm) thick and polypropylene films of
`
`0.001 - 0.003 inch (0.025 - 0.076 mm) thick perform well. Illustratively, vessel body 19 is
`
`made of a clear material so that the vessel body 19 is capable of transmitting
`
`approximately 80% - 90% of incident light.
`
`To perform simultaneous reactions of multiple samples, the vessels 18 are
`
`15
`
`illustratively arranged to form an array or row 20 ofreaction vessels 18, as shown in FIG.
`
`2B. In the illustrated embodiments, the vessels 18 are arranged with 9 mm or 6 mm
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`spacing to mimic the spacing found on standard 96-well or 384-well microtiter plates.
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`However, it is within the scope of this disclosure to include a row 20 of vessels 18 having
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`other suitable spacing. FIG. 2B illustrates the row or array 20 of vessels 18 to includes
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`20
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`twelve reaction vessels 18. It is within the scope of this disclosure, however, to include
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`other configurations having other numbers of vessels 18 for use with system 10 of the
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`present disclosure. When used with fluroescence, for example, for real-time monitoring, a
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`bottom edge 14 of sealed sides 22 may be blackened to reduce bleed-over of fluroescent
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`light from one sample vessel 18 to the next.
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`25
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`As mentioned above, vessels 18 (or row 20 of vessels 18) are used with
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`thermocycling subassembly 12, shown in FIGS. 3-5. Illustratively, as shown in FIG. 3,
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`thermocycling subassembly 12 includes a mounting support 32 having a first wall 48 and a
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`second wall 50 spaced apart from first wall 48 and coupled to first wall 48 by spacers 30.
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`Illustratively, there are four spacers 30 coupled to each of the first and second walls 48, 50
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`30
`
`of support 32. Mounting support 32 further includes mounts 51 coupled to first wall 48 so
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`that mounting support 32 may be coupled to support structure 36, as shown in FIG. 6, for
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`example.
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`Illustratively, thermocycler subassembly 12 further includes four heaters: a first
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`heater 25, a second heater 26, a third heater 27, and a fourth heater 28, as shown in FIG. 3
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`5
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`and as shown diagrammatically in FIGS. lA-lE. Each heater 25, 26, 27, 28 includes a
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`head 54 having a first surface 56 facing the one or more vessels 18, as is discussed below,
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`and an opposite surface 58. Illustratively, each head 54 is generally rectangular in shape,
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`as shown in FIG. 3. However, it is within the scope of this disclosure to include a head
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`having any suitable shape for thermally contacting vessels 18.
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`10
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`Each heater 25, 26, 27, 28 further includes a heater element 60 coupled to the
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`contact surface 56 of each heater 25, 26, 27, 28. Heater element 60 illustratively may be a
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`thin-film metal heater or one or more circuit-board based heaters, or a combination of the
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`two. Metallic (e.g. copper) wires or traces may be etched into the circuit-board based
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`heaters. Each heater head 56 is illustratively a metal plate so that heat produced by heater
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`15
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`elements 60 may be uniformly dispersed or distributed. Each head or metal plate 56,
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`however, is an optional component of the heaters 25, 26, 27, 28. Circuit-board based
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`heater elements provide heating by controlling the voltage across the metallic traces and
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`provide temperature sensing by measuring the resistance of the metallic traces. It is within
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`the scope of this disclosure, however, to include other types of temperature sensors. A
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`20 microprocessor can be used to read the calibrated temperature of the circuit-board and
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`control the voltage to achieve the desired temperature. It is also within the scope of this
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`disclosure to include an outer un-etched copper layer of each heater element 60 to increase
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`temperature uniformity. Thick metal heaters and Peltier devices are also an option if the
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`device need not be small. Optional active heating can be performed at the higher
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`25
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`temperature zone(s) by applying heat purges, or the like, prior to or during sample contact.
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`Optional active cooling could be used at the lower temperature zone(s) by use of heat
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`sinks 52 or the like, which are to be in contact with the heating elements (shown in FIGS.
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`ID and IE) for appropriate durations of time, as is discussed below.
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`As shown in FIG. 3, and diagrammatically in FIGS. lA-lE, each of the first and
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`30
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`second heaters 25, 26 is rigidly coupled to first wall 48 of mounting support 32 by a shaft
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`33. Each shaft 33 is rigidly coupled to first wall 48 so that first and second heaters 25, 26
`
`remain stationary throughout the temperature cycling process, as is described below. Each
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`of the second and third heaters 27, 28 are movably coupled to second wall 50 of mounting
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`support 32 by a shaft or linear bearing 34. As shown in FIGS. 4A-4C, each shaft 34 is
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`5
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`rigidly coupled to respective heaters 27, 28 so that heaters 27, 28 are urged to move with
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`each respective shaft 34 relative to wall 50.
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`Further, second and third heaters 26, 27 create a first, upper temperature zone 66
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`and first and fourth heaters 25, 28 create a second, lower temperature zone 68.
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`Illustratively, the upper zone 66 is provided for denaturation of the sample 16 while the
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`10
`
`lower zone 68 is provided for primer annealing and extension. The heaters of each zone
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`66, 68 are programmed to maintain a certain predefined temperature for the heating and
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`cooling of mixture 16 within each vessel 18. As such, zone 66 (including second and third
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`heaters 26, 27) is maintained at a different temperature than zone 68 (including first and
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`fourth heaters 25, 28). The upper and lower zones 66, 68 of heaters are diagrammatically
`
`15
`
`shown in FIGS. lA-lE, for example, and are discussed in greater detail below. Further, it
`
`is within the scope of this disclosure to include a thermocycling subassembly 12 having
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`additional temperature zones (and thus more heaters) than upper and lower zones 66, 68
`
`described herein. For example, third and fourth temperature zones 70, 72 are illustratively
`
`shown in FIG. lA.
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`20
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`Thermocycler subassembly 12 further includes a first stepper motor 29 and a
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`second stepper motor 31, as shown in FIG. 3. First stepper motor 29 is coupled to and
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`controls third heater 27 positioned in the first, upper zone 66. Second stepper motor 31 is
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`coupled to and controls fourth heater 28 positioned in the second, lower zone 68. Each
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`stepper motor 29, 31 is provided to move the respective heaters 27, 28 in a linear path
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`25
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`along an axis lying along the length of shafts 34. Although stepper motors 29, 31 are
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`shown, it is within the scope of this disclosure to include any suitable type of
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`electromechanical mover or actuator such as servo motors, geared motors, solenoids,
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`piezo-electric devices, etc., for example.
`
`FIG. IA diagrammatically illustrates a single soft-sided reaction vessel 18
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`30
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`sandwiched between heaters 25, 26, 27, 28 for use with system 10, and specifically with
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`subassembly 12, of the present disclosure. As mentioned above, the heaters create two or
`
`more temperature zones: first, upper zone 66 and second, lower zone 68. Each of the
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`third and fourth heaters 27, 28 is movable between an opened and a closed position
`
`because ofrespective stepper motors 29, 31. As shown in FIG. IA, all sets of heaters are
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`5
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`illustratively in an open position for ease in loading vessels 18 therein. As described
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`below and shown in FIGS. lB-lE, the reaction mixture 16 inside the reaction vessel 18 is
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`incubated in a particular temperature zone when the particular heater within that
`
`temperature zone is held in the opened position while all o