US 20060088931 A1
Flexible vessels for thermal cycling a sample are provided, the flexible vessels comprising a flexible body having first and second portions coupled together and in fluid communication with each other, wherein the flexible body is configured for insertion into a thermal cycler such that the first portion is received into a first temperature zone and the second portion is received into a second temperature zone of the thermal cycler. Systems comprising the thermal cycler and flexible vessel are also provided.
1. A flexible vessel for thermal cycling a sample, the flexible vessel comprising
a soft-sided reaction vessel comprising,
a receptacle for receiving the sample, and
a flexible body coupled to the receptacle, the flexible body having first and second portions coupled together and in fluid communication with each other, wherein the flexible body is configured for insertion into a thermal cycler such that the first portion is received into a first temperature zone and the second portion is received into a second temperature zone of the thermal cycler.
2. The vessel of
3. The vessel of
4. The vessel of
5. The vessel of
6. A flexible vessel for thermal cycling a sample, the flexible vessel comprising two faces of flexible material that are sealed together to form a plurality of flexible bodies, each flexible body separated from its adjacent flexible body by a sealed side, and each flexible body having a first portion and a second portion, the first and second portions coupled together and in fluid communication with each other, wherein the flexible bodies are configured for insertion into a thermal cycling apparatus such that the first portion of each flexible body is received into a first temperature zone and the second portion of each flexible body is received into a second temperature zone of the thermal cycler.
7. The vessel of
8. The vessel of
9. The vessel of
10. The vessel of
11. The vessel of
12. The vessel of
13. The vessel of
14. A thermal cycling system comprising
the reaction vessel of
the first temperature zone of the thermal cycler is configured for receiving the first portion of the reaction vessel, the first temperature zone comprising a first heater, the first temperature zone movable between an open orientation in which the first heater affects the temperature of the reaction mixture contained within the first portion, and a closed orientation in which the reaction mixture is forced from the first portion and the first heater does not substantially affect the temperature of the reaction mixture, and
the second temperature zone of the thermal cycler is configured for receiving the second portion of the reaction vessel, the second temperature zone comprising a second heater, the second temperature zone movable between an open orientation in which the second heater affects the temperature of the reaction mixture contained within the second portion, and a closed orientation in which the reaction mixture is forced from the second portion and the second heater does not substantially affect the temperature of reaction mixture.
15. The thermal cycling system of
16. The thermal cycling system of
17. The thermal cycling system of
18. The thermal cycling system of
This application is a continuation of U.S. patent application Ser. No. 10/478,453, filed Nov. 21, 2003, which is U.S. national counterpart application of international application serial No. PCT/US02/22543, filed Jul. 16, 2002, which claims priority to U.S. Provisional Application No. 60/305,632, filed Jul. 16, 2001.
The present invention relates to a thermal cycling device and method that 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.
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.
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.
A real-time thermal cycling apparatus or system 10 is provided, as shown in
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
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 vessel body 19. Each plastic fitment attaches to the top of a respective vessel body 19 and 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.
Additionally, an illustrative polypropylene fitting or plastic cap 23 (shown in
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 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 vacuum sealed inside a protective bag for long-term stability. See U.S. Provisional Application No. 60/374,730, filed Apr. 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 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 of 0.02-20 W/m*degK. Because vessel body 19 is thin, it does not effectively transfer heat between portions of 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-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 38 can be left without the foil. In the example of PCR, film laminates composed of polyester (Mylar, Dupont, Wilmington Del.) 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 illustratively arranged to form an array or row 20 of reaction vessels 18, as shown in
As mentioned above, vessels 18 (or row 20 of vessels 18) are used with thermocycling subassembly 12, shown in
Illustratively, thermocycler subassembly 12 further includes four heaters: a first heater 25, a second heater 26, a third heater 27, and a fourth heater 28, as shown in
Each heater 25, 26, 27, 28 further includes a heater element 60 coupled to the contact surface 56 of each heater 25, 26, 27, 28. Heater element 60 illustratively may be a thin-film metal heater or one or more circuit-board based heaters, or a combination of the two. Metallic (e.g. copper) wires or traces may be etched into the circuit-board based heaters. Each heater head 56 is illustratively a metal plate so that heat produced by heater elements 60 may be uniformly dispersed or distributed. Each head or metal plate 56, however, is an optional component of the heaters 25, 26, 27, 28. Circuit-board based heater elements provide heating by controlling the voltage across the metallic traces and provide temperature sensing by measuring the resistance of the metallic traces. It is within the scope of this disclosure, however, to include other types of temperature sensors. A microprocessor can be used to read the calibrated temperature of the circuit-board and control the voltage to achieve the desired temperature. It is also within the scope of this disclosure to include an outer un-etched copper layer of each heater element 60 to increase temperature uniformity. Thick metal heaters and Peltier devices are also an option if the device need not be small. Optional active heating can be performed at the higher temperature zone(s) by applying heat purges, or the like, prior to or during sample contact. Optional active cooling could be used at the lower temperature zone(s) by use of heat sinks 52 or the like, which are to be in contact with the heating elements (shown in
As shown in
Further, second and third heaters 26, 27 create a first, upper temperature zone 66 and first and fourth heaters 25, 28 create a second, lower temperature zone 68. Illustratively, the upper zone 66 is provided for denaturation of the sample 16 while the lower zone 68 is provided for primer annealing and extension. The heaters of each zone 66, 68 are programmed to maintain a certain predefined temperature for the heating and cooling of mixture 16 within each vessel 18. As such, zone 66 (including second and third heaters 26, 27) is maintained at a different temperature than zone 68 (including first and fourth heaters 25, 28). The upper and lower zones 66, 68 of heaters are diagrammatically shown in
Thermocycler subassembly 12 further includes a first stepper motor 29 and a second stepper motor 31, as shown in
After an appropriate duration, heater 27 is moved to the open position so that zone 66 is opened and heater 28 is moved to the closed position so that zone 68 is closed, thus moving the reaction mixture 16 into the upper portion of vessel 18 and into full thermal contact with second and third heaters 26, 27 of upper zone 66, as shown in
Illustratively, subassembly 12 features an active area between the heaters 110 mm wide and 50 mm high. This is large enough to accommodate twelve reaction vessels 18 (such as array 20) of 100 μl, or 9 mm of spacing. However, it is within the scope of this disclosure to include a device having another suitably sized active area for vessels 18.
As shown in
In the operation of the two-temperature thermal cycling system 10 illustrated, a strip or row 20 of reaction vessels 18 is loaded into the active area of subassembly 12, as shown in
While the illustrated embodiment of the thermocycling subassembly 12 shown in
One way to accomplish rapid uniform heating and cooling of the reaction mixture 16 is to maintain a small distance between heaters 25 and 28 and between heaters 26 and 27 (or, vessel receptacle gap 35) in the active temperature zone. However, it is also conceivable that rapid temperature uniformity of the reaction mixture 16 can be achieved by agitation of the system 10. Illustratively, for temperature transitions used in two-temperature PCR systems, a vessel receptacle gap 35 of about 0.1 mm to about 2 mm is preferred, with 0.25 to 1 mm being the most preferred, for the active temperature zone when respective heaters are in the opened position for effective heating or cooling of the reaction mixture. When respective heaters are in the closed position, illustratively a vessel receptacle gap 37 is brought as close as possible to the thickness of the fully collapsed sample vessel, illustratively approximately 0.1 to 0.15 mm. Furthermore, illustratively, the speed of closing and opening of the heaters 29 within each respective zone is relatively fast. Again, in the case of two-temperature PCR systems, a closing and opening speed of 5 mm/s to 0.01 mm/s is preferred, with about 1 mm/s being the most preferred. However, it is within the scope of this disclosure for heaters 27, 28 to open and close at other suitable speeds. Further, it is understood that in some applications, slower temperature transitions may be preferred, with concomitant slower closing and opening speeds.
Looking now to
The first, upper set 129 of bladders includes upper bladders 144, 146 coupled to movable heater 27 and the second, lower set 131 of bladders includes lower bladders 148, 150 coupled to movable heater 28. Bladders 144, 146, 148, 150 are each illustratively manufactured by heat-sealing a polyethylene/polypropylene laminate film onto a pneumatic fitting 128. The seals are arranged such that rounded, rectangular areas are created and positioned adjacent the heaters. Illustratively, each bladder 144, 146, 148, 150 (and each respective heater 25, 26, 27, 28 coupled thereto) generally runs the length of apparatus 110 so that each bladder 144, 146, 148, 150 affects all vessels 18 within the array 20 of vessels 18. Further each bladder 144, 146, 148, 150 is illustratively coupled to two pneumatic fittings 128, although only one fitting 128 is shown in cross-section in
In operation, air is forced into the fittings 128 to inflate each plastic film bladder 144, 146, 148, 150 thus forcing the heater elements 60 coupled to each movable heater 27, 28 into contact with vessels 18 and respective heater elements 60 of stationary heaters 25, 26 to force the liquid sample 16 within each vessel 18 into the other heating zone. Apparatus 110 may include a rigid mechanical support 126 coupled to each set 129, 131 of bladders and an insulator 133 coupled to each support 126. Illustratively, mechanical support 126 is made of a metal or carbon fiber composite strips, however, it is within the scope of this disclosure for support 126 to be made of any suitable material. As shown, one insulator 133 is coupled to heater 27 and another insulator 133 is coupled to heater 28. Each insulator 133 is made of an insulating material so that the temperature of each respective heater 27, 28 may be more consistently maintained.
As shown in
As shown in
A controller board (not shown) is also provided and includes a heater board retraction spring, MAC valves, and receptacles for fittings 128. Apparatus 110 is illustratively battery operated and includes an internal pneumatic system, described above, including first and second sets 129, 131 bladders 144, 146 and 148, 150. Illustratively, first set 129 of bladders 144, 146 acts as a first mover or actuator of the system and second set 131 of bladders 144, 146 acts as a second mover or actuator of the system. A disposable twenty-five gram carbon dioxide cylinder 132 is illustratively used to drive the pneumatic system. While
As shown in
Illustrative automatic calling software may be provided to analyze the samples by a multi-test analysis method described in U.S. patent application Ser. Nos. 10/074,178 and 10/117,948, herein incorporated by reference, with the following modifications and additions. Multiple points (for example, 10, 20, 30, or 50 points) are interrogated across a bottom 15 of each individual sample vessel 18 to acquire multiple fluorescence values, and the median of those fluorescence values for each time point is used as input to the algorithm. Data points from portions of the sample vessel 18 that are close to seal 22 are preferably not used due to edge effects in fluorescence. Prior to taking the median, the software also compares the individual fluorescence values with data acquired from the cycle preceding the present acquisition, and ignores those values that are significantly different in value. This allows the software to ignore portions of the sample vessel 18 which further generate erroneous fluorescence signal due to the appearance, or drift, of air bubbles and other interfering particles in the reaction. It is understood that other numerical methods besides taking a median value may be sued to reduce the many measurements taken across the bottom to a single fluorescence value for each sample. These include Fourier transformation, averaging, fitting to known functions, and stored standards. Finally, in an illustrated embodiment, the classification of a sample (i.e. “positive” or “negative” for the presence of an analyte) is reported to the user only if the automated calls have registered the expected results in the positive and negative controls, and optionally, only if duplicate reactions or alternative gene loci provide concordant results with the sample. Illustratively, if the automated calls are inconsistent with the expected calls in the positive and/or negative controls, then the software will report to the user that the reaction needs to be repeated. If the result of the duplicate reaction, or the alternative gene loci, is inconsistent with the sample, the software will report the inconsistency and will not call the sample “positive” or “negative”. Apparatus 110 displays the result in the display screen 136 and allows users to look deeper at the specific reactions if they choose.
As mentioned above, apparatus 110 further includes gas chamber 132 for providing compressed gas to the first and second sets 129, 131 of pneumatic bladders 144, 146, 148, 150. As shown in
Looking now to
Subassembly 112 also includes an eleven-valve manifold coupled to the bladders 144, 146, 148, 150 to regulate the carbon dioxide gas into and out of each bladder 144, 146, 148, 150. Although an eleven-valve manifold is disclosed herein, it is within the scope of this disclosure to include a manifold having another suitable number of valves to operate bladders 144, 146, 148, 150. The carbon dioxide gas within gas chamber 132 is regulated to 30 psi and is switched through the manifold to minimize electrical losses from the valves. First set 129 of pneumatic bladders 144, 146 forces movable heater 27 toward stationary heater 25 while second set 131 of pneumatic bladders 148, 150 forces movable heater 28 toward stationary heater 26 in order to force samples 16 within each vessel 18 between lower and upper temperature zones 66, 68.
In operation, the illustrative sets 129, 130 of bladders 144, 146 and 148, 150 are able to produce a rocking motion on respective movable heaters 27 and 28 to allow mechanical mixing of the samples 16 within each temperature zone 66, 68. Mixing the samples 16 by the rocking motion of the heaters 27, 28 increases temperature uniformity within each sample 16 and aids in positioning each sample 16 for optimal fluorescence measurement at the bottom of each respective vessel body 19.
Subassembly 112 further includes a sealing mechanism 142, shown in
The foregoing description of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms described, and obviously many other modifications are possible in light of the above teaching. The embodiments were chosen in order to explain most clearly the principles of the invention and its practical applications, thereby to enable others in the art to utilize most effectively the invention in various other embodiments and with various other modifications as may be suited to the particular use contemplated.