EP1252931A1 - Dispositif de traitement par cycle thermique pour l'amplification de séquences d' acides nucléiques - Google Patents

Dispositif de traitement par cycle thermique pour l'amplification de séquences d' acides nucléiques Download PDF

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Publication number
EP1252931A1
EP1252931A1 EP01401073A EP01401073A EP1252931A1 EP 1252931 A1 EP1252931 A1 EP 1252931A1 EP 01401073 A EP01401073 A EP 01401073A EP 01401073 A EP01401073 A EP 01401073A EP 1252931 A1 EP1252931 A1 EP 1252931A1
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EP
European Patent Office
Prior art keywords
fluid
tanks
support
transfer
temperature
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01401073A
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German (de)
English (en)
Inventor
Ivo Glynne Gut
Noah Peter Christian
David Serge Malinge
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Centre National de Genotypage
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Centre National de Genotypage
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Publication date
Application filed by Centre National de Genotypage filed Critical Centre National de Genotypage
Priority to EP01401073A priority Critical patent/EP1252931A1/fr
Publication of EP1252931A1 publication Critical patent/EP1252931A1/fr
Withdrawn legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Rigid containers without fluid transport within
    • B01L3/5085—Rigid containers without fluid transport within for multiple samples, e.g. microtitration plates
    • B01L3/50851—Rigid containers without fluid transport within for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/54—Heating or cooling apparatus; Heat insulating devices using spatial temperature gradients

Definitions

  • the present invention relates to thermal cycle devices, and in particular those used for high-throughput analysis of biological samples mainly by amplification of nucleic acid sequences, using for example the techniques of polymerase chain reaction, ligase chain reaction, primer extension, enzymatic cleavage of deoxy- and ribonucleic acids or the like.
  • PCR Polymerase Chain Reaction
  • SNP Single Nucleotide Polymorphism
  • the PCR is now almost routinely used in biochemical laboratories, and serves to illustrate the methods by which thermal cycling is typically achieved.
  • the very first methods for this thermal cycling involved placing the required components for PCR in a test tube, small beaker, or a flask and sealing the top. This vessel was then placed in a thermostatically controlled bath to achieve equilibrium with the bath temperature. Cycling between three temperatures (close to 100 °C to denature the DNA, 55 °C to anneal a primer to a single stranded template, and 75°C to promote elongation of a DNA copy) was then accomplished by transferring this vessel between three baths.
  • Peltier-heated and cooled thermal cycle devices became popular. These units had the particular advantage that the heating and cooling of a sample occurred on a single thermostatically controlled plate. Thus samples could be placed into a small tube, sealed, and then the reaction could progress on a single unit with no transfer to other locations for the heating and cooling cycles. As a consequence of the lack of any mechanical intervention, systems were also rapidly integrated with computerized control devices. Because there was no mechanical motion of the sample during the PCR, it was also learned that one could seal a sample by placing mineral oil on top of the sample, thus further simplifying the mechanical aspects of small-volume liquid handling.
  • High-throughput machines have an increased demand for robustness and rapid and automated handling when compared to a research cycle device. While research cycle devices have relied on Peltier elements, high-throughput instruments typically require plate sealing and immersion in a bath. Several machines have been recently described that use a different approach than the Peltier element thermal cycle device. US patent n°5-504 007 describes a closed fluid system with two tanks. The fluid is transferred through a plastic or ceramic block, which allows for thermal equilibration to a new temperature. A modular high-throughput device is described in US patent 5 601 141, where an integral temperature sensor and controller is incorporated on each block.
  • All thermal cycle devices of the Peltier variety comprise a large area underneath the thermal block that contains a large heat-sink cooled by air.
  • instant invention provides a thermal cycle device comprising:
  • the invention may also show at least one of the following features:
  • the invention also provides a process for thermal cycling of samples, comprising the steps of:
  • This process may be used for amplification of nucleic acid sequences.
  • the samples intended to be cycled are disposed in a plurality of vessels that are thermostatically controlled by a temperature regulation block and lid.
  • Each sample vessel is sealed by a sealing tape, a viscoelastic lid, a pressure plate, oil and/or other low density, low-volatility liquid placed above the desired sample.
  • oil is used to seal the top of the sample.
  • a pressure plate could be positioned above the sample already sealed by viscoelastic means, and that this lid could furthermore be thermostatically regulated by means of a Peltier (thermoelectric) heating/cooling device.
  • the device may comprise one or several such blocks.
  • Criteria for high-throughput operation by the device are robustness, simplicity in design, a high density of blocks presentable to external handling systems, high thermal stability and predictable thermal performance, and extensibility of design in incorporating additional elements.
  • Previous designs in thermal cycle devices fail in one or more of the aforementioned criteria.
  • the form factor of a commercially available thermal cycle device is such that the thermal control block is a small fraction of the overall size of the unit, and thus the space efficiency is poor for an automated system.
  • a heating and cooling block 2 is provided as shown on figure 1. It comprises in instant case a hollow metal element having a circuit 4 therein comprising cavities 6, 8 so that one or multiple heat or sink fluids may be pumped and circulated through the block.
  • the block 2 is made of a highly thermal conductive material but low thermal mass material such as stainless steel, anodized aluminum, Inconel, or Monel could be used.
  • the upper external shape of this block is designed, here, to accept a microtiter plate 10 (such as a conventional 96-well, 384-well, or 1536-well format) as illustrated on figures 2 and 3.
  • the plate 10 comprises an array of identical vertical receptacles 12 for receiving the samples 7.
  • the upper part of the block 2 has a shape complementary to the shape of the lower part of the plate 10.
  • the lower part of the plate has truncated cones defined by the receptacles 12, spaced one from the others.
  • the upper part of the block has housings 11 for receiving the cones. These housings 11 are defined by and between reliefs 13 of the block. Some cavities 8 of the block extend up inside the reliefs 13 for a better control of the sample temperature by the circulated fluid. Accordingly, the upper part of these cavities 8 is conical or partially conical, similarly to the reliefs 13.
  • the final product allows 30 to 60 similar plates 10 to be processed simultaneously.
  • it could comprise an equivalent number of blocks 2. But some blocks could each accommodate several plates.
  • the proof-of-principle and will therefore show a test rig that uses between one and four plates 10 only.
  • the block could otherwise be arranged to receive individual sample tubes.
  • the block is furthermore arranged to minimize the mass of the block.
  • the circuit 6 of the block has an internal construction designed to accept a flow of fluid permitting to afford maximal thermal transfer from the fluid to the wells with minimal temperature gradients across the block.
  • the block and fluidics connecting the block to the tanks as we will see below are designed to maximize thermal transfer from the fluid to the block and to promote even thermal distribution across the block. In our preferred embodiment, this is due in the block to a large internal surface to internal volume ratio and a non-linear flow pattern.
  • the embodiment of the device comprises mixing valves in fluid communication with the tanks and with the block.
  • a baffle system may be inserted that also promotes turbulent mixing in the block.
  • the block 2 comprises fins 14 placed along the underside of the block into the circuit 6 to maximize the thermal contact of the block with the contained fluid. This ensures that the temperature of the block will quickly equilibrate with the temperature of the contained solution.
  • the device contains tanks or reservoirs of fluid 16, 18, 20, here in the number of three, where fluid is heated and/or cooled to maintain a constant temperature in each tank.
  • each tank comprises conventional heating or cooling means 28 in this regard.
  • the fluid temperatures are different in the respective tanks.
  • fluid tanks 16, 18, 20 having respective temperatures of 54, 72, and 98 °C can be used for a PCR thermal cycling. These temperatures are not those used for any PCR. Of course, other temperatures could be encompassed.
  • HS1, HS2 and HS3 are heat sources represented by the tanks 16, 18, 20, and HD1, HD2 and HD3 are heat drains or waste streams.
  • .TR block is the temperature regulation block 2 or an array of blocks that contains suitable channels for the fluid flows. As we shall see below, each heat drain HDi in fact communicates with a corresponding one of the heat source HSi.
  • FIG 4 shows more precisely the arrangement of the fluidics in case the device comprises n blocks 2.
  • Each block 2 (TRB1...TRBn) is in fluid communication with the three tanks 16, 18, 20 so that it can receive fluid from any one of the three tanks and transfer fluid to any of these by different respective fluidics 25, 27. Accordingly, each block 2 is connected to each tank 16, 18, 20 by two pipes 25, 27. The blocks are connected in parallel to the tanks. The tanks are also connected in parallel to the blocks.
  • the device includes one or several manifolds comprising solenoid valves 22 permitting to control the flow of fluid in each of these pipes 25, 27.
  • the device is controlled, as will be explained below, so that the waste stream from each block 2 is recycled into one of the temperature regulated tanks 16, 18, 20, hence being regenerated to the source stream. Moreover, it is recycled in the one tank which temperature is closest to the temperature of the fluid evacuated from the block. Very often, the chosen tank will be the source stream's tank. By directing the waste stream into the tank having the closest temperature, maximum efficiency and thermal stability can be maintained in the heating and cooling system. Such a system has three tremendous advantages over a thermoelectric system. First, the area below the thermal block does not require a chamber that is thermally equilibrated to a second temperature, which is a requirement of a thermoelectric system. Thus, considerable space savings can be achieved in an array, or bank, of such blocks.
  • thermoelectric units the thermal and electrical efficiency of a combined external heating or cooling system is much better than in individual thermoelectric units, and one such unit may serve a large array of these blocks. Furthermore, the thermal conductivity and thermal transfer characteristics of a pumped liquid is far greater at the temperature block, and thus the temperature regulation. Third, the reliability of such a system is much greater than an array of high-cost, low-efficiency thermoelectric elements and is thus better suited to a parallel system.
  • a plurality of temperature blocks or block arrays are configured in this manner, thus affording temperature control for a large number of thermal blocks with a minimum of ancillary heating and cooling systems.
  • the external tanks for heating and cooling are externally heated and/or refrigerated units, such as those commercially available from Lauda, Julabo, Fisher Scientific, Neslab, and others. This choice is not limited to one particular model, as the pumping speed, tank size, heating rate, cooling rate, and thermal stability must be matched to the system.
  • a custom system may be constructed that incorporates the essential elements of such a thermally controlled bath device.
  • a shunt valve 23 may be opened in the apparatus at any time.
  • Each tank may be associated with such a valve 23.
  • the valve directly connects a feed pipe 25 to a waste pipe 27.
  • This shunt valve can be used to equilibrate components in the system to the appropriate tank temperature, to asses the systematic temperature drops from passive radiation, conduction, or convection in the system, or to maintain fluid pressure to a predetermined level in the system. Because this shunt valve may serve a large number of temperature blocks in the system, it has a higher conductance than the manifold valve in each block.
  • Our preferred embodiment comprises six banks each containing six thermally controlled blocks 2, each having a fluid channel of approximately 50 ml.
  • the combined volume of all blocks is 1.8 L.
  • Each recirculation heather/chiller contains 8L of fluid with a 10 L/min pump.
  • Full replacement of all plates 10, assuming an isothermal program, is accomplished in 9.6 seconds.
  • a thermal gradient ranging from 74 to 98 °C would have a heating rate of 2.5 °C per second. If only one bank were changed, this would be 15 °C per second.
  • an actively controlled system is used.
  • each thermal block 2 contains a temperature sensor 24, for example a simple semiconductor sensor, that is in close thermal contact with the block.
  • each block contains a low-cost microcontroller 26 or other suitable control device to maintain some autonomy within the control electronics of the system.
  • control functions may be centralized to a higher-capability microcontroller, a programmable logic controller, a microcomputer, or any other analogic or digital device capable of computation and control.
  • Our preferred embodiment is also shown on figure 6, more precisely, as having for each block 2i a low-cost microcontroller unit MCUi 29 capable of monitoring the temperatures of the inlet and outlet streams and the block i, and of controlling said streams by means of the six regulation valves 22 associated with this block along one of the following control schemes: simple logic, fuzzy logic, proportional integral/differential (PID) logic, linear or nonlinear optimization algorithms, or any combination thereof.
  • a low-cost microcontroller unit MCUi 29 capable of monitoring the temperatures of the inlet and outlet streams and the block i, and of controlling said streams by means of the six regulation valves 22 associated with this block along one of the following control schemes: simple logic, fuzzy logic, proportional integral/differential (PID) logic, linear or nonlinear optimization algorithms, or any combination thereof.
  • a centralized processor 30 may be provided to inform the individual block processors MCU of stream temperatures, desired programmed temperatures, and the status of sample loading, unloading, and simple temperature programs. Furthermore, this processor 30 controls a stepper motor 34 of the device for the sample presentation system. This processor 30 also interfaces with an external processor, controller, or microprocessor that is responsible for the assembly line control. Synchronization between processors, sample verification (by barcode, transponder, or other means), temperature programming instructions, and other essential communication functions are carried out through this interface. This interface 30 replaces a display and keypad that are typically provided on an off-the-shelf thermal cycle device, and reduces unneeded duplication of major control systems. This arrangement furthermore allows for an uninterrupted queue of samples to be loaded, cycled, and unloaded from the system.
  • a typical isothermal temperature is achieved as follows. For regulation at 72 °C of one block, if the actual temperature of the block is at 54°C, then the waste stream for 54 °C (communicating with tank 16) is left opened, the inlet for 54 °C (communicating with the same tank) is closed, and the inlet for 72 °C (communicating with tank 18) is opened. Thus, in block 2, the fluid at 54 °C is progressively replaced with fluid at 72 °C arriving from the corresponding tank. As the block nears 72 °C, the waste stream for 54 °C is closed while simultaneously or at a variable delay the waste stream for 72 °C is opened. It should be understood that, in order to maintain efficient pumping, at least one waste stream must be open during pumping.
  • the circulation of fluid from the tank to the block and from the block to the tank is preferably not interrupted.
  • This example shows the control of the block temperature by receiving the fluid from one tank and wasting the other fluid to another tank.
  • a fluid consisting of a convenient mixture of fluids from at least two of the tanks, especially but not only in order to obtain a temperature ranging between the temperatures of two of the tanks.
  • a fluid consisting of a convenient mixture of fluids from at least two of the tanks, especially but not only in order to obtain a temperature ranging between the temperatures of two of the tanks.
  • another inlet valve 22 may be opened to allow fluid of a different temperature to be mixed with the first one into the system: if the block is monitored to be at 71.5 °C whereas it should be at 72°C, then a small fraction of 98 °C water from tank 20 may be admitted in the block mixed with the water at 72°C arriving from tank 18.
  • the preferred embodiment uses pulse modulation, whereby the open time of the 98 °C valve is modulated in time to affect regulation. Throughout this process, the waste stream for the 72 °C outlet remains open.
  • control is performed by means of the aforementioned control logic.
  • This logic may incorporate means for time-dependant programmatic temperature changes, programmed overshoot to ballistically arrive at a desired temperature, or other means for arriving at an optimal temperature profile.
  • a continuous operation load balancing system consisting of a fluid level sensor 33 in each thermal tank and a leveling system that may be a pump such as a peristaltic pump, impeller, or any other means to maintain the level of each fluid tank.
  • An alternative system contains three thermally isolated systems in which an overflowing tank immediately spills into another of the tanks. In this example, if the 54°C tank is overflowing, this would flow into the 72°C tank by passive means.
  • Fluids for such a heating/cooling system must be of sufficient viscosity, thermal conductivity, and compatibility for this flow system.
  • These fluids may include silicone oil, mineral oil, water, or any other low-viscosity fluid whose characteristics encompass a safe operating range for the system.
  • Julabo thermal liquid type H5S has a working temperature ranging from -40°C to 120°C, with a viscosity in this temperature ranging from 23 mm 2 /s to greater than 5 mm 2 /s.
  • water be used for transferring heat to/from the plates. This is based on the good thermal properties of water, the relatively low cost associated with water equipments and the ability to process multiple plates using the same energy supply.
  • water should be in direct contact with the plate to optimize heat transfer and speed of operation.
  • scientists tend to dislike dealing with wet lab elements. Therefore the use of aluminum block will be preferred.
  • the water is then used as a source of energy to heat up/cool down this block on which the plate is sitting.
  • an array of six banks each containing six thermal blocks 2 are arranged in a circular manner and the location to load or unload is rotated to close proximity of the assembly line.
  • a bank of 36 plates may be arranged in a vertical square format, and a suitable external X/Y/Z robotic arm may load or remove the desired plate 10.
  • FIG. 1 is a top view of the plate arrangement for four plates, where A through D are microtiter plate holders (blocks 2) that are controlled by the aforementioned control systems housed in the central region E.
  • the blocks A to D are arranged around the control system, in the same plane.
  • Arrow 32 shows the direction of rotation of the housing containing the blocks A through D, and it should be noted that while a clockwise direction is drawn, counterclockwise rotation is also possible.
  • FIG. 8 shows the elevation of such a unit as would be seen from an assembly line, the unit presenting 6 superimposed blocks to the assembly line at respective levels 1-6. Only one elevation of blocks is presentable to the assembly line at any given time, thus some flexibility in random access to the plates is sacrificed to maintain the compact nature of the system. It should be noted, of course, that other groups of blocks could be made accessible from other areas in the system. Thus, if the system was placed at a crossroads in an assembly line, two groups of plates would be accessible without rotation of the unit.
  • the invention provides a compact, simple, and reliable means for high-throughput thermal cycling procedures.
  • An arrangement of the systems stacked and/or on a turntable system permits the compact arrangement of a large number of thermostatically controlled blocks.
  • the temperature regulation blocks are mounted on a tray that may be moved into and out of the remainder of the system.
  • a possible sequential transfer of samples to and from the apparatus allows for continuously uninterrupted operation of the apparatus.
  • the registration of loading and unloading from the apparatus ensures proper identification and tracking of samples.
  • the apparatus may be used for high-throughput thermal cycling procedures such as PCR, LCR, primer extension or the like.
  • another embodiment of the invention may use only two tanks, one very hot the other very cold. Water from these tanks would then be mixed using electrically controlled 3-port modulating valves. The temperature of the hot tank would be close to boiling temperature of water while the other tank would be as cold as possible to allow optimized control of the mixed water temperature. But calculations show that the power required to keep the cold bath at a suitable temperature (>10kW) would not be acceptable in many cases.
  • Two fluid tanks 18 and 20 are heated to temperatures of 75°C and 98°C respectively, thus higher than the denaturing and elongating respective temperatures used for the PCR.
  • a third fluid tank 16 is cooled to a temperature of 50°C thus slightly lower than the annealing temperature.
  • Three pumps 40 are used, one for each tank, as well as pressure relief valves 42 which are situated in parallel with each pump 40 to ensure that constant pressure is achieved in the circuit.
  • Two 3-port mixing valves 44 are used to achieve accurate control of the temperature.
  • the first one may mix the fluids arriving from tanks 18 and 20.
  • the second one may mix the later mixture with the fluid of the third tank 16.
  • Water is pumped through the block 2 and back to the tanks through two 3-port on/off valves 46.
  • Valves 46 are arranged in the waste path in the same way as the valve 44 in the feed path. Accordingly, waste water is distributed by the first valve 46 to the tank 20 or to the group of tanks 16, 18. In the later case, the water is distributed by the second valve 46 to the tank 16 or to the tank 18. Again, the waste tank is chosen each time as having the closest temperature to the temperature of the evacuated water.
  • the temperature of the water in the block 2 is accurately controlled thanks to a thermocouple 50 reading the temperature of water coming out of the block.
  • the block 2 holds four plates 10.
  • blocks would be removable resulting in the need for an on/off valve that allows the circuit to be closed when no block is present or being processed.
  • Means 60 are provided for overflow of water from one tank to another in case the level in one tank is two high.
  • valves 23 and means 60 permit transfer of fluid from one of the tanks to one of the others directly or indirectly but without passing through the blocks.
  • thermal cycle devices also contain elements that are not necessary in our embodiments and include a user accessible control panel and/or display device, readily re-assignable temperature programs, and other sophisticated programmatic features. These elements are of a relatively high level and are more suited for individual machines, research and development on a small scale. They add sophistication and expense that limit robustness and reliability for a high-throughput system. Similarly, some sophisticated state-of-the-art machines contain elements such as thermal gradients that are more applicable to research and development and are thus not included in our preferred embodiments. But one skilled in the art will of course realize that if a high-throughput assay were developed that required the use of a thermal gradient, this gradient could be incorporated into the present invention by means of multiple heating and/or cooling units in one block.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
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EP01401073A 2001-04-26 2001-04-26 Dispositif de traitement par cycle thermique pour l'amplification de séquences d' acides nucléiques Withdrawn EP1252931A1 (fr)

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EP01401073A EP1252931A1 (fr) 2001-04-26 2001-04-26 Dispositif de traitement par cycle thermique pour l'amplification de séquences d' acides nucléiques

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EP01401073A EP1252931A1 (fr) 2001-04-26 2001-04-26 Dispositif de traitement par cycle thermique pour l'amplification de séquences d' acides nucléiques

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7030340B2 (en) 2003-06-04 2006-04-18 Siemens Aktiengesellschaft Thermocycler

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3808942A1 (de) * 1988-03-17 1989-09-28 Bio Med Gmbh Ges Fuer Biotechn Inkubator, insbes. fuer die polymerase-ketten-methode
US5290101A (en) * 1992-12-03 1994-03-01 At&T Bell Laboratories Liquid thermal cycling methods and apparatus
WO1995018676A1 (fr) * 1994-01-11 1995-07-13 Abbott Laboratories Appareil et procede de cyclage thermique de dosages d'acides nucleiques
US5508197A (en) * 1994-07-25 1996-04-16 The Regents, University Of California High-speed thermal cycling system and method of use

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3808942A1 (de) * 1988-03-17 1989-09-28 Bio Med Gmbh Ges Fuer Biotechn Inkubator, insbes. fuer die polymerase-ketten-methode
US5290101A (en) * 1992-12-03 1994-03-01 At&T Bell Laboratories Liquid thermal cycling methods and apparatus
WO1995018676A1 (fr) * 1994-01-11 1995-07-13 Abbott Laboratories Appareil et procede de cyclage thermique de dosages d'acides nucleiques
US5508197A (en) * 1994-07-25 1996-04-16 The Regents, University Of California High-speed thermal cycling system and method of use

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7030340B2 (en) 2003-06-04 2006-04-18 Siemens Aktiengesellschaft Thermocycler

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