EP1252931A1

EP1252931A1 - Thermal cycle device for amplification of nucleic acid sequences

Info

Publication number: EP1252931A1
Application number: EP01401073A
Authority: EP
Inventors: Ivo Glynne Gut; Noah Peter Christian; David Serge Malinge
Original assignee: Centre National de Genotypage
Current assignee: Centre National de Genotypage
Priority date: 2001-04-26
Filing date: 2001-04-26
Publication date: 2002-10-30

Abstract

The thermal cycle device comprises:

a support (2) for samples ;
at least two fluid tanks (16, 18, 20) ;
means (28) for keeping the fluid of each tank at a given respective temperature, the temperatures associated with the respective tanks being different; and
means (25) for transferring fluid from the tanks to the support.

Description

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.
Various biochemical manipulations on DNA and RNA involve the repeated heating and cooling of samples. PCR (Polymerase Chain Reaction) has been identified as a key step in most SNP (Single Nucleotide Polymorphism) genotyping protocols.
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.
Subsequent to these early PCR reactions, 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. A series of US patents 5 475 610, 5 602 756, 5 710 381, 5 725 381, and 6 015 534 describe a system where fluid is used for cooling and a thin heater is used for heating. Open fluidic systems have also been described. In one case (US patent 5 942 432), a turbulent fluid flow is directed to a sample holder.
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.
These devices are not amenable to high-throughput applications, where a large number of samples must be thermally cycled with a minimum of space while maintaining robustness. Because all devices presented to an assembly-line may consume valuable resources (floor space, assembly line space, electrical power, etc.), it is of considerable interest to (a) maximize space in the assembly line, (b) maximize efficiency of the heating or cooling system, (c) minimize the complexity of the resulting device, and (d) allow for continuous uninterrupted operation (i.e. non-batch processing) of the apparatus to maximize throughput and duty cycle of the machine. However, dozens of currently available PCR thermal cycle devices are needed to achieve the fast/high-throughput operations required. Most of these systems use Peltier heating elements and air-based cooling units resulting in a lot of heat being ventilated into the lab. The use of conventional thermal cycle devices is therefore not possible, as it would require the installation of an air conditioning system to limit the risk of failure for the thermal cycle devices. The space required for these systems would also not be appropriate for lab applications.
It is thus our goal in this current invention to present a system that is adapted for high-volume, high-throughput applications and amenable to a high-throughput assembly line. Maximal space efficiency and reliability are thus primary goals.
In this view, instant invention provides a thermal cycle device comprising:

a support for samples;
at least two fluid tanks;
means for keeping the fluid of each tank at a given respective temperature, the temperatures associated with the respective tanks being different; and
means for transferring fluid from the tanks to the support.

The invention may also show at least one of the following features:

the number of fluid tanks is at least three;
it comprises means for transferring fluid from the support to the tanks, the device being arranged so that, each time a fluid transfer from the support to one of the tanks is required, the transfer means transfers the fluid from the support to the one tank which temperature is, at the beginning of the transfer, closest to a temperature of the fluid which transfer is required;
the transfer means is arranged to transfer to the support fluid from any of the tanks, one at a time;
the transfer means is arranged to transfer to the support a mixture of fluids from two or at least two of the tanks;
the transfer means is arranged so that proportions of the fluids in the mixture are adjustable;
the transfer means is arranged so that proportions of the fluids in the mixture are modulated during transfer;
it comprises means for transfer of fluid from one of the tanks to the other tank or another of the tanks without passing through the support;
it comprises a plurality of supports, the transfer means being arranged to transfer fluid from the tanks to any of the supports;
the support comprises means to promote turbulent flow of fluid through the support;
the means to promote turbulent flow comprises at least one fin;
the support comprises housings for accommodating the samples and fluid cavities extending between the housings; and
the support is adapted to receive a microtiterplate containing the samples.

The invention also provides a process for thermal cycling of samples, comprising the steps of:

keeping fluids of at least two tanks at respective different temperatures; and
successively transferring fluid from each tank to a support holding samples in order to control a temperature of the samples.

This process may be used for amplification of nucleic acid sequences.
Other features and advantages will appear in the following description of various embodiments, with reference to the drawings in which:

figure 1 is a partial cross sectional view of a block according to one embodiment of the invention ;
figures 2 and 3 are views respectively from above and in cross section of a microtiter plate usable with this embodiment of the invention;
figures 4, 5 and 6 are schematic views of this embodiment;
figure 7 and 8 respectively show the spatial arrangement of blocks in the embodiment and a variation of the later ; and
figure 9 is a diagrammatic view of another embodiment of the invention.

In the following embodiments, 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. In a preferred embodiment, oil is used to seal the top of the sample. But it will be apparent that alternatively 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.
An important element of our device is the temperature regulation support or block (hereafter referred to as "the block"). 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. For example, 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.
To meet the aforementioned requirements, 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.
It is suggested that the final product allows 30 to 60 similar plates 10 to be processed simultaneously. Thus, it could comprise an equivalent number of blocks 2. But some blocks could each accommodate several plates. However, here we only disclose 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.
We see that, in the embodiment illustrated on figure 1, 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. After the mixing valves and prior to the thermal block with reference to fluid flow, a baffle system, not shown, may be inserted that also promotes turbulent mixing in the block. In figure 1, 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.
With reference to figure 4, 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. For example, 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.
In figure 5, 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.
Figure 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. Second, 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. Alternatively, a custom system may be constructed that incorporates the essential elements of such a thermally controlled bath device.
If a particular temperature is not used, 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. Thus 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. Assuming 100% efficiency in transfer of the heat to the surrounding environment (the blocks and the plates), 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. To control the changes in temperature, an actively controlled system is used.
Control of the temperature is accomplished as follows. Refering to figure 4, each thermal block 2 contains a temperature sensor 24, for example a simple semiconductor sensor, that is in close thermal contact with the block. In addition, each block contains a low-cost microcontroller 26 or other suitable control device to maintain some autonomy within the control electronics of the system. Anyone skilled in the art will acknowledge that 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 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. Beside, once the block has reached the desired temperature in association with one tank, 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.
It is also possible to fill the block with 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. For example, if during an isothermal equilibration the temperature of the block 2 deviates from the fluid stream temperature arriving from one tank whereas the desired temperature is the temperature of this tank, 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.
While this mixing could be done by variable conductance valves, 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. Such 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.
In another example, for isothermal control at 95 °C, a corresponding mixture of 98°C fluid from tank 20 is combined with 72°C fluid, and then is exhausted in the 98°C waste stream. Similarly, for control at 55°C, the combination of 54°C and 72°C water is introduced and then exhausted to the 54°C waste stream.
Anyone skilled in the art will immediately recognize that by such a control system any temperature may be reached by just two temperature tanks, though in the preferred embodiment as recited above we use three to minimize the thermal pollution of the waste stream into the fluid re-equilibration tank and the recycling of this stream into the inlet stream.
Because one stream can exhaust into another temperature waste stream, a continuous operation load balancing system is used, 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. For example, 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 mm²/s to greater than 5 mm²/s. Thus pumping through an array of thermal blocks can be achieved while still allowing for level regulation of the three temperatures.
It is suggested that 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.
Ideally, water should be in direct contact with the plate to optimize heat transfer and speed of operation. However, 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.
In our preferred embodiment, 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. Alternatively, 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. Anyone skilled in the art will realize that any arrangement could be used without changing the important features of our invention (simplicity, reliability, and compactness). Yet essential in this design is to maximize space efficiency in the assembly line format, and for this we prefer an embodiment where the plates are arranged as follows and illustrated on figure 7. This figure 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.
Each block 2 of figure 7 could be replaced by a column of blocks. Accordingly, figure 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. Because the system is designed for repetitive high-throughput PCR work, it is anticipated that the thermal cycling processes will take longer than rotation and/or loading/unloading steps, and thus this system provides the best solution for space efficiency. Because of the number of blocks, their close relationship to the assembly line, and the previously described processing system, this description allows for the sequential loading of plates at one or several positions during the operation of thermal cycling. Thus the system allows for the continuous operation of thermal cycling on an assembly line.
In this embodiment of the invention, if six banks containing six plates each accommodating a 384-well microtiter plate are thermally regulated using thermostatically regulated baths with pumps, 13824 samples may be thermally cycled for high-throughput assays within a single 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.
We explained above the use of three tanks, for example each at one of the temperatures required for the PCR, and a number of on/off valves to direct the water from these tanks to the blocks. But experiments may show that the temperature of the blocks can not be easily achieved using a single supply (i.e. at one given temperature). Instead it is recommended that water be mixed from different hotter/cooler sources to achieve the temperature wanted as already observed above.
Accordingly, 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.
Therefore, we disclose hereafter another preferred embodiment combining the two approaches, i.e. the three water baths at temperatures slightly higher/lower than the required temperatures and the use of mixing valves. This embodiment is illustrated on figure 9.
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. Here, the block 2 holds four plates 10. In the final unit, 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.
In the embodiments of figures 4 and 9, 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.
Current state of the art in 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.

Claims

A thermal cycle device comprising a support (2) for samples (7), characterized in that it comprises:

at least two fluid tanks (16, 18, 20);

means (28) for keeping the fluid of each tank at a given respective temperature, the temperatures associated with the respective tanks being different; and

means (25) for transferring fluid from the tanks to the support.
The device of claim 1, characterized in that the number of fluid tanks (16, 18, 20) is at least three.
The device of any of claims 1 to 2, characterized in that the transfer means (25) is arranged to transfer to the support fluid from any of the tanks, one at a time.
The device of any of claims 1 to 3, characterized in that the transfer means (25) is arranged to transfer to the support a mixture of fluids from two or at least two of the tanks.
The device of claim 4, characterized in that the transfer means (25) is arranged so that proportions of the fluids in the mixture are adjustable.
The device of any of claims 4 to 5, characterized in that the transfer means (25) is arranged so that proportions of the fluids in the mixture are modulated during transfer.
The device of any of claims 1 to 6, characterized in that it comprises a plurality of supports, the transfer means (25) being arranged to transfer fluid from the tanks to any of the supports.
The device of any of claims 1 to 7, characterized in that it comprises means (27) for transferring fluid from the support (2) to the tanks (16, 18, 20), the device being arranged so that, each time a fluid transfer from the support to one of the tanks is required, the transfer means transfers the fluid from the support to the one tank which temperature is, at the beginning of the transfer, closest to a temperature of the fluid which transfer is required.
The device of any of claims 1 to 8, characterized in that it comprises means (23, 60) for transfer of fluid from one of the tanks to the other tank or another of the tanks without passing through the support.
The device of any of claims 1 to 9, characterized in that the support (2) comprises means (14) to promote turbulent flow of fluid through the support (2).
The device of claim 10, characterized in that the means to promote turbulent flow comprises at least a fin (14).
The device of any of claims 1 to 11, characterized in that the support (2) comprises housings (11) for accommodating the samples and fluid cavities (8) extending between the housings.
The device of any of claims 1 to 12, characterized in that the support is adapted to receive a microtiterplate (10) containing the samples (7).
A process for thermal cycling of samples, comprising the steps of:

keeping fluids of at least two tanks (16, 18, 20) at respective different temperatures; and

successively transferring fluid from each tank to a support (2) holding samples (7) in order to control a temperature of the samples.
The process of claim 14, characterized in that it is used for amplification of nucleic acid sequences.