GB2226161A - Programmable waterbath apparatus for control of rapid temperature cycling - Google Patents

Abstract

A waterbath comprising an open-topped tank or trough (10) containing an immersion heater (12), cooling coil (14) and thermostat temperature sensors (16a, 16b, 16c) has a programmable control system adapted to switch the heater and cooling coil on and off to provide rapid, automatic temperature cycling through a predetermined temperature/time profile comprising successive stages each characterised by a particular user-selected temperature and a dwell period during which the bath remains at that temperature. The apparatus is especially suitable for use in carrying out a polymerase chain reaction (PCR) process for amplifying nucleic acid sequences.

Description

TEMPERATURE CONTROL APPARATUS AND USES THEREOF Technical Field This invention relates to the field of temperature control apparatus, particularly temperature control apparatus for cycling the temperature of a reaction vessel or vessels, e.g. a tube or tubes, through a series of different, selectable, temperature levels.

Background Art More specifically, the apparatus of the present invention is especially, although not exclusively, useful for carrying out the so-called polymerase chain reaction (PCR) process for amplifying nucleic acid sequences, as described for example by Saiki et al in Science, 230, 1530-1534 (1985) and in Genome Analysis, A practical approach, 1988 (Ed. G. Davies) published by IRL Press, Eynsham, Oxford.

This process provides a means for amplifying the amount of a given nucleic acid sequence in a sample by performing a particular reaction known as a polymerase chain reaction which employs temperature sensitive enzymes. The reaction process comprises a number of successive steps; each step generally requires the temperature of the reaction mix to be maintained at a particular temperature value or level for a particular time. Thus, the process usually involves maintaining the reaction mix at a first temperature for a first time, then at a second, higher, temperature for a second time period, and lastly at a third, yet higher temperature, for a third time period.By cyclically repeating the three steps or stages the reaction of this process can proceed reptitiveiy to achieve a large amplification in the amount of te nucleic acid sequence in the sample.

For this amplification it is therefore necessary to cycle the reaction mixture through the three temperatures (sometimes only two different temperatures need be used), to be able to select each of those temperatures, and to select the time for which each temperature is maintained.

One form of apparatus for use in carrying out automatically this process and the sequence of operations involved has been proposed in which an aluminium block is drilled or cast to receive reaction tubes, the block being adapted to be heated and cooled respectively by a heater and a refrigerator. To define the temperature profile through which the reaction tubes are to be taken, the apparatus is provided with a computer. The apparatus is, however, unnecessarily expensive and complex.

Disclosure of Invention The present invention basically provides temperature control apparatus for cycling the temperature of a heat transfer medium between at least two different selectable temperature levels, said apparatus comprising: a bath for containing, in use, the heat transfer medium in the form of a liquid, for example water, within which can be positioned a reaction vessel or other body in thermal contact with said liquid, heating means for raising the temperature of the bath liquid, cooling means for reducing the temperature of the bath liquid, temperature sensing means for sensing the temperature of the liquid within the bath or of a body in said liquid and for providing a signal indicative of such temperature, and a programmable control system embodying control means responsive to the temperature indicative signal from said temperature sensing means and operable to switch on and to switch off said heating means and said cooling means in such a manner as to enable the apparatus, in use, to perform automatically a sequence of operations in which the temperature of the bath liquid is cycled repetitively through a predetermined temperature/time profile set by the user in programming the control system, each cycle through said temperature/time profile comprising a plurality of successive stages, in each stage the heating or cooling means being operated to bring the bath to a particular user-selected temperature at which it is then held or allowed to remain for a particular period of time, the temperature of the bath then being altered in the following stage to bring it to another user-selected value at which the bath is likewise held or allowed to remain for a further particular period of time.

In preferred embodiments, as already indicated the apparatus, including the temperature/time profile of the bath liquid obtainable by appropriate setting of the programmable control system, is suitable for use in carrying out a conventional polymerase chain reaction (PCR) process, as hereinbefore referred to, for amplifying nucleic acid sequences. The preferred temperature cycle comprises three stages defining a temperature/time profile which has three different userselected temperature levels.

The control provided by the control system over both heating and cooling of the bath liquid has been found to be necessary for obtaining a temperature/time profile suitable for the polymerase chain reaction process referred to. As is well known, this process generally requires (i) a difference between the highest selected temperature and the lowest selected temperature la each temperature cycle which is greater, often substantially greater, than 25to, (ii) steep temperature rises and falls so that the transition times in changing from one user-selected temperature level to another userselected temperature level are small, e.g. substantially less than say, three minutes, and (iii) a rapid rate of temperature cycling, considerably less than 15 minutes for example for each complete cycle, so that a sufficient number of cycles to provide a high degree of amplification can be executed in a reasonable time.

Usually it is to be expected that in performing a PCR process a user will wish or need to carry out between 25 and 50 cycles or more in order to achieve the required degree of amplification.

Usually, the preferred heat transfer medium liquid used in the bath will be water, and the heating means for raising the temperature of the bath liquid will comprise a heater in thermal contact therewith whilst the cooling means for reducing the temperature of the bath liquid will comprise a tubular heat exchanger element inside the bath, for example a coil of metal tubing, adapted to be connected to a supply of cold liquid coolant, e.g. tap water, to enable a flow of the cold liquid coolant to be passed therethrough. Conveniently the bath comprises an open-topped tank or trough of substantially rectangular form in horizontal cross-section whilst the abovementioned tubular heat exchanger element of the cooling means comprises a vertically orientated coil of metal tubing which is located in the upper part of the bath and which closely follows the contour of the walls thereof.

In this arrangement the heater preferably comprises an immersion heater element or elements located in the lower part of the bath.

It has been found that in order to obtain suitable temperature/time profiles, at least for use in carrying out the aforesaid PCR process, the size of the bath and the form, configuration and relative positioning of the components therein, such as the above-mentioned cooling coil and immersion heater element or elements, are important. In general the bath requires to be of relatively small capacity as compared with most conventional temperature control liquid baths used in laboratory work, holding less than 1 litre of water for example, which results in a substantial proportion of the space within the bath being occupied by the bulk of the heat exchanger element of the cooling means and by the immersion heater element or elements of the heating means.Although such a crowded space in a small bath, which must also accommodate reaction vessels or other bodies being processed, would present problems in arranging for any mechanical stirring within the bath, surprisingly it has been found that satisfactory temperature stability and uniformity can be obtained without providing any means, e.g. a mechanical stirrer, for stirring the liquid in a bath as herein described.

In general, the size and capacity and heat loss characteristics of the bath will be such that, when filled with water as the liquid heat transfer medium and operating in an environment within the range of usual laboratory ambient temperatures, heating means provided by an immersion heater having a total rating as low as 1.3KW can be sufficient to obtain a temperature/time profile suitable for said PCR process in which the highest temperature level is in a range that is high enough to bring about denaturation of nucleic acid, as is required in said process.

Preferably, the programmable control system includes timing means adapted to enable the user, in programming the control system, to preselect said particular period of time for each stage of said t erstu.re cycle, and the sequence of operations, especially for carrying out the aforesaid PCR process, is such that during the temperature cycling operation, immediately after the stage in which the highest selected temperature is reached, the bath is cooled in the following stage in a single step to the lowest selected temperature.

The timing means may comprise three manually settable timing relays each for timing a respective constant temperature part of the cycle and each of which is started running when the liquid bath reaches the respective constant temperature. In one embodiment three separate thermostats may be provided, one for each temperature, each connected to activate one of the timing relays and comprising circuits including separate temperature sensors, e.g. thermocouple junctions or liquid expansion thermostats in the liquid bath.

Preferably an additional timing means is provided to be activated once the bath has passed through the required number of cycles so that the temperature of the bath remains at a desired level for a set time at the end of the reaction process.

Conveniently, a separate temperature probe is also provided to monitor independently the temperature inside the bath and/or reaction tubes and a display may be provided on the apparatus to display that temperature.

The control system is also preferably programmable by the user to set the desired total number of complete temperature cycles to be performed in the sequence of operations and the control system operates automatically to terminate further temperature cycling operation of the apparatus when said total is reached. For this purpose the control system may include a counter which is incremented for each complete cycle and means are provided for comparing the value recorded by such counter after each increment with a record of the required total number of complete cycles entered manually by the user.

Although the control system for controlling the operation of the heating and cooling means and the temperature cycling of the apparatus may comprise a network of electromechanical relays and/or timers and thermostats as described, if preferred it may alternatively be composed of electronic components or solid state devices, e.g. electronic timing relays or counters, and may include a suitably programmed microprocessor and associated interfacing circuitry.

It will also be appreciated that during the particular periods of time in each temperature cycle that provide "constant temperature steps", it may be sufficient simply to leave the bath with both the heating and cooling means switched off and, provided the rate of heat loss from the bath is not too high, the temperature of the bath liquid will remain sufficiently close to the selected value as to be considered constant for all practical purposes. If desired however, the control system could also be arranged to operate thermostatically to maintain the bath temperature more closely constant at the required selected temperature during these particular time periods, e.g. by intermittently switching on the heating means for short intervals to counteract natural heat loss.

The invention also extends to uses of the apparatus herein disclosed and, in particular, it includes a method of performing a polymerase chain reaction process for amplifying nucleic acid sequences using such apparatus.

Brief Description of the Drawings By way of example, the invention will be further described with reference to the accompanying drawings in which: FIGURE 1 is a front elevational view showing a control panel of a control system of an apparatus in accordance with a first embodiment of the invention; FIGURE 2 is a diagrammatic perspective view showing the general arrangement of the main components of the apparatus of the embodiment of FIGURE 1 other than those of the associated control system; FIGURE 3 a graph showing, in a diagrammatic idealised form, temperature cycling operation of the apparatus as when in use in carrying out a PCR process for amplifying nucleic acid sequences;; FIGURE 4 is a graph similar to FIGURE 3 showing an example, derived from actual measurements, of the temperature cycling operation of apparatus in accordance with the present invention; FIGURE 5 is a schematic diagram illustrating the operation of the control system of the apparatus of this first embodiment; FIGURE 6 is a perspective view illustrating a typical form of reaction tube rack for use with the apparatus; and FIGURE 7 is a perspective view illustrating a further embodiment of the apparatus in accordance with the invention.

Mode(s) for Carrying Out the Invention Referring first to FIGURES 1 to 5 of the drawings, the apparatus as shown diagrammatically in FIGURE 2 comprises a bath 10 intended in use to be filled with water, an immersion type electric heating element or elements 12, a cooling coil 14, and temperature monitoring sensors 16a, 16b and 16c.

The bath 10 is conveniently in the form of an opentopped rectangular metal tank or trough which is preferably lagged to reduce heat loss. In a typical example where the apparatus is intended primarily for carrying out the PCR process previously referred to for amplifying nucleic acid sequences and is designed to operate up to a temperature approaching boiling point, e.g. 90-95"C, the bath may have a length of approximately 180mm and a height and depth each of approximately lOOmm.

For this size, in use it would be filled with approximately 700ml of water, much of the remaining space within the bath being taken up by the bulk of the heater element(s) 12, the cooling coil 14, the temperature sensors 16a, 16b, 16c, and, in use, by the reaction vessel or vessels immersed within the water. Although liquids other than water may be used to fill the bath, in general water, more particularly distilled or de-ionised water, is cheap, readily available, non-toxic and has satisfactory heat transfer characteristics for the normal temperature range within which the apparatus is usually required to operate.

The heater element or elements 12 (only one is shown but in practice a pair of elements may be provided each, for example, of 650 watts rating) is or are positioned towards the lower part of the bath 10.

The cooling coil element 14 is a coil of four turns of metal tubing, e.g. narrow bore copper pipe of approximately 6mm outside diameter, which is positioned with its axis orientated vertically in the upper part of the hath and which closely follows the rectangular contour of the walls thereof. In use, tap water is conveniently used as the coolant medium since this is readily available and is generally at an ambient temperature substantially lower than the lowest temperature needed for the polymerase chain reaction for which this embodiment of the apparatus is intended. The tap water coolant medium enters the cooling coil 14 through a water inlet 20 which connects to a tap (not shown) and exits through a water outlet 22.The cold tap water may be passed through the coil 14 at a typical rate of about 2 to 3 litres a minute, and its flow is controlled by an electrically operated on/off solenoid valve (not shown) in the inlet line.

The temperature sensors 16a, 16b, 16c are provided for monitoring the temperature within the bath and, in this embodiment, are positioned alongside one wall below the cooling coil 14 in a generally mid position of the bath. These sensors are conveniently in the form of liquid expansion thermostats, although other types of sensors may of course be used if desired. They provide temperature measurement signals that are fed to the control system.

As indicated, in use a reaction vessel or vessels containing the material or constituents of the reaction mixture for the process being carried out in the apparatus will generally be positioned and immersed, either partially or totally, in the water in the upper part of the bath within the unobstructed region encircled by the cooling coil 14. For carrying out the above mentioned PCR process at least, the reaction vessel or vessels will usually comprise one or more capped or screw topped plastic microcentrifuge or sample tubes, of 0.5ml or 1.5ml capacity for example, supported in a rack or holder fitted in the open top of the bath so as to depend at least partially into the water. A convenient form of rack 18 fitted with a few such tubes 17 is illustrated in FIGURE 6.

In order that the temperature at different locations within the bath and/or within reaction tubes immersed therein may be monitored, a separate temperature probe on a flexible lead may be provided if desired, as indicated at 54 in FIGURE 2.

Control devices incorporated in a programmable control system fpr controlling the flow of water through the cooling coil 14 and for controlling energisation of the heater(s) 12 are conveniently housed within the front part of the apparatus which is indicated diagrammatically at 15 in FIGURE 2, and there is provided a front control panel 13 as shown in FIGURE 1.

The apparatus will usually be programmed to cycle the temperature of the water in the bath 10 through a predetermined temperature/time profile set by the user that includes a plurality of successive stages, in each stage the heating or cooling means being operated to bring the bath to a particular selected temperature at which it is then held or allowed to remain for a particular period of time, the temperature of the bath liquid then being altered in the following stage to bring it to another selected value at which the bath liquid is likewise held or allowed to remain for a further particular period of time.

As shown in FIGURE 1, the control panel is provided with three controls, 23, 25 and 27, for setting three selected temperature levels, together with three controls 29, 31 and 33, for setting the period of time for which the bath is to be maintained at each of the set temperature levels. Indicator lights 35, 37 and 39 are also provided to indicate when each of these three temperature levels has been reached during operation, and a further indicator light 41 is provided to show a preset number of cycles has been completed. There are two cycle counters, 43 and 44, the one 43 being arranged to indicate the number of cycles completed and the other, 44, being a countdown counter set initially to indicate the total number of cycles to be carried out.In use, the operator will set the required total number of cycles on the counter 44 and the apparatus will then operate automatically, cycling the temperature as programmed, until counter 43 shows the required total and counter 44 shows zero.

Another indicator 21 shows whether or not the power to the apparatus has been switched on, whilst further indicators 42 and 46 show whether the water bath is being heated or cooled, and an indicator 45 shows whether or not cycling is in progress. A reset button 51 is also provided for starting the cycling operation after having first programmed the apparatus, or for resetting the apparatus to the initial condition if for any reason operation is interrupted or prematurely terminated.

As already indicated, for carrying out the PCR process for amplifying nucleic acid sequences the apparatus would usually be set to cycle rapidly and repeatedly through three preselected temperatures and time periods for which an idealised temperature/time profile is shown in the diagram of FIGURE 3. Part of an actual temperature/time profile as measured for such apparatus during cycling operation, through a temperature range of about 50 to 95 as in carrying out a PCR process, and with the temperature measurements being made within the vicinity of a reaction vessel in the upper part of the bath, is shown in the graph of FIGURE 4.

In this particular embodiment, an electromechanical type of control system is provided for controlling the operation, utilising readily available thermostats, electromechanical relays and timers. FIGURE 5 represents a schematic diagram of the control system which operates as hereinafter described.

Upon first switching on the apparatus a first timing relay TR1 is activated which, after it has timed out, switches on the heater 12 via a power relay (not shown). This then heats the bath through a first temperature level T1 required by the process until a second temperature T2, detected by sensor 16b, has been reached. This marks the commencement of a cycle. The heater 12 is then switched off, the counter 43 is incremented by one and a second timing relay TR2 is activated. When the latter has timed out the heater 12 is again switched on and heating continues until a third temperature T3, detected by sensor 16c, is reached when heater 12 is again switched off and a third timing relay TR3 is then activated.After this third timing relay TR3 has timed out, supply of cooling water to the cooling coil is switched on (via a solenoid valve in the water supply line) and the bath is cooled until the first temperature T1, detected by sensor 16a, is reached. The supply of cooling water is then switched off, the first timing relay TR1 is again activated and, after timing out, the heater 12 is again switched on until temperature T2 is again reached and the sequence of operations is repeated. Cycling through these temperatures T2, T3 and T1 then continues for the required total number of cycles (n) preset on counter 44, i.e. until the temperature T2 is reached for the (n+l)th time. At this point, when both counters 43 and 44 show equal readings, the heater and the supply of coolant are each switched off and a fourth timing relay TR4 is activated. This represents the commencement of a post-treatment period which is usal1y desirable and which lasts for a preset time, e.g.

five minutes, until relay TR4 times out. During this period the bath temperature remains substantially at temperature T2, but when timing relay TR4 finally times out it switches on the flow of cooling water to the cooling coil 14 and the bath is cooled down towards the tap water coolant temperature until finally the apparatus is switched off.

It will be appreciated that with this arrangement, in each stage whilst the respective timing relay is timing out, both the heater 12 and the flow of cooling water with the cooling coil 14 are cut-off but, although the temperature of the bath is not thermostatically maintained constant, the temperature in the bath does in fact remain substantially constant so long as the time period is not excessive, heat loss from the bath during this time generally being relatively small. In practice, the temperature during these "constant temperature" intervals or steps does of course tend to change to some extent, as will be seen in the graph of FIGURE 4, but usually the extent of temperature change is small and is insufficient to affect adversely the performance of a polymerase chain reaction process.

However, if the changes in temperature during these "constant temperature" steps of the cycle are likely to cause problems, as for example if there are relatively long time periods between each transition from one temperature level to another, in a modified embodiment the heating and cooling means may be arranged to be thermostatically controlled to maintain the bath temperature close to the selected temperature value during the entire time period between successive temperature transitions.

Thus, the temperature of the bath can then be maintained substantially constant even if heat is lost or gained from the surroundings at a rate which otherwise would result in temperature changes of an undesirably high order.

As previously mentioned the control system, instead of being of an electromechanical design as herein described, may alternatively be of an electronic design including a microprocessor together with other solid state electronic components and associated interfacing circuitry. An embodiment of waterbath apparatus in accordance with the invention, incorporating a programmable electronic control system of this kind, is illustrated by way of example in FIGURE 7. This shows the apparatus complete as a compact self-contained unit, including at the top the rectangular tank or trough of the bath 10' containing a cooling coil 14', temperature sensing element 16' and a heater element (not visible) and including, at the front, a control panel 13' with various electronic data entry keypads, displays and indicators.

Various other modifications may of course also be made within the scope of the invention which is not to be construed as being limited by the illustrative examples or by the terms and expressions used herein merely in a descriptive or explanatory sense. The invention also includes all novel and inventive features and aspects herein disclosed, either explicitly or implicitly and either singly or in combination with one another.

Claims (20)

1. Temperature control apparatus for cycling the temperature of a heat transfer medium between at least two different selectable temperature levels, characterised in that said apparatus comprises a bath (10) for containing, in use, the heat transfer medium in the form of a liquid, for example water, within which can be positioned a reaction vessel or other body in thermal contact with said liquid, heating means (12) for raising the temperature of the bath liquid, cooling means (14) for reducing the temperature of the bath liquid, temperature sensing means (16a,16b,16c) for sensing the temperature of the liquid within the bath or of a body in said liquid and for providing a signal indicative of such temperature, and a programmable control system embodying control means responsive to the temperature indicative signal from said temperature sensing means (16a,16b,16c) and operable to switch on and to switch off said heating means (12) and said cooling means (14) in such a manner as to enable the apparatus, in use, to perform automatically a sequence of operations in which the temperature of the bath liquid is cycled repetitively through a predetermined temperature/time profile set by the user in programming the control system, each cycle through said temperature/time profile comprising a plurality of successive stages, in each stage the heating or cooling means being operated to bring the bath to a particular user-selected temperature at which it is then held or allowed to remain for a particular period of time, the temperature of the bath then being altered in the following stage to bring it to another user-selected value at which the bath is likewise held or allowed to remain for a further particular period of time.

2. Temperature control apparatus as claimed in Claim 1 wherein the programmable control system includes timing means adapted to enable the user, in programming the control system, to preselect said particular period of time for each stage of said temperature cycle.

3. Temperature control apparatus as claimed in Claim 1 or 2 wherein said sequence of operations is such that during the temperature cycling operation, immediately after the stage in which the highest selected temperature is reached the bath is then cooled in a single step in the following stage to the lowest selected temperature.

4. Temperature control apparatus as claimed in any of the preceding claims wherein the control system is programmable by the user to set the desired total number of complete temperature cycles to be performed in the sequence of operations and the control system operates automatically to terminate further temperature cycling operation of the apparatus when said total is reached.

5. Temperature control apparatus as claimed in Claim 4 wherein the control system includes a counter (43) which is incremented for each complete cycle and means are provided for comparing the value recorded by such counter after each increment with a record of the required total number of complete cycles entered manually by the user.

6. Temperature control apparatus as claimed in any of the preceding claims wherein the cooling means for reducing the temperature of the bath liquid comprises a tubular heat exchanger element inside the bath, for example a coil of metal tubing, adapted to be connected to a supply of cold liquid coolant e.g. tap water, to enale a flow of the cold liquid coolant to be passed therethrouh.

7. Temperature control apparatus as claimed in Claim 6 wherein the control system includes on/off valve means for controlling the flow of said cold liquid coolant through the heat exchanger element.

8. Temperature control apparatus as claimed in Claim 6 or 7 wherein the bath comprises an open-topped tank or trough of substantially rectangular form in horizontal cross section and the tubular heat exchanger element comprises a vertically orientated coil of metal tubing which is located in the upper part of the bath and which closely follows the contour of the walls thereof, whilst the heating means for raising the temperature of the bath liquid comprises an immersion heater element or elements located in the lower part of the bath.

9. Temperature control apparatus as claimed in Claim 8 wherein a substantial proportion of the space within the bath is occupied by the bulk of the heat exchanger element of the cooling means and by the immersion heater element or elements of the heating means.

10. Temperature control apparatus as claimed in any of the preceding claims characterised in that the bath is not provided with any mechanical stirring means.

11. Temperature control apparatus as claimed in any of the preceding claims wherein the control system also operates to thermostatically maintain the bath temperature constant during said particular periods of time in each cycle when the temperature is at a respective user-selected value or level.

12. Temperature control apparatus as claimed in any of the preceding claims wherein the stages of each temperature cycle define a temperature/time profile suitable for carrying out a conventional polymerase chain reaction (PCR) process for amplifying nucleic acid sequences in said apparatus.

13. Temperature control apparatus as claimed in any of Claims 1 to 11- wherein each temperature cycle comprises three stages defining a temperature/time profile which has three different user-selected temperature levels and which is suitable for carrying out a conventional polymerase chain reaction (PCR) process for amplifying nucleic acid sequences in said apparatus.

14. Temperature control apparatus as claimed in Claim 12 or 13 wherein the size and capacity and heat loss characteristics of the bath are such that, when filled with water as the liquid heat transfer medium and operating in an environment at an ambient room temperature, heating means provided by an immersion heater having a total rating as low as 1.3KW can be sufficient to obtain a temperature/time profile suitable for said PCR process in which the highest temperature level is in a range that is high enough to bring about denaturation of nucleic acid, as is required in said process.

15. Temperature control apparatus as claimed in any of Claims 12 to 14 wherein, during temperature cycling operation throughout the full range of said temperature/time profile, the time of each transition in which the temperature is altered from one user-selected level to another in the temperature cycle is less than three minutes.

.

16. Temperature control apparatus as claimed in any of Claims 12 to 15 in which the difference between the highest selected temperature and the lowest selected temperature in each temperature cycle is at least 25"C.

17. Temperature control apparatus as claimed in any of Claims 12 to 16 in which the maximum period of each complete temperature cycle is less than fifteen minutes.

18. Temperature control apparatus as claimed in any of Claims 12 to 17 in which the control system is programmed to provide, after completion of the preset total number of temperature cycles, a post-treatment period in which the bath liquid temperature remains substantially constant at an intermediate user-selected value for a preset extended period of time.

19. Temperature control apparatus substantially as herein described and illustrated with reference to the accompanying drawings.

20. A method of performing a polymerase chain reaction process for amplifying nucleic acid sequences characterised in that it is carried out in a reaction vessel or vessels positioned in the liquid bath provided by programmable temperature control apparatus as claimed in any of Claims 12 to 19.