WO1995015671A1

WO1995015671A1 - Apparatus and process for performing a chemical reaction

Info

Publication number: WO1995015671A1
Application number: PCT/GB1994/002660
Authority: WO
Inventors: Ronnie Aurun Ray; Andrew Jeremy Wakefield
Original assignee: Inceltec Ltd
Priority date: 1993-12-03
Filing date: 1994-12-01
Publication date: 1995-06-08
Also published as: AU1194995A; GB9324843D0

Abstract

There is disclosed an apparatus and process for performing chemical reaction using microwave emissions. The apparatus includes a cabinet (1) and a heating means (2) for generating microwave energy within the cabinet. A cooling means (3) also provided within the cabinet is controllable to initiate a cooling cycle after cessation of, or during a specific period of a heating cycle. The process thereby includes successively subjecting a sample within the cabinet to microwave emission and cooling cycles.

Description

APPARATUS AND PROCESS FOR PERFORMING A CHEMICAL REACTION

The invention relates to an apparatus for performing a chemical reaction, and particularly to an apparatus for performing nucleic acid reactions automatically at different energy settings.

Many chemical reactions such as nucleic acid reactions require a predetermined cycle of heating and cooling steps to be performed with a minimum of intervention. For example there are now a number of methods known for the amplification and transcription of nucleic acid sequences. These include the polymerise chain reaction (PCR) for amplifying DNA sequences, reverse transcription-PCR (RT- PCR) for amplifying RNA sequences, the self-sustained sequence replication (3SR) for amplifying RNA and DNA sequences, the ligase chain reaction to amplify DNA as well as to discriminate a single base mutation and the primed in situ extension (PRINS) reaction. These processes are now coming into more general use and are particularly suited to utilisation of the apparatus in accordance to the present invention.

Known methods for heating the samples used in such reactions include the use of heating elements such as for example electrically powered hot plates or the like, to provide the required heat energy for the reactions to occur. Such conventional forms of heating apparatus suffer from a number of problems. For example, the duration of heating that is required to transmit the required heat energy has disadvantageous effects on the sample. In this regard, the duration of heating required can cause excessive evaporation from the sample. Also the structure of the cell and hence, its morphology, can be destroyed, and the proteins in the sample can become denatured.

Moreover, the duration of heating required is a restrictive factor which compromises the speed and efficiency at which the reactions can be performed.

According to a first aspect of the present invention therefore there is provided a microwave heating and cooling apparatus comprising; a cabinet, a heating means for generating microwave energy within the interior of said cabinet, control means therefor and a sample support disposed within said cabinet; characterised in that said cabinet further comprises cooling means to cool the sample and in that the control means is adapted to initiate a cooling cycle after cessation of, or during a specific period of, a heating cycle.

The invention provides therefore a microwave heating and cooling apparatus suitable for liberated or intra-cellular nucleic acid reactions but is also suited to other chemical reactions in which radiation energy is input and dissipated in accordance with a predetermined cycle.

In this connection, it has been found that the use of microwaves to provide the required excitation of the sample molecules has many advantages. Microwaves provide a very rapid and direct manner of molecular excitation. Thus the duration of subjection of a sample to microwave emissions to achieve an equivalent effect as heating by more conventional methods is much reduced. Thus a cycling time for a reaction can be significantly reduced since even a small reduction in time for a single heating cycle will afford a significant time saving over a number of cycles. Further microwaves do not destroy the sample morphology but rather preserve it with associated preservation of the sample DNA sequences. The shorter duration of heating also provides for less evaporation from the sample. A further advantage of microwaves concerns the feature that they have the properties of radiant energy, e.g. reflection, refraction and focusing. Thus they can, for example, be readily focused to concentrate their paths at a particular point. It follows that their use reduces the occurrence of false positives or negatives.

Preferably said apparatus according to the invention is adapted to perform a plurality of successive heating and cooling steps according to a preset control input. In a preferred arrangement the cooling means may comprise a cooling fluid arranged to circulate between the sample or sample reactant and a cooling unit. Alternatively the cooling means may be adapted to provide a current of cool and/or dry air over the sample or sample support thereby to subject the sample to forced cooling.

The sample support may include means for oscillating or rotating the sample during the heating cycle and during the cooling cycle so that both the heating and cooling steps are evenly effected. Preferably means are provided for varying the relative humidity of the interior of the cabinet such that it can be increased during the heating step. Liquid bath means may be provided to assist controlled evaporation.

In a preferred form of the invention (for example as shown in Figure 3) a first sample surface such as a glass or plastic surface is placed onto the sample support, and may include a well formed therein or thereon. Said well may be associated with a microwave transparent cover slip. It will be appreciated that the well may be formed either by utilisation of a biocompatable semi-adhesive filler such as a gum, glue or nailpolish, or may be etched or moulded into the first surface itself. Alternatively, the sample comprises a container and lid composed of microwave transmitting, microwave resistant, material (Figure 4) . In a particularly preferred form of the invention the well or container may comprise a target nucleic acid either liberated or within cells (or tissues) and a nucleic acid synthesis mixture. The apparatus may be arranged such that the cyclic heating and cooling steps are repeated according to a preset computer program thereby to synthesise a target nucleic acid sequence. Alternatively cell suspensions or liberated nucleic acid may be placed within a microwave heat or cold transmitting, preferably enclosed, container.

Specifically therefore the arrangement may comprise a device which generates radiant energy within a closed space, the radiant energy generated is an electromagnetic radiation generally in the microwave spectrum extending from 3000 to 100 megacycles. Other useful ranges may include 2000 to 300 megacycles or 1000 to 700 megacycles. The microwave energy is controlled in the device to provide rapid energy cycle changes and is associated with a device to permit rapid cooling. The device capable of rapid cooling may include any suitable form of heat exchanger, for example an air conditioning or other direct refrigeration device adapted to rapidly cool the sample. The effect of rapid heating and cooling of the sample is to significantly shorten the cycling times and hence allow energy initiated synthesis of nucleic acids including PCR.

The cabinet generally encloses spaces of 0.0156 to 0.0317 m³ (0.55 to 1.12 cubic feet) and in accordance with the usual practice in microwave arrangements, a sample support may be provided to ensure that microwave energy is distributed evenly or the device may comprise a microwave focusing arrangement. The sample support should be removable for easy cleaning and should be either rotatable through 360°C, or should oscillate, or both rotate and oscillate.

The device is capable of controlling rapid changes in temperature and may include a manual dial as well as auto touch controls for accurate timing and programming. The control means should be capable, if required, of initiating a sequence of cycling in accordance with appropriate input and controlled power output. In this way the generation of energy can be programmed at different power levels for different lengths of time in a specific sequence; the sequence may be repeated continuously or cycled. In a preferred embodiment, the apparatus includes a computer for controlling at least the heating and cooling means.

The use of a microprocessor allows each of the steps of the process for performing the reactions with the apparatus to be carefully, efficiently and accurately controlled.

The target nucleic acid sequence may be a DNA or RNA sequence. When the target sequence is a DNA sequence, PCR, 3SR, a ligase chain reaction or another DNA amplification reaction may be used. When the target sequence is an RNA sequence RT-PCR, 3SR or another RNA amplification reaction may be used. Alternatively, one nucleic acid sequence may by synthesised using another nucleic acid sequence as a template (eg reverse transcription or PRINS reaction) .

The sample of interest may be placed onto the surface of the rotating or oscillating sample support allowing even distribution of heat. The sample may be disposed in a plastics or glass microscope slide formed as set forth above and may comprise components of a reaction mixture for intra-cellular nucleic acid reactions. Alternatively, the sample of interest may include cells and/or tissues in suspension or liberated nucleic acid along with components for intracellular reactions within a container.

It will be appreciated that more than one sample can be subject to heating and cooling sequences at the same time.

In accordance with the conventional practice in this field the energy generated may be recorded by a device for detecting and measuring microwave energy. Cooling inputs can be recorded by suitable measuring devices inserted in accordance with known methods in this field; preferably in a location where said measuring device is protected from microwave energy generated within the cabinet.

According to a second aspect of the present invention, there is provided a process for performing amplification and/or replication reactions using microwaves, the process comprising the steps of;

(a) subjecting a sample for amplification or replication to microwave emissions for a predetermined period;

(b) subjecting the sample to rapid cooling; and

(c) successively repeating steps (a) and (b) .

Preferably, the process performs PCT reactions in situ and the sample is disposed in a microwave transparent enclosure in the presence of liquid reactants.

Conveniently, the microwaves are concentrated in a predetermined region of the enclosure.

In the preferred embodiments, cooling is effected using a cooling fluid introduced at or adjacent the sample and/or reactant.

Preferably, the process includes inducing a vacuum in the enclosure at least whilst subjecting the sample to microwave emissions.

Conveniently, the process is controllable by means of a computer. The invention will now be further described, by way of illustration only, with reference to the accompanying drawings; wherein

Figure 1 shows schematically apparatus in accordance with a preferred embodiment of the invention;

Figure 2 shows a vertical cross section in diagrammatic form a microwave arrangement in greater detail in accordance with a preferred embodiment of the present invention;

Figure 3 shows a vertical section through a microwave transparent first surface including a well formed therein or thereon, said well being associated with a microwave transparent coverslip in accordance with the present invention;

Figure 4 shows a vertical cross section through a sample container in accordance to the present invention;

Figure 5 shows a diagrammatic view of an arrangement for measuring microwave radiation; and

Figure 6 shows a grammatically in plan a further arrangement for measuring microwave radiation.

Apparatus for performing reactions in accordance with a preferred embodiment of the present invention is shown in Figures 1 and 2.

The apparatus includes cabinet (1) that is insulated against microwave leakage. As shown in Figure 2, the cabinet includes a magnetron (2) for generating microwave emission, a sample support (4) on which is received a container (11) having in a reactant chamber (5) , a cooling means (3) and monitoring devices (7) , for example. temperature gauges which may be direct as shown in this figure or indirect as shown in Figure 2.

The cabinet is also served by means (13) for creating a vacuum therein. Reactant chemicals (a-h) are stored in a suitable storage unit (14) and can be transferred to the reactant chamber when desired under the control of microprocessor (12) . The choice of reactant can be controlled by way of control valve block Cl which is linked to the microprocessor. Also paraffin wax for introduction into the container (11) is stored in storage unit (15) , the use thereof being controllable by way of control valve block C2, which again is coupled to the microprocessor. A heater H is used to heat the wax when required to the appropriate temperature, e.g.65 °, operation of the heater being controllable by the microprocessor. Pumps P are used to circulate the various substances in the apparatus and are also controllable by the microprocessor.

The microprocessor is also linked to the magnetron, the cooling means and the monitoring devices so that the performance of the reactions can be automatically and accurately carried out. The computerisation of the process increases the efficiency thereof and also allows the process to be carried out in a contamination free environment.

The use of such an arrangement allows greater control of the whole reaction process. In particular, it provides greater control of the magnetron cycling time and further variation of the magnetron operating power.

The capability of being able to set up a vacuum within the cabinet allows the process to be carried out at even lower temperatures, thereby enhancing the advantages of lower temperatures discussed above in reaction to the use of the microwaves. The cabinet (1) itself constitutes an insulated cabinet of the sort normally associated with microwave enclosures and well known as such. The cabinet may be provided with in a fully computerised set up as described above and/or may include a control panel (8) with control input actuators

(9) as known in the art and shown in Figure 2. A different arrangement of programmes in accordance with the chemical reactions required may be programmed using the actuators or may of course be attained by way of the microprocessor arrangement shown in Figure 1. The cabinet also provides a distribution fan (10) disposed in the upper portion of the cabinet (1) to evenly distribute microwave radiation (16) from the magnetron (2) over a sample support which is preferably of a turntable nature (4) . Sample support (4) is driven by means of a sample support drive or axis.

The cabinet (1) is also provided with a cooling means shown generally at (3) . In the embodiment shown, the cooling means comprises a heat transfer unit including a sleeve (6) disposed about the reactant chamber (5) , the sleeve being connected to a liquid input and a liquid output. Liquid cooled by cooling unit (3) is fed to the sleeve where it is urged to circulate to the output. In the process of passing from the relatively hot liquid input to the output, heat is transferred from the reactant to the cooling liquid, whereby the reactant is cooled. The warmed cooling liquid passes from the liquid output to the cooling means whereby the cooling process is continued. Of course, any suitable cooling means may be employed.

With reference to Figure 3 a cell preparation or tissue section (intracellular target nucleic acid) (17) for analysis may be disposed within a well (18) formed on a first surface which is preferably a slide (19) of glass or plastic. The reactants (20) may then be disposed therein whereupon a cover slip (21) is placed in contact with a bio-compatible semi-adhesive filler such as a gum, glue or nailpolish (22) .

Alternatively the sample comprises a container and lid as shown in Figure 4. The container (23) and lid (25) are both composed of microwave transmitting and microwave stable material. The container contains cell or tissue suspension or liberated nucleic acid (intracellular or free nucleic acid respectively) .

The control panel (8) or microprocessor (12) is actuated to provide, for example, 40 cycles of heating and cooling. The power input to the magnetron is suitably controlled as is the efficiency of the heat exchanger (3).

Accordingly with the door (not shown) to the cabinet (1) closed the process is actuated by pressing the correct actuator (9) on the control panel (8) or providing the required instruction via the microprocessor. The sample support (4) rotates in the field of the microwave radiation which is evenly distributed throughout the interior of the cabinet by means of fan (10) . Alternatively or additionally the turntable (4) may oscillate through a defined angular segment so as to impart mixing action to the contents of the well (18) or container (23) . After a predetermined period of heating the preliminary reaction will have commenced. It may then be necessary to cool the sample before a reaction, for example, a nucleic acid reaction, is able to proceed and/or before another sequence of polymerization, synthesis and/or amplification is initiated.

The measurement of microwave energy within the cabinet may be effected by any suitable means, for example as shown in Figures 5 or 6. In the embodiment of Figure 5, microwave energy is absorbed by black material, and the amount of absorbed energy can then be measured. Microwave energy within the cabinet is allowed to pass across a rapid microwave energy transmitting material (preferably plastics) (26) so as to be absorbed by black material (29) present on one side of each blade of a fan (27) . The other side of each blade of this fan is formed of a less microwave absorbent material (for example a white material) . The different degree of energy absorbtion will cause the fan to rotate and the rate of rotation can be measured readily by any suitable known means indicated by reference 28.

An alternative method of measuring microwave energy using energy absorbtion by black material is shown in Figure 6. In this method energy is absorbed by a black material (35) . The absorbed energy is then transmitted as heat across the rapid heat transmitting heat (and microwave) stable material (30) formed of a material such as a plastic, which heats a fluid within a heat transmitting heat and microwave stable pipe (31) formed of, e.g. plastics. The temperatures of the heated fluid at input and output may be detected by known methods to the exterior of the microwave casing by devices (32) and (34) . Fluid may be added and withdrawn at a controlled rate if required via valve (33) connected to a reservoir; the input temperature is measured by well known methods. The difference in temperature (T2- Tl) between the measuring device (34) and the measuring device (32) may therefore be ascertained and is proportional to the amount of microwave energy absorbed by the black material. Of course, any suitable alternative means for measuring the microwave energy transferred may be utilised.

Accordingly at that point, and in accordance with control inputs from the control panel (9) or microprocessor (12) , the magnetron (2) shuts down or reduces output and the cooling means (3) is initiated to cool the cabinet (1) .

The sample (Figure 1) is therefore subjected to rapid cooling for a predetermined period. When the predetermined period has expired the cycle is repeated, for example, for up to 40 cycles. This allows the amplification reaction, for example, to proceed to a conclusion in a much shorter time than would previously have been required and without significant skilled intervention. This allows for the ready provision of amplified specimens for tests and can be effected by relatively less qualified staff using the apparatuses in accordance to the present invention.

Claims

1. A microwave heating and cooling apparatus comprising:- a cabinet, a heating means for generating microwave energy within the interior of said cabinet, control means therefor, and a sample support disposed within said cabinet; characterised in that said cabinet further comprises cooling means to cool the sample and in that the control means is adapted to initiate a cooling cycle after cessation of, or during a specified period of, a heating cycle.

2. An apparatus according to Claim 1 wherein said device is adapted to perform a plurality of successive heating and cooling steps according to a preset control input.

3. An apparatus according to either of Claims 1 or 2 wherein said cooling means is adapted to provide a current of cool and/or dry air toward the sample support.

4. An apparatus according to any of Claims 1 to 2 wherein said cooling means comprises a cooling liquid arranged to circulate between the sample or sample reactant and a cooling unit.

5. An apparatus according to any of Claims 1 to 4 including means for rotating and/or oscillating the sample support during the heating cycle and optionally during the cooling cycle.

6. An apparatus according to any of Claims 1 to 5 for varying the relative humidity of the interior of the cabinet during the heating step.

7. An apparatus according to any of Claims 1 to 6 wherein the sample comprises a microwave transparent first surface including a well formed therein or thereon, said well being associated with a microwave transparent cover slip.

8. An apparatus according to any of Claims 1 to 6 wherein the sample is disposed in a microwave transparent container.

9. An apparatus according to any of Claims 1 to 8 wherein the well comprises a target nucleic acid, present intracellularly (tissues or cell preparations) or extracelluarly (free, extracted or cell or tissue lysate) and a nucleic acid synthesis mixture including a transcription or PRINS or amplification mixture and wherein the heating and cooling cycling steps are repeated according to a preset program thereby to synthesise a target nucleic acid sequence.

10. An apparatus according to any preceding claim including a computer for controlling at least the heating and cooling means.

11. A process for performing amplification and/or replication reactions using microwaves, the process comprising the steps of

a) subjecting a sample for amplification and/or replication to microwave emissions for a predetermined period;

b) subjecting the sample to rapid cooling; and

c) successively repeating steps a) and b) .

12. A process as claimed in claim 11 wherein the process performs PCR reactions in situ and the sample is disposed in a microwave transparent enclosure in the presence of liquid reactants.

13. A process as claimed in claim 11 or 12 wherein the microwaves are concentrated in a predetermined region adjacent the sample.

14. A process as claimed in any of claims 11 to 13 wherein cooling is effected using a cooling fluid circulated at or adjacent the sample and/or reactant(s).

15. A process as claimed in any of claims 11 to 14 wherein the process includes inducing a vacuum about the sample at least whilst subjecting the sample to microwave emissions.

16. A process as claimed in any of claims 11 to 15 wherein the steps (a) and (b) are controllable by means of a computer in accordance with a preset programme.