WO1990005329A1 - Appareil de reglage de la temperature et ses applications - Google Patents

Appareil de reglage de la temperature et ses applications Download PDF

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Publication number
WO1990005329A1
WO1990005329A1 PCT/GB1989/001339 GB8901339W WO9005329A1 WO 1990005329 A1 WO1990005329 A1 WO 1990005329A1 GB 8901339 W GB8901339 W GB 8901339W WO 9005329 A1 WO9005329 A1 WO 9005329A1
Authority
WO
WIPO (PCT)
Prior art keywords
temperature
bath
control apparatus
liquid
control system
Prior art date
Application number
PCT/GB1989/001339
Other languages
English (en)
Inventor
Bryan Clifford Sykes
Original Assignee
Grant Instruments (Cambridge) Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Grant Instruments (Cambridge) Limited filed Critical Grant Instruments (Cambridge) Limited
Publication of WO1990005329A1 publication Critical patent/WO1990005329A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • G—PHYSICS
    • G05—CONTROLLING; REGULATING
    • G05D—SYSTEMS FOR CONTROLLING OR REGULATING NON-ELECTRIC VARIABLES
    • G05D23/00—Control of temperature
    • G05D23/19—Control of temperature characterised by the use of electric means
    • G05D23/1919—Control of temperature characterised by the use of electric means characterised by the type of controller

Definitions

  • 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.
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • 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.
  • the reaction of this process can proceed repetitively to achieve a large amplification in the amount of the 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.
  • 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
  • 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.
  • PCR polymerase chain reaction
  • the preferred temperature cycle comprises three stages defining a temperature/time profile which has three different user- selected 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.
  • this process generally requires (i) a difference between the highest selected temperature and the lowest selected temperature in each temperature cycle which is greater, often substantially greater, than 25°C, (ii) steep temperature rises and falls so that the transition times in changing from one user-selected temperature level to another user- selected 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.
  • 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.
  • the preferred heat transfer medium liquid used in the bath will be water
  • 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.
  • the bath comprises an open-topped tank or trough of substantially rectangular form in horizontal cross-section whilst the above- mentioned 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.
  • the heater preferably comprises an immersion heater element or elements located in the lower part of the bath.
  • 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.
  • 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.
  • 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.
  • 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, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIGURE 7 is a perspective view illustrating a further embodiment of the apparatus in accordance with the invention.
  • the apparatus as shown diagram atically 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 open- topped rectangular metal tank or trough which is preferably lagged to reduce heat loss.
  • the bath may have a length of approximately 180mm and a height and depth each of approximately 100mm.
  • the bath 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.
  • 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 bath and which closely follows the rectangular contour of the walls thereof.
  • 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 syste .
  • 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.
  • 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.
  • rack 18 fitted with a few such tubes 17 is illustrated in FIGURE 6.
  • 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 for 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 diagrammati ⁇ ally 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.
  • 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 pre ⁇ set number of cycles has been completed.
  • 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.
  • 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.
  • an idealised temperature/time profile Is shown in the diagram of FIGURE 3.
  • FIGURE 5 represents a schematic diagram of the control system which operates as hereinafter described.
  • 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 Tl 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.
  • 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.
  • timing relay TR4 is activated. This represents the commencement of a post-treatment period which is usually 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.
  • the heating and cooling means may be arranged to be thermo ⁇ statically controlled to maintain the bath temperature close to the selected temperature value during the entire time period between successive temperature transitions.
  • 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 re ⁇ sult in temperature changes of an undesirably high order.
  • control system instead of being of an electromechanical design as herein descr ⁇ ibed, may alternatively be of an electronic design including a microprocessor together with other solid state electronic components and associated interfacing circuitry.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Automation & Control Theory (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Un bain-marie comprenant une cuve ou auge à toit ouvert (10), contenant un thermoplongeur (12), un refroidisseur tubulaire (14) et des détecteurs de température à thermostat (16a, 16b, 16c), est doté d'un système de commande programmable conçu pour enclencher et déclencher le thermoplongeur et le refroidisseur de manière à obtenir rapidement et automatiquement des cycles de température grâce à un profil temps/température prédéterminé comportant des étapes successives, caractérisées chacune par une température particulière choisie par l'utilisateur et par une période d'arrêt pendant laquelle le bain-marie reste à cette température. Cet appareil est conçu en particulier pour les réactions en chaîne de polymérase (PCR), pour amplifier les séquences d'acides nucléiques.
PCT/GB1989/001339 1988-11-10 1989-11-10 Appareil de reglage de la temperature et ses applications WO1990005329A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB888826364A GB8826364D0 (en) 1988-11-10 1988-11-10 Improvements in/relating to temperature sequencing
GB8826364 1988-11-10

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WO1990005329A1 true WO1990005329A1 (fr) 1990-05-17

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0463648A2 (fr) * 1990-06-29 1992-01-02 Patrick Collin Pomeroy Procédé et dispositif pour le développement de substances transférées sur un support solide
US5270183A (en) * 1991-02-08 1993-12-14 Beckman Research Institute Of The City Of Hope Device and method for the automated cycling of solutions between two or more temperatures
US5819842A (en) * 1991-12-05 1998-10-13 Potter; Derek Henry Method and apparatus for temperature control of multiple samples
WO2000029115A1 (fr) * 1998-11-16 2000-05-25 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Dispositifs et procedes de temperage d'echantillons
WO2003075111A1 (fr) * 2002-03-06 2003-09-12 Samsung Electronics Co., Ltd. Procede de commande de la temperature et appareil de pilotage d'une puce par amplification en chaine par polymerase (pcr)
WO2009129777A2 (fr) * 2008-04-21 2009-10-29 Hirt Zerspanungstechnik Gmbh Dispositif de réchauffage d'un objet au moyen d'un bain d'eau
CN103041883A (zh) * 2011-10-15 2013-04-17 四川汇利实业有限公司 能够监测水温的水浴锅
CN103041885A (zh) * 2011-10-15 2013-04-17 四川汇利实业有限公司 能够监测温度的搅拌式恒温水浴振荡器
RU2506624C2 (ru) * 2012-04-27 2014-02-10 Федеральное государственное бюджетное учреждение "Арктический и Антарктический научно-исследовательский институт" (ФГБУ "ААНИИ") Термостат для калибровки и проверки океанографических приборов
CN116719365A (zh) * 2023-07-03 2023-09-08 深圳海关食品检验检疫技术中心 一种pcr温度控制装置及控制方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8807297D0 (en) * 1988-03-26 1988-04-27 Dean P D G Intelligent heating block
CN105779973A (zh) * 2014-12-23 2016-07-20 中国电子科技集团公司第十八研究所 一种用于化学镀的恒温水浴装置
CN105647795A (zh) * 2016-02-25 2016-06-08 李亮亮 基于pcr技术的新型微生物酶快速检测装置
CN111568164B (zh) * 2020-05-22 2021-10-08 海信集团有限公司 一种蒸烤箱

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US2645461A (en) * 1948-08-26 1953-07-14 Socony Vacuum Oil Co Inc Thermoregulator
US3323578A (en) * 1964-12-11 1967-06-06 Gen Motors Corp Method and apparatus for controlling temperatures
US3348556A (en) * 1965-05-27 1967-10-24 Interlab Inc Temperature regulation of fluid baths
EP0236069A2 (fr) * 1986-02-25 1987-09-09 The Perkin-Elmer Corporation Appareil et méthode pour réaliser l'amplification automatique de séquences d'acides nucléiques et essais utilisant des étapes de chauffage et refroidissement

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
US2645461A (en) * 1948-08-26 1953-07-14 Socony Vacuum Oil Co Inc Thermoregulator
US3323578A (en) * 1964-12-11 1967-06-06 Gen Motors Corp Method and apparatus for controlling temperatures
US3348556A (en) * 1965-05-27 1967-10-24 Interlab Inc Temperature regulation of fluid baths
EP0236069A2 (fr) * 1986-02-25 1987-09-09 The Perkin-Elmer Corporation Appareil et méthode pour réaliser l'amplification automatique de séquences d'acides nucléiques et essais utilisant des étapes de chauffage et refroidissement

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Perkin Elmer Cetus : GeneAmp DNA Amplification Reagent Kit. Printed in U.S.A. Perkin -Elmer Cor Order No. L-1060 September 1987 Printed in U.S.A. Perkin Elmer Corporation see the whole document *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0463648A2 (fr) * 1990-06-29 1992-01-02 Patrick Collin Pomeroy Procédé et dispositif pour le développement de substances transférées sur un support solide
EP0463648A3 (en) * 1990-06-29 1992-05-06 Patrick Collin Pomeroy Development of post-transferred material on solid supports: method and apparatus
US5270183A (en) * 1991-02-08 1993-12-14 Beckman Research Institute Of The City Of Hope Device and method for the automated cycling of solutions between two or more temperatures
US5819842A (en) * 1991-12-05 1998-10-13 Potter; Derek Henry Method and apparatus for temperature control of multiple samples
WO2000029115A1 (fr) * 1998-11-16 2000-05-25 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Dispositifs et procedes de temperage d'echantillons
WO2003075111A1 (fr) * 2002-03-06 2003-09-12 Samsung Electronics Co., Ltd. Procede de commande de la temperature et appareil de pilotage d'une puce par amplification en chaine par polymerase (pcr)
WO2009129777A2 (fr) * 2008-04-21 2009-10-29 Hirt Zerspanungstechnik Gmbh Dispositif de réchauffage d'un objet au moyen d'un bain d'eau
WO2009129777A3 (fr) * 2008-04-21 2011-10-27 Hirt Zerspanungstechnik Gmbh Dispositif de réchauffage d'un objet au moyen d'un bain d'eau
US8642930B2 (en) 2008-04-21 2014-02-04 Pfm Medical Ag Device for heating an object by means of a water bath
CN103041883A (zh) * 2011-10-15 2013-04-17 四川汇利实业有限公司 能够监测水温的水浴锅
CN103041885A (zh) * 2011-10-15 2013-04-17 四川汇利实业有限公司 能够监测温度的搅拌式恒温水浴振荡器
RU2506624C2 (ru) * 2012-04-27 2014-02-10 Федеральное государственное бюджетное учреждение "Арктический и Антарктический научно-исследовательский институт" (ФГБУ "ААНИИ") Термостат для калибровки и проверки океанографических приборов
CN116719365A (zh) * 2023-07-03 2023-09-08 深圳海关食品检验检疫技术中心 一种pcr温度控制装置及控制方法

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GB2226161A (en) 1990-06-20
GB8826364D0 (en) 1988-12-14
GB8925459D0 (en) 1989-12-28
AU4744890A (en) 1990-05-28

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