EP2827991B1

EP2827991B1 - Thermal cycler and method for heating and cooling

Info

Publication number: EP2827991B1
Application number: EP13714243.6A
Authority: EP
Inventors: Stephan Kling; Michael Heil
Original assignee: Qiagen GmbH
Current assignee: Qiagen GmbH
Priority date: 2012-03-23
Filing date: 2013-03-22
Publication date: 2019-05-29
Anticipated expiration: 2033-03-22
Also published as: EP2827991A1; WO2013139970A1

Description

The invention pertains to the field of laboratory equipment for chemical, biological or for biotechnological methods which require a specific temperature, especially repeated heating and cooling. In particular the invention relates to a thermal cycler for use in the point-of-need analysis comprising a reaction chamber, a heating means and a fan. Further, the invention relates to a method for heating and cooling a reaction chamber of a thermal cycler.
Many laboratory methods require handling in such a way that the process or a particular step in the process is dependent on analysis and/or treatment of the respective reaction mixture.
One example for a process which requires specific handling of vessels and analysis of parameters is the polymerase chain reaction (PCR). PCR is a technique in molecular biology that allows polynucleotide sequences to be amplified. The basic principle of PCR is well known in the art and is described in many books, including, PCR: A Practical Approach M. J. McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methods and Applications by Innis, et al., Academic Press (1990), and PCR Technology: Principals and Applications for DNA Amplification H. A. Erlich, Stockton Press (1989). PCR is also described in many U.S. patents, including U.S. 4,683,195 ; 4,683,202 ; 4,800,159 ; 4,965,188 ; 4,889,818 ; 5,075,216 ; 5,079,352 ; 5,104,792 ; 5,023,171 ; 5,091,310 ; and 5,066,584 .
Each PCR cycle usually comprises three basic discrete temperature steps, a denaturation step, an annealing step and an extension step. Denaturation of DNA is typically performed at around takes place at around 90 to 95°C, annealing a primer to the denatured DNA is typically performed at around 40 to 60°C, and the step of extending the annealed primers with a polymerase is typically performed at around 70 to 75°C. Therefore, during a PCR cycle the temperature of the reaction mixture must be varied repeatedly. The PCR technique has a wide variety of biological applications, including but not limited for example, DNA sequence analysis, probe generation, cloning of nucleic acid sequences, site-directed mutagenesis, detection of genetic mutations, diagnoses of viral infections, molecular "fingerprinting" and the monitoring of contaminating microorganisms in biological fluids and other sources.
The PCR is usually carried out in a laboratory apparatus called thermal cycler or PCR cycler.
In addition to PCR, other in vitro amplification procedures, including ligase chain reaction as disclosed in U.S. Patent No. 4,988,617 to Landegren and Hood , are known and used in the prior art. More generally, several important methods known in the biotechnology, such as nucleic acid hybridization and sequencing, are dependent upon changing the temperature of solutions containing sample molecules in a controlled fashion and also require monitoring.
Conventional techniques rely on use of individual wells or tubes cycled through different temperatures. For example, a number of thermal "cyclers" used for DNA amplification and sequencing are disclosed in the prior art in which a temperature controlled element or "block" holds a reaction mixture, and wherein the temperature of the block is varied over time. An advantage of these devices is that a relatively large number of samples can be processed simultaneously, e.g. 96 well plates are commonly employed.
Different designs of such thermal cyclers are known including devices for the thermal cycling of multiple samples. A common format of such devices uses a block of heat conductive material which has a plurality of channels or cavities for receiving vessels - such as reaction tubes or plates - in which the desired reactions are executed. Monitoring of temperature is relatively easy in such devices since a temperature sensor can be associated with the block.
However, such block devices suffer various drawbacks, e.g. in that they are relatively slow in cycling the reaction mixtures, they are relatively energy intensive to operate and detection of parameters of the reaction mixture in situ is difficult. In an effort to avoid several of these disadvantages, thermal cyclers have been developed in which a plurality of containers for holding reaction mixture(s) is supported on a rotor which is rotated in a controlled temperature environment. Temperature cycling is effected by heating and cooling of the environment. One of the thermal cyclers comprising a rotor is the "Rotor-Gene" of QIAGEN. A further example of such a thermal cycler is disclosed in WO 98/49340 A1 .
WO 2007/054747 A1 , US 2002/0123131 A1 , WO 03/000428 A1 , and EP 1649931 A1 disclose thermal cyclers comprising a reaction chamber, a heating means, and an air supply means.
The present invention seeks to solve the problem of providing an improved thermal cycler and a method for heating and cooling a reaction chamber of a thermal cycler.
The problem is solved by a thermal cycler comprising a reaction chamber, a heating means and air supply means as defined in claim 1. The thermal cycler comprises means for mixing air with different temperatures.
The present invention is based on the finding that mixing air with different temperatures will affect the time required to obtain a specific temperature for heating as well as for cooling. According to the invention a thermal cycler can be realized which is capable of heating and cooling to specific temperatures in a short time. The temperatures of the cycles can be obtained precisely and with almost no overshoot.
According to the invention a "thermal cycler" is a laboratory apparatus or device for carrying out thermal cycles with regard to a reaction process, especially a polymerase chain reaction. The thermal cycler is capable of raising and lowering the temperature of an environment, e.g. a reaction chamber, in which micro-environments are provided in discrete, pre-determined steps. A thermal cycler according to the invention can preferably obtain and maintain temperatures in the range of room temperature and 95°C.
It will be appreciated from the above that the term thermal cycler is preferably directed to a thermal cycler for nucleic acid amplification. Reaction vessels, vials or tubes (the micro-environments) may be supported on a rotatable circular carousel or rotor rotatably mounted within the environment.
The term "reaction chamber" according to the invention is intended to encompass any environment, i.e. any space in which micro-environments may be placed. The reaction chamber is not necessarily required to be closed. The reaction chamber may be any space which is at least partially defined to hold the reaction mixture and can be in contact with the air.
The term "environment" according to the invention is intended to encompass any temperature-controlled environment established in the thermal cycler, e.g. in a (insulated) reaction chamber of the thermal cycler. E.g. the temperature in said environment can be controlled for a process or a step within a process to be carried out at a specific temperature. Temperature cycling can be achieved in the respective thermal cycler by heating and cooling the environment by air flow.
In the environment, e.g. the reaction chamber, micro-environment may be provided. The term "micro-environment" according to the invention relates to a volume in which a reaction mixture can be filled. Preferably, the volume of the micro-environment is in the range of 0.01 to 2.0 ml, preferably 0.1 to 0.6 ml. The term "reaction mixture" according to the invention is intended to encompass an amount of liquid, solution or suspension intended to be reacted and/or subjected to qualitative or quantitative determination of any of its properties, such as the presence or absence of a component, the concentration of a component, etc. The reaction mixture is subjected to cyclic heating and/or cooling, especially in accordance to carry out a polymerase chain reaction.
The term "heating means" according to the invention is intended to encompass any means which provides Joule heating, ohmic heating or resistive heating, in which the passage of an electric current through a conductor releases heat. The heating means may be in the shape of a heater coil.
The term "temperature adjusted air" according to the invention relates to air which has been cooled by cooling means or heated by the heating means. The term "temperature adjusted air" is used in contrast to the term ambient air (at room temperature) which has been sucked into the thermal cycler but has not been affected by the heating means or cooling means.
The dimensions of the thermal cycler can be small and the elements provided for heating and cooling then do not lead to a substantial increase of the outer dimensions of the thermal cycler. Even if the thermal cycler is portable as required in the field of point-of-need analysis, the assembly or build-up of the thermal cycler is simple.
The position of the heating means with regard to the air flow is chosen to be behind the reaction chamber such that the "status" of the heating means is decoupled from the purpose whether to cool or to heat the reaction chamber.
According to the invention the heating means is positioned behind the air supply means with regard to the flow of air. Thus, the heating means is arranged in blowing direction of the air supply means and the heating means is arranged behind the reaction chamber with regard to the blowing direction. Therefore, an air flow path can be established in which the reaction chamber is arranged behind air supply means. Further, in the air flow path the heating means is arranged behind the reaction chamber with regard to the flow of air. The described air flow path can lead to an advantageous supply of air after which has passed the heating means and enters the reaction chamber. Additionally, an efficient control of temperature can be obtained. Further, the heating means can be heated regardless whether the reaction chamber is to be heated or not with almost constant conditions of air flow. Almost constantlv blowing air onto the heating means can produce an appropriate turbulence and result in a long life time of the heating means.
The term "point-of-need analysis" relates to the location of use in that the reaction mixtures in the micro-environments are subjected to the thermal cycling on the spot or on-site. Thus, the reaction mixtures or samples do not have to be transported into a laboratory with stationary equipment. Instead, the thermal cycler can be transported to the point-of-need, i.e. to the location where the samples or reaction mixtures are provided. Therefore, the thermal cycler according to the invention is preferably portable and, thus, small of volume and simple to handle which is required by an analysis on the spot or on-site.
Preferably, the air with different temperatures can be temperature adjusted air on one hand and ambient air on the other hand.
The air supply means is a fan. Preferably, means are arranged to adjust the direction of the temperature adjusted air flow around the reaction chamber. Additionally to the mixing of the air, adjusting of the direction will lead to a further decrease of time for cooling and heating to a specific temperature.
In a preferred embodiment means for adjusting the direction of the temperature adjusted air flow are provided, wherein the heating means is arranged behind the fan and the reaction chamber in the air flow path. The position of the heating means with regard to the air flow can lead to a further decrease of time required to obtain a specific temperature in the reaction chamber of the thermal cycler.
The means for mixing and adjusting the temperature adjusted air comprise PTFE and PVDF.
Preferably, in the thermal cycler at least a first and a second path of air flow are provided, wherein the first air flow path is a circulation of air, in which the air circulates between fan, reaction chamber and heating means, and the second air flow path comprises a part of the first air flow path between the fan and the heating means, wherein the second air flow path comprises a suction of ambient air into the reaction chamber through an inlet and an exhaust of air through an outlet, wherein the heating means is arranged in the second air flow path between the reaction chamber and the outlet. The circulation of air according to first air flow path is a closed loop of air flow such that the air which passed the heating means passes again the fan. Further, with the specification that the second air flow path comprises a part of the first air flow path, a small thermal cycler can be obtained. Further the assembly of the deflection means may be simple and some of the deflection means can be used for the first and the second air flow path. In principle it becomes possible that a combination of the first and the second air flow path can be obtained. Further, the circulation of air in the thermal cycler leads to a decrease of required energy due to the pre-heated air and the reaction chamber can be heated fast and efficient.
In a preferred embodiment the reaction chamber is at least partly limited by an air guide plate for guiding the air through the reaction chamber from a center area to a circumferential area. Preferably, the guide plate limits the reaction chamber with regard to the top exterior wall of the thermal cycler. The reaction chamber may be the space between a base and the air guide plate. Such an arrangement is simple and the thermal cycler may be constructed with the heating means and the fan above the reaction chamber, the heating means and the fan comprised in a unit or module. The unit or module may be part of the closure of the thermal cycler.
In a preferred embodiment the fan is an axial-flow fan and arranged in substantial rotational symmetry with regard to the reaction chamber. This will lead to a rotational symmetric arrangement of the built-up of the thermal cycler which results in an advantageous relationship of the volume and the base area of the thermal cycler. Preferably the base of the reaction chamber is in the shape of a circle.
Further, it is preferred that the inlet is provided in rotational symmetry with regard to the fan and the reaction chamber. A rotational symmetric inlet for ambient air may lead to a homogenous distribution of air flow and change of temperature with regard to the center of the reaction chamber.
In a preferred embodiment the heating means is provided in close proximity to the outlet. The influence of the heating means which is positioned in close proximity to the outlet is minimized for the cooling step of the reaction chamber. Preferably, the inlet and the outlet are arranged in close proximity to each other. Preferably the inlet and the outlet are oriented in a direction substantially perpendicular to the base of the thermal cycler or the base of the reaction chamber. The inlet and the outlet may be formed in a closure of the thermal cycler, the closure further comprising the heating means and the fan such that the heating means and the fan are arranged in the closure as a unit. The closure may be a cap of plastic material with high heat resistance. Preferably, the closure is double-walled which reduces thermal loss and the temperature on the outside of the closure.
The term "close proximity" according to the invention is intended to encompass that the inlet and the outlet are adjacent to each other without interfering air flows such that the suctioned air is substantially ambient air and not the air passed through the outlet.
In a preferred embodiment the deflection means comprises an air flow mixing valve for a continuous change between the first and the second position. With the air flow mixing valve it is possible to operate the thermal cycler not only in a mode with the first air flow path or the second air flow path but a mixture between those two paths. A mixture of cool air (ambient air, second air flow path) and heated air (first air path) allows a fast and accurate temperature control of the reaction chamber and the reaction mixtures. The heating means can be held on high temperature which results in a fast temperature increase in case of heating the reaction chamber. The air flow mixing valve may be integrated in the closure.
It is advantageous to keep the number of movable parts within the thermal as low as possible. Therefore, the deflection means arranged between the heating means and the air supply means is the only movable part for guiding air within the thermal cycler. Further elements for establishing the air flow paths can be incorporated in the closure or the housing of the thermal cycler. This can prolong the life time of the thermal cycler or simplify maintenance. Thus, the air path between the air supply means and the reaction chamber can be clear and without movable parts with regard to guidance of the air flow. The air path between the air supply means and the reaction chamber can be unchanged over time. The same can hold for the air path between the reaction chamber and the heating means.
In a preferred embodiment the first and the second air flow path each have a length of 4 to 8 cm. Preferably, the length is in the range of about 5 to 7 cm, especially preferred the length is about 7 cm. The mentioned values are intended as approximate values. The mentioned values might vary ±1 cm or ±10%.
Due to the length of the first and second air flow path a very small thermal cycler can be achieved. It is possible that only a small air volume and a small surface in the air flow paths needs to be tempered. This will lead to even shorter heating and cooling cycles due to the compact dimensions of the thermal cycler.
Further, a method for heating and cooling a reaction chamber of a thermal cycler is provided. The reaction chamber is heated and cooled by mixing temperature adjusted air and ambient air.
Preferably the direction of the temperature adjusted air flow around the reaction chamber is adjusted.
In a preferred embodiment the air according to at least a first and a second path of air flow is guided, wherein the first air flow path is established as a circulation of air, in which the air circulates between fan, reaction chamber and heating means, and the second air flow path is established as comprising a part of the first air flow path between the fan and the heating means, wherein according to the second air flow path suction of ambient air into the reaction chamber through an inlet and diverting air through an outlet is carried out, wherein the heating means is arranged in the second air flow path between the reaction chamber and the outlet.
Other objects, features, advantages and aspects of the present application will become apparent to those skilled in the art from the following description and appended claims.
Examples of the invention will now be described with reference to the accompanying drawings in which:

Figure 1 is a schematic side view of an embodiment of a thermal cycler according to the invention operated in a heating mode;
Figure 2 is a schematic side view of the thermal cycler according to Figure 1 in a cooling mode;
Figure 3 is a schematic side view of the thermal cycler according to Figure 1 in a mixing mode;
Figure 4 is a perspective front view of the thermal cycler according to Figure 1 with opened closure;
Figure 5 is schematic side view of a further embodiment of a thermal cycler according to the invention operated in a heating mode;
Figure 6 is a schematic side view of the thermal cycler according to Figure 5 in a cooling mode; and
Figure 7 is a schematic side view of the thermal cycler according to Figure 5 in a mixing mode.

Figure 1 is a schematic side view of a thermal cycler according to the invention. The thermal cycler is for use in the point-of-need analysis. The thermal cycler comprises a reaction chamber 1, a heating means 2 and a fan 3. In the reaction chamber 1 micro-environments in the form of reaction vessels, vials or tubes 4 may be located. The reaction chamber 1 is located between a base 8 (see Figure 4) with regard to the bottom and an air guide plate 9 which extends from the fan 3 to a circumferential area of the reaction chamber 1.
The fan 3 is an axial-flow fan and the heating means 2 is formed as a heating coil. The fan 3 is arranged in rotational symmetry to the reaction chamber 1 and the heating means 2. Thus, the fan 3, the reaction chamber 1 and the heating means 2 have a coinciding center line. The middle area of the reaction chamber is open and the fan 3 can blow air into the reaction chamber 1. The reaction chamber 1 is not limited to the top at the circumferential or peripheral area so that air blown from the fan 3 into the reaction chamber 1 can exhaust the reaction chamber 1 at the peripheral area to the top in the direction of the heating means 2. A channel to the top from the reaction chamber 1 is formed by the inner surface of the circumferential side wall of a closure 10 and a curved guide plate 12.
Deflection means are provided for adjusting a path of air flow. With regard to Figure 1 an air flow for heating the reaction chamber 1 is described. The fan 3 circulates the air in the thermal cycler. The circulating air passes the fan 3, the reaction chamber 1 and the heating means 2. The heating means 2 heats the air which passes the heating means 2. The air flow is in toroidal ring form substantially symmetric to the center of the fan 3. The air flow path established for heating is indicated by dotted line 6. In the air flow path shown in Figure 1, the heating means 2 is arranged behind the reaction chamber 1 with regard to the fan 3.
Figure 2 describes cooling of the reaction chamber 1 in the thermal cycler. By means of an air flow mixing valve an inlet 6 and an outlet 11 are opened. Further, the air flow mixing valve closes the air passage between the heating means 2 and the fan 3. Through the inlet 6 the fan 3 is capable to aspirate ambient air into the thermal cycler. The ambient air passes the fan 3 and is blown into the reaction chamber 1 by the fan 3. The air flows from the middle area of the reaction chamber 1 to the peripheral area of the reaction chamber 1 and up to the heating means 2. In close proximity to the heating means the outlet 11 is formed and the air exhausts the thermal cycler. The air flow path for cooling is indicated by a dotted line denoted by reference sign 7. The air flow path 7 for cooling comprises a part of the first air flow path 5 between the fan 3 and the heating means 2.
In Figure 3 it is shown that the thermal cycler is operated in a mixing mode in which the inlet 6 and the outlet 11 are partially opened. Further, the passage between the heating means 2 and the fan 3 is at least partially open. The air which passes the heating means 2 flows partially to the fan 3 (circulation according to air flow path 5) and partially to the outlet 11 (air flow path 7). Thus, the air flow mixing valve is in an intermediate position between the first position and the second position.
Figure 4 shows the thermal cycler with opened closure 10. As can be seen from Figure 4 the cooling and heating arrangement is formed as a unit or module located in the closure 10 of the thermal cycler.
Figure 5 is a schematic side view of a thermal cycler according to a further embodiment of the invention. The thermal cycler comprises a reaction chamber 1, a heating means 2 and two fans 3a, 3b. The reaction chamber 1 is located between a base 8 with regard to the bottom and an air guide plate 9. The fans 3a, 3b are formed as cross-flow blower and the heating means 2 is formed as a heating coil.
In the heating mode the fan 3b circulates the air in the thermal cycler. The temperature adjusted air flows around the thermal cycler.
In the cooling mode shown in Figure 6, the fan 3a arranged in an inlet opening channel for suctioning ambient air into the thermal cycler blows the ambient air into the reaction chamber 1. The inlet opening channel extends from the inlet 6. The ambient air will not pass the heating means 2 before entering the reaction chamber 1. The air that has passed the reaction chamber 1 reaches the heating means 2 and then exits the thermal cycler through an outlet 11.
Figure 7 shows a mixing mode. The inlet 6 is at least partially opened as well as the outlet 11. Ambient air enters the thermal cycler and cools the reaction chamber 1. The temperature adjusted air that passed the heating means 2 is at least partially diverted to the reaction chamber 1. Thus, the outlet 11 is at least partially open to allow air flow of the temperature adjusted air to the reaction chamber 1 as well as through the outlet 11. The mixing valve is on a position between the heating mode and the cooling mode.

Claims

A thermal cycler comprising a reaction chamber (1), a heating means (2), air supply means, and an air flow mixing valve for mixing air with different temperatures, characterized in that the reaction chamber (1) is arranged in blowing direction of the air supply means which is a fan and the heating means (2) is arranged behind the reaction chamber (1) with regard to the blowing direction for heating and cooling of the reaction chamber.
The thermal cycler according to claim 1, characterized in that the air with different temperatures is temperature adjusted air and ambient air.
The thermal cycler according to claim 1 or 2, characterized in that air flow mixing valve is arranged to adjust the direction of temperature adjusted air flow around the reaction chamber (1).
The thermal cycler according to claim 1 or 3, characterized in that deflection means are provided for adjusting the direction of the temperature adjusted air flow, wherein the heating means (2) is arranged behind the air supply means and the reaction chamber (1) in the air flow path.
The thermal cycler according to any one of claims 1 to 4, characterized in that at least a first and a second path (5, 7) of air flow are provided, wherein the first air flow path (5) is a circulation of air, in which the air circulates between air supply means, reaction chamber (1) and heating means (2), and the second air flow path (7) comprises a part of the first air flow path (5) between the air supply means and the heating means (2), wherein the second air flow path (7) comprises a suction of ambient air into the reaction chamber (1) through an inlet (6) and an exhaust of air through an outlet (11), wherein the heating means (2) is arranged in the second air flow path (7) between the reaction chamber (1) and the outlet (11).
The thermal cycler according to any one of claims 1 to 5, characterized in that the thermal cycler is for use in the point-of-need analysis.
The thermal cycler according to any one of claims 1 to 6, characterized in that the reaction chamber (1) is at least partly limited by an air guide plate (9) for guiding the air through the reaction chamber (1) from a center area to a circumferential area.
The thermal cycler according to any one of claims 1 to 7, characterized in that the air supply means is an axial-flow fan and arranged in substantial rotational symmetry with regard to the reaction chamber (1).
The thermal cycler according to any one of claims 5 to 8, characterized in that the inlet (5) is provided in rotational symmetry with regard to the air supply means and the reaction chamber (1).
The thermal cycler according to any one of claims 5 to 9, characterized in that the heating means (2) is provided in close proximity to the outlet (11).
The thermal cycler according to any one of claims 4 to 10, characterized in that the deflection means comprises the air flow mixing valve for a continuous change between the first and the second position.
The thermal cycler according to any one of claims 5 to 11, characterized in that the first and second air flow path each have a length of 4 to 8 cm.
A method for heating and cooling a reaction chamber (1) of a thermal cycler, comprising the steps of heating and cooling the reaction chamber (1) by mixing air with different temperatures, characterized by establishing an air flow path in which the reaction chamber (1) is arranged behind air supply means which is a fan and a heating means (2) is arranged behind the reaction chamber (1) with regard to the flow of air.
The method according to claim 13, characterized by using ambient air and temperature adjusted air as air with different temperatures.
The method according to claim 14, characterized by adjusting the direction of the temperature adjusted air flow around the reaction chamber (1).
The method according to any one of claims 13 to 15, characterized by guiding air to create an air flow path, in which the air passes heating means (2) after having passed the reaction chamber (1).
The method according to any one of claims 13 to 16, characterized by guiding air according to at least a first and a second path of air flow, wherein establishing the first air flow path (5) as a circulation of air, in which the air circulates between air supply means, reaction chamber (1) and heating means (2), and establishing the second air flow path (7) comprises a part of the first air flow path (5) between the air supply means and the heating means (2), wherein according to the second air flow path (7) comprises suction of ambient air into the reaction chamber (1) through an inlet (5) and diverting air through an outlet (11), wherein the heating means (2) is arranged in the second air flow path (7) between the reaction chamber (1) and the outlet (11).