WO2008065423A1

WO2008065423A1 - Thermal cycler

Info

Publication number: WO2008065423A1
Application number: PCT/GB2007/004606
Authority: WO
Inventors: David James Squirrell; Ross Jones
Original assignee: Enigma Diagnostics Limited
Priority date: 2006-11-30
Filing date: 2007-11-30
Publication date: 2008-06-05
Also published as: GB0623910D0

Abstract

Apparatus for thermally cycling the contents of a reaction vessel, said apparatus comprising an electrically conducting polymer arranged to act as a resistance heater for a reaction vessel, electrical contacts arranged to supply electrical current or potential to said polymer from a power supply, and a rigid foam material arranged in direct contact with at least one of said electrical contacts. The apparatus is particularly useful for polymerase chain reactions and for conducting analysis of the melting points of nucleic acids.

Description

Thermal Cycler

The present invention relates to apparatus for carrying out. chemical reactions in which rapid heating and cooling is required, in particular to thermal cyclers which are intended for use in carrying out reactions such as the polymerase chain reaction (PCR), as well as vessels and elements for use in these apparatus.

Reactions requiring thermal cycling, such as DNA amplification methods like the Polymerase Chain Reaction (PCR) are well known and widely used in the art. PCR is a procedure for generating large quantities of a particular DNA sequence and is based upon DNA' s characteristics of base pairing and precise copying of complementary DNA strands. Typical PCR involves a cycling process of three basic steps.

Denatυration : A mixture containing the PCR reagents (including the DNA to be copied, the individual nucleotide bases (A, T, G, C), suitable primers and polymerase enzyme) are heated to a predetermined temperature to separate the two strands of the target DNA.

Annealing : The mixture is then cooled to another predetermined temperature and the primers locate their complementary sequences on the DNA strands and bind to them.

Extension : The mixture is heated again to a further predetermined temperature. The polymerase enzyme (acting as a catalyst) joins the individual nucleotide bases to the end of the primer to form a new strand of DNA which is complementary to the sequence of the target DNA, the two strands being bound together.

Each stage of the reaction is carried out at a different temperature. For many applications of the PCR technique it is desirable to complete the sequence of cycles in the minimum possible time. In particular for example where respiratory air or fluids or foods for human and animal stock consumption are suspected of contamination rapid diagnostic methods may save considerable money if not health, even lives.

A wide variety of thermal cycling devices, specifically designed for carrying out reactions in which sequential heating and cooling occurs, such as the PCR are available. These include devices such as solid block heaters which are heated by electrical elements or thermoelectric devices inter alia, as well as apparatus heated by halogen bulb/turbulent air arrangements . The vessels may be cooled by thermoelectric devices, compressor refrigerator technologies, forced air or cooling fluids . These vessels therefore are designed to provide for sequential heating an cooling of a sample . They may therefore be used in any situation where such conditions are required, including for example, the conducting of a melting point analysis.

Generally however, the heating and cooling parameters of the thermal cycler limit the speed at which a reaction such as PCR can be carried out .

An improved, compact and adaptable reaction vessel, which allowed for rapid thermal cycling, and apparatus containing it was described in WO 98/24548, the content of which is incorporated by reference. A particular apparatus which may incorporate reaction vessels of this type is also described in WO 2005/019836, the content of which is also incorporated herein by reference.

The reaction vessels described here utilise electrically conducting polymer (ECP) to form reaction vessels and in particular PCR vessels which are effectively "self-heating".

The vessels have proved themselves to be very useful and robust and able to achieve significant enhancements in the speed of reaction such as PCR. The ECP is an ideal material for providing controlled heating and cooling as it has a low thermal mass and therefore, can heat and cool rapidly.

However, it has been noted that efficiency of the reaction can be hindered as a result of heat flows through the electrical contacts .

WO 2004/045772 describes reaction vessels and apparatus where flexible compressible sheets are covered with conducting layers which act as contacts. However, in use, the sheets themselves become compressed, expelling air and increasing the thermal conductivity of the sheet .

According to the present invention, there is provided apparatus for thermally cycling the contents of a reaction vessel, said apparatus comprising an electrically conducting polymer arranged to act as a resistance heater for a reaction vessel, electrical contacts arranged to supply electrical current or potential to said polymer from a power supply, and a rigid foam material arranged in direct contact with at least one of said electrical contacts.

The rigid foam material acts as an insulator, preventing heat flows and particularly heat loss through the electrical contacts. As a result, a more uniform temperature can be maintained within the reaction vessel. By utilising specifically a rigid foam in preference to a conventional solid insulator, such as a solid plastics insulator, the performance of the apparatus was significantly enhanced. Thermal gradients set up within the reaction vessel were significantly reduced.

It is believed that this may be due to the fact that foam-like materials have a lower thermal mass than solids. Therefore, in addition to providing insulation preventing the flow of heat into and out of the reaction vessel through the electrical contact, they do not significantly hinder the heating and cooling process.

In contrast, solid insulators with significant thermal mass were found to heat up and cool down slowly (rather than not at all) , thereby acting as sources or sinks of heat depending on their temperature relative to the sample. This was found to be disadvantageous in this context.

Suitable rigid foam materials include metal, glass, carbon, polymer, ceramic foams or composites made of several of these.

Particular examples of such foam materials are polymeric foams such as rigid polyurethane or polystyrene foams .

In a particular embodiment, the foam material is a ceramic foam. Ceramic foams generally comprise inorganic, non-metallic materials (such as metal oxides, suicides, nitrides, carbide or borides) with a crystalline structure, which have usually been processed at a high temperature at some time during their manufacture .

Many such foams are now commercially available. They have been developed mainly for the aerospace industry where their utility as insulators is a result of their light weight.

Rigid foams comprise solids which have many gas bubbles trapped within them. They are not compressible, flexible or elastomeric. Because the foams used in the invention are rigid and so not-pliablein nature, which provides a good support for support the electrical contacts. Furthermore, the thermal conductivity remains constant because the air content of the material is not altered when it is pressed against the contacts . Various forms and types of rigid foam materials are known. Some are known as "refractory" foams . They are made by various methods depending upon the nature of the material used. For instance, rigid foams comprising polymers may be readily prepared including foaming agents into the preparation process, as is well understood in the art. Syntactic foams and self- foamed materials such as foam glass may be prepared in a similar way.

Other rigid foams may utilise a foamed polymer as the basic starting material and materials are essentially coated onto these or onto carbonaceous skeletons formed by pyrolysis of the polymer foam. For example, ceramic foams can be produced by coating a polymer foam or a carbon skeleton derived from it, with an appropriate binder and ceramic phases, and then sintering at elevated temperatures . Metallic foams may be formed by electrolytically depositing the metal onto a polymer foam, utilizes an electrodeless process for the deposition of a metal onto the polymer foam precursor via electrolytic deposition.

Suitably the rigid foam material is of a material, which has no fluorescent or phosphorescent properties, even when illuminated with a light source. This means that it may not interefere with the fluorescent signalling or labelling systems that are frequently utilised for detecting the products of an amplification reaction. Such systems may be used to detect the product of amplification either at the end point of the reaction, or, increasingly, in "real-time" as the reaction progresses. These systems, which include the well known "Taqman™" system as well as other systems such as those described for example in Homogeneous fluorescent chemistries for real-time PCR. Lee, M.A., Squirrell, D.J., Leslie, D. L. and Brown, T. in Real-time PCR: an essential guide, J.Logan, K.Edwards & N.Saunders eds . , Horizon Scientific Press,

Wymondham, p.31-70, 2004, the content of which is incorporated herein by reference. For instance, generic methods utilise DNA intercalating dyes that exhibit increased fluorescence when bound to double stranded DNA species. Fluorescence increase due to a rise in the bulk concentration of DNA during amplifications can be used to measure reaction progress and to determine the target molecule copy number. Furthermore, by monitoring fluorescence with a controlled change of temperature, DNA melting curves can be generated, for example, at the end of PCR thermal cycling.

However, when the material has a degree of fluorescence, this may be obviated by dying, coating or inking the foam before use.

In a particular embodiment, the electrical contacts themselves are made of a material which has a lower thermal conductivity than copper which is conventionally used for electrical contacts. For example, copper has a thermal conductivity of 399W/mK, whereas a metal such as stainless steel has a conductivity of about 16 W/mK. Therefore, stainless steel is clearly a preferred option for electrical contacts in this situation.

It is advisable also, for the surface area of the electrical contacts to be as small as possible.

The rigid foam material is arranged in contact with at least one and preferably both of the electrical contacts. Suitably sufficient foam material is arranged so that in use, it effectively isolates the electrical contacts from environmental effects . This will generally be achieved by arranging the rigid foam material in contact with at least the remote edges of the electrical contacts .

If desired, a solid insulator material may also be provided and arranged to contact the rigid foam material. Suitable solid insulator materials are well known in the art, and include polymeric or fibrous materials . In particular the solid insulator is a polymeric insulator such as an acetal homopolymer resin such as Delrin™, acrylonitrile-butadiene- styrene terpolymer (ABS) or polytertrafluroethylene (PTFE) .

The solid insulator may be arranged to contact a substantial portion, for example at least one side of the rigid foam material. This additional insulation will protect the rigid foam material itself from changes in environmental temperature and so enhance the overall reliability of the system. Suitably the additional solid insulator is provided so as to effectively isolate the reaction vessel from the external environment when in use . The precise arrangement of the solid insulator therefore will vary depending upon the nature of the reaction vessel and the manner in which it is used.

Preferably the contact or contacts are held in place abutting against the rigid foam material by means of a biasing means, such as a spring for instance a leaf or coil spring, or the like, arranged to urged one of the contact or the rigid foam material towards the other. The precise nature, arrangement and position of the biasing means will depend upon the particular apparatus being used, and would be apparent to a skilled person. Preferably the biasing means is not in direct contact with electrical contacts themselves. However, they may be in contact with the rigid foam material or a support for the contact or the rigid foam material, provided the overall effect is to urge the contact and the rigid foam material together when in position within the apparatus. Conventional spring mounting arrangements will be usable.

The reaction vessels which are utilised in the apparatus may be of any shape or size, and will depend upon the particular purpose. Thus they may include tubes, flasks, slides, chips or the like. The vessels may be constructed from the ECP themselves or the ECP may be arranged proximal to a pre-existing vessel.

For example, the reaction vessel may take the form of a reagent container such as a glass, plasties or silicon container, with electrically conducting polymer arranged in close proximity to the container. In one embodiment of the vessel, the polymer is provided as a sheath which fits around the reaction vessel, in thermal contact with the vessel. The sheath can either be provided as a shaped cover which is designed to fit snugly around a reaction vessel or it can be provided as a strip of film which can be wrapped around the reaction vessel and secured.

The polymer sheath arrangement means that close thermal contact is achievable between the sheath and the reaction vessel. This ensures that the vessel quickly reaches the desired temperature without the usual lag time arising from the insulating effect of the air layer between the reaction vessel and the heater.

Furthermore, a polymer sheath can be used to adapt apparatus using pre-existing reaction vessels. In particular, a strip of flexible polymer film can be wrapped around a reaction vessel of various different sizes and shapes.

Where a sheath is employed it may be advantageous for it to be perforated or in some way reticulated. This may increase the flexibility of the polymer and can permit even readier access by a cooling medium if the polymer is not itself used to effect the cooling.

In another embodiment of the invention, the polymer is provided as an integral part of the reaction vessel. The reaction vessel may itself be made from the polymer by extrusion, injection moulding or similar techniques. Alternatively, the reaction vessel may be manufactured using a composite construction in which a layer of the conducting polymer is interposed between layers of the material from which the vessel is made or in which the internal or external surfaces of the reaction vessel is coated with the polymer, or again in which the vessel is basically made of the polymer coated with a thin laminate of a PCR compatible material. Such vessels may be produced using overmoulding, lamination and/or deposition such as chemical or electrochemical deposition technigues as is conventional in the art.

Vessels which comprise the polymer as an integral part may provide particularly compact structures with low thermal mass suitable for rapid heating and cooling.

In order to provide for efficient heating and cooling, it is preferred that the chamber has a high surface area to volume ratio such that rapid heat exchange can occur. One example of such a chamber is a capillary tube . These are ideal for the rapid heating or cooling of small volumes of fluid samples.

In a particular embodiment therefore, the reaction vessel comprises a capillary tube, which is suitably a glass capillary tube, having a coating of ECP. An end of the capillary tube is suitably sealed so as to form a closed vessel. The other end of the capillary tube may be connected to an expanded support section, which can act as a funnel to allow filling of the tube. This support is suitably made of the ECP itself for ease of manufacture.

However, due to the surface tension of a fluid sample it can be difficult to load the fluid sample into a capillary tube. It is therefore optionally preferred that the support section is adapted for use in a centrifuge device so that a fluid sample can be introduced into the capillary tube by centrifugal force. Generally this will mean that the support is provided with elements that can engage the centrifuge device in pivotal manner, such that the vessel can swing freely whilst rotating and fluid can be driven into the capillary tube. Such a device is described in detail in WO 2005/019836.

When the reaction vessel is a capillary tube, the electrical contacts are suitably ring electrodes, arranged one at each end of the tube. The ring adjacent to the expanded support section may contact the support at the point at which the expansion in cross sectional area occurs.

Electrically conducting polymers are well known in the art and may be obtained from a variety of commercial sources. Examples of such polymers are disclosed for instance in US Patent No . 5106540 and US Patent No. 5106538 but there are many others available. Many are made of polymeric materials which contain carbon powder at varying levels to provide conductivity.

Suitable conducting polymers can provide temperatures up to 300⁰C and so are well able to be used in PCR processes where the typical range of temperatures is between 30° and 100⁰C.

Preferably the polymer has a high resistivity for example in excess of lOOOohm.cm. The temperature of the polymer can be readily controlled by controlling the amount of electric current passing through the polymer or the electrical potential of the system, allowing it to be held at a desired temperature for the desired amount of time.

Furthermore, the rate of transition between temperatures can be readily controlled after calibration, by controlling the power supply used to deliver an appropriate electrical current. The power supply may for example be under the control of a computer programme, which is arranged to control the potential or current to provide the required rate of transition.

Apparatus as described above which further comprises means for applying a current or electrical potential to the electrical contacts in a manner so as to induce a desired thermal cycling regime within the reaction vessel, forms a particular embodiment of the invention.

The apparatus may also comprise elements for the automatic handling and processing of samples, such as are required for the particular samples and reactions being conducted. A particular example of such apparatus is described in WO2005/019836.

In a further aspect, the invention provides a method for conducting a thermal cycling reaction, said method comprising placing a sample to be thermally cycled in a reaction vessel of an apparatus as described above, applying a current or electrical potential to the electrically conducting polymer, for example under the control of a computer, by way of the electrical contacts so as to induce in the sample the desired sequence of thermal cycles .

The apparatus may also be used for conducting a melting point analysis, for example where a nucleic acid is combined with a signalling system which indicates whether or not the nucleic acid is in double stranded form, such as a DNA intercalating dye that exhibits increased fluorescence when bound to double stranded DNA species. By monitoring fluorescence with a controlled change of temperature, DNA melting curves can be generated, for example, at the end of PCR thermal cycling. Such methods form a further aspect of the invention.

The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings in which:

Figure 1 illustrates apparatus embodying the invention;

Figure 2 is a graph of static thermal measurements made along the length of a tube heated in a comparative apparatus; Figure 3 is a graph of static thermal measurements made along the length of a tube heated in an apparatus of Figure 1;

Figure 4 is a graph or dynamic thermal measurements made along the length of a tube in an apparatus of Figure 1 during thermal cycling;

Figure 5 are graphs showing post-PCR melting plots with the electrodes in the comparative apparatus described above (a) or in the apparatus of Figure 1; and

Figure 6 are graphs showing a comparison of PCR amplification plots carried out in the comparative apparatus described above (a) and in the apparatus of Figure 1 b) .

In the illustrated device, a reaction vessel (1) such as is described and used in WO2005/019836 is provided. The vessel (1) comprises a capillary tube (2 (a)) which is coated with an electrically conducting polymer (2(b)). The base (3) of the tube (2 (a)) is sealed with a transparent seal, so that the contents of the tube can be viewed through the base (3) .

The remote end of the tube (2) is integral with an expanded support section (4) which is arranged to be held within a centrifuge device.

A lower ring electrical contact (5) is provided in the region of the base (3) of the tube (2) . An upper electrical contact

(6) is provided around the outside of the support section (4) which carries an integral electrical contact able to feed current to the electrically conducting polymer (2b) of the tube

(2a) on application of a suitable electrical potential.

A ring of a rigid foam material (7) (a polyurethane engineering foam is provided in contact with the lower edge portion of the upper electrical contact (6) . The lower electrical contact (5) is held on a shaped support (8), also of the rigid foam material, and arranged to abut an second integral contact which projects from the electrically conducting polymer (2b) of the tube (2a) .

The rigid foam material is arranged to minimise heat transfer from the electrical contacts, but does not interfere with the contact to the electrical supply (not shown) .

A ring (9) of solid insulator material such as Delrin is provided adjacent the ring of solid foam insulator material (7) in contact with it. The ring (9) effectively surrounds the rest of the upper electrical contact (6) but is not in direct contact with it. As a result, it acts as an insulator from environmental effects coming from above, but does not act as either a heat sink or heat source in relation to the electrical contact itself.

Similarly, the shaped support (8) of a solid foam insulator material is itself supported on a ring (10) of solid insulator material such as Delrin so as to provide similar protection for the lower electrical contact (5). The solid insulator rings (9, 10) are provided with conduits for electrical connection and spring-mounted in housing (11) - The spring mounting has the effect of urging the rigid foam supports (7, 8) against the contacts (6, 5 respectively).

In use, the solid insulator rings (9, 10) combined with the apparatus in which the vessel is held define a chamber for the tube (2) which is effectively isolated from the environment.

However, the electrical contacts themselves are in contact with the solid foam material of low thermal mass .

When arranged in this way, the apparatus could be utilised in a polymerase chain reaction in a far more effective and reliable manner, as compared to devices which had no or alternative arrangements of insulator material.

Steady state temperature measurements using apparatus as shown in Figure 1 and a similar apparatus in which the polyurethane foam was omitted and so where the electrical contacts rested on the Delrin, were carried out. This was done by setting a target temperature of 50⁰C and measuring the temperature profile with a thermistor that was raised in steps from the bottom of the capillary. The results for the comparative apparatus is shown in Figure 2 and the results for the apparatus embodying the invention are shown in Figure 3. It can be seen that the thermal gradient was reduced to less than 3°C along the length of the tube with the central portion being within 1°C of the target temperature. (Note that the temperature axis covers a narrower range in Figure 3 than in Figure 2) .

Clearly, mounting the electrode contacts directly onto Delrin as a support material as in the comparative apparatus is therefore not ideal because, although a thermally insulating material, the thermal mass of Delrin relative to that of the ECP gives rise to significant heat-sinking effects.

Although PCR amplification can be achieved with a gradient as illustrated in Figure 2 because a portion of the sample within the tube will be at permissible temperatures, but the situation is far from ideal and amplification efficiency is sub-optimal. Thus the arrangement of the invention provides for more effective PCR reactions.

This was confirmed in a series of experiments .

A PCR assay, (a "Dual Hyb" real-time PCR for DNA from Bacillus globigii) , was run in both the comparative apparatus lacking the foam material and the apparatus of Figure 1. Fluorescence from the FAM FRET donor (dotted line) was quenched as specific product was formed whilst fluorescence from the Cy5 FRET acceptor rose, (n.b. the gain setting for the 570nm Cy5 channel was 32x higher than for the 520nm FAM channel) .

Figure 4 illustrates the temperature gradient during thermal cycling with the electrodes mounted onto the polyurethane engineering foam in the apparatus of Figure 1. The measurements during thermal cycling were made at the bottom, middle and top of the capillary tube with a three thermistor assembly. As above, the temperature cycling profile was: heat from 50⁰C to 80⁰C at 10°C per second; hold at 80⁰C for 20 seconds; cool from 80⁰C to 5O⁰C at 10⁰C per second; hold at 50⁰C for 20 seconds; repeat. Under dynamic conditions, as shown in Figure 4, the gradients were about 3°C at 80⁰C and 2⁰C at 50⁰C. Given that in these measurements only the gradient extremes were determined, the temperature for the majority of the sample would have been very close (ie <1°C) to the programmed temperature .

The results give rise to greater efficiency of the PCR amplification with higher signals and fewer cycles required before the fluorescence becomes measurable (ie lower "Ct" values ) .

Figure 6 gives a comparison of PCR amplification plots with the electrodes in (a) Delrin mounts or (b) polyurethane .engineering foam mounts .

The results from the rigid foam-supported electrodes (6(b)) showed that PCR amplification was more efficient, with a take of point at 20 cycles, versus take off at 25 cycles with the Delrin-supported electrodes. A comparison of post-PCR melting plots with the electrodes in

(a) Delrin mounts or (b) polyurethane engineering foam mounts is provided in Figure 5.

The same reactions shown in Figure 5 (a&b) were analysed post- PCR using slow melts (50⁰C to 95°C at 0.1⁰C per second). The melt profile of the reaction products with the Delrin electrode supports (a) was noisy because the signals were low (because less product was formed) and more than one peak is apparent. The melt profile from the reaction with foam electrode supports

(b) shows a single sharp peak indicating that the required thermal homogeneity had been achieved.

Claims

1. Apparatus for thermally cycling the contents of a reaction vessel, said apparatus comprising an electrically- conducting polymer arranged to act as a resistance heater for a reaction vessel, electrical contacts arranged to supply electrical current or potential to said polymer from a power supply, and a rigid foam material arranged in direct contact with at least one of said electrical contacts.

2. Apparatus according to claim 1 which further comprises biasing means, arranged to urge a contact towards said rigid foam material.

3. Apparatus according to claim 1 or claim 2 wherein the rigid foam material is a metal, glass, carbon, polymer, or ceramic material or is a composite of these.

4. Apparatus according to claim 3 wherein the rigid foam material is a ceramic foam.

5. Apparatus according to claim 3 wherein the rigid foam material is a polymeric foam.

6. Apparatus according to any one of the preceding claims wherein the rigid foam material is non-fluorescent.

7. Apparatus according to any one of claims 1 to 5 wherein the rigid foam material is dyed, coated or inked to substantially eliminate fluorescence therefrom.

8. Apparatus according to any one of the preceding claims wherein the electrical contacts are made of a metal which has a thermal conductivity of less than 399W/mK.

9. Apparatus according to claim 8 wherein the electrical contacts are of stainless steel.

10. Apparatus according to any one of the preceding claims wherein the rigid foam material is arranged in contact with both of the electrical contacts.

11. Apparatus according to any one of the preceding claims further comprising a solid insulator material which is arranged to contact the rigid foam material.

12. Apparatus according to any one of the preceding claims wherein the reaction vessel is a capillary tube.

13. Apparatus according to claim 12 wherein the electrical contacts are ring electrodes, provided one at each end of the capillary tube .

14. Apparatus according to any one of the preceding claims which further comprises means for applying a current or electrical potential to the electrical contacts in a manner so as to induce a desired thermal cycling regime within the reaction vessel.

15. Apparatus according to any one of the preceding claims which further comprises elements for the automatic handling and processing of samples.

16. A method for conducting a thermal cycling reaction, said method comprising placing a sample to be thermally cycled in a reaction vessel of an apparatus according to any one of the preceding claims, applying a current or electrical potential to the electrically conducting polymer by way of the electrical contacts so as to induce in the sample the desired sequence of thermal cycles .

17. A method according to claim 16 wherein the current or electrical potential applied is controlled by a computer.

18. A method according to claim 16 or claim 17 wherein the thermal cycling reaction is a polymerase chain reaction.

19. A method for determining a melting point of a nucleic acid, said method comprising placing a nucleic acid sample and a signalling system able to provide a distinguishable signal in the presence of single stranded nucleic acid as compared to the signal produced in the presence of double stranded nucleic acid, in a reaction vessel of an apparatus according to any one of claims 1 to 14, applying a current or electrical potential to the electrically conducting polymer by way of the electrical contacts so as to heat or cool the sample whilst monitoring the temperature and the signal from the signalling system, and determining the temperature at which the signal changes.

20. A method according to claim 19 wherein the signalling system comprises an intercalating dye.