WO2004024330A2

WO2004024330A2 - Thermocycler and sample holder

Info

Publication number: WO2004024330A2
Application number: PCT/GB2003/003940
Authority: WO
Inventors: Mark Ebdon; Brian Wilkinson
Original assignee: Quanta Biotech Limited
Priority date: 2002-09-12
Filing date: 2003-09-12
Publication date: 2004-03-25
Also published as: AU2003264755A1; GB0221167D0; AU2003264755A8; WO2004024330A3

Abstract

A thermal engine for a thermocycler, the thermal engine comprising a first heating and cooling element, a sample holder receiver in thermal contact with the first heating and cooling element, and a second heating and cooling element situated adjacent the sample holder receiver and removed therefrom so as to allow, in use, the placement of a sample holder between, and in thermal contact with, the first and second heating and cooling elements. Also provided is a low volume sample holder for use in a thermocycler, the sample holder having a plurality of wells for holding samples, wherein the number of wells is a multiple of 96 and the spacing between the centres of neighbouring wells is approximately 4.5 mm or 2.25 mm, the wells being arranged in a substantially rectangular pattern of a multiple of 12 wells in length and a multiple of 8 wells in width.

Description

Control Apparatus

This invention relates to apparatus and consumables for the control of chemical reactions. In particular, it concerns automated or semi-automated apparatus and parts therefor for the thermal control of nucleic acid polymerisation and hybridisation reactions.

Many chemical and biochemical reactions performed in vitro require close control of their temperature to ensure predictability and reproducibility of their outcome. This is especially true in the use of the polymerase chain reaction (PCR) for the amplification of polynucleic acid sequences. In PCR, and particularly quantitative PCR (QPCR), it is essential to control the temperatures of samples in order that the intended number of amplifications are carried out accurately in each sample and consistently between samples. The accepted way of achieving such temperature control is to perform the PCR reactions in a thermocycler.

A typical thermocycler comprises a programmable control module having a user interface, together with a thermal engine under the control of the control module. The thermal engine is where samples are placed for thermocycling or incubations requiring high thermal accuracy and uniformity and typically comprises one or more thermoelectric heating and cooling elements in intimate contact both with a heat sink and a fixed sample block. Samples are mounted in disposable glass or plastics vials, capillaries, microplates, microscope slides or micro arrays, onto or into the sample block and are typically clamped into the sample block by an overhead mechanism provided in the thermal engine. The thermal engine may also contain an overhead heat source, usually low-powered and coarsely-controlled, to provide a non-condensing environment around the sample containers, thus avoiding potential temperature control problems associated with condensation of evaporating aqueous samples during heating. Such an overhead heat source does not directly heat the samples themselves and does not actively cool the samples.

Current PCR and QPCR instrumentation typically utilises sample blocks suitable for receiving sample holders with sample chamber volumes of around 200 μl to 500μl. A typical individual sample volume for a PCR or QPCR reaction, however, is 100 μl or less 1 and is often 25 μl or less. The use of such oversized sample chambers has the following disadvantages:

a) sample volume may change significantly during thermocycling due to evaporation of the sample. This affects the kinetics of the reaction and, since the volume change often depends on the location of individual samples within a multiple sample holder (the heating of the sample holder being typically subject to such phenoma as edge losses and hot spots), a non-uniform treatment of samples results;

b) the size and mass of the sample block necessary to accommodate the sample holders leads to a lack of temperature control and uniformity throughout the block resulting in different samples encountering different temperatures for different time periods;

c) when the reaction products are detected and/or quantified by optical means (e.g fluorimetry), the large sample block format is problematic since it presents a large field to be excited and/or imaged. Thus, different samples encounter differential excitation and differences in the recorded signal are obtained depending on the position of the sample in the sample holder. This problem is exacerbated when using multiple sample holders since it is frequently the case that either the sample holder or the excitation and/or detection components of the optical detection/quantification means must be physically moved to bring them into registration with each other, the result of which is a time difference between the recording of different samples and thus the introduction of further error;

d) the large sample block requires a considerable power input to the thermal engine for the necessary temperature transitions to be achieved; and

e) the large mass and surface area of the sample block makes temperature transitions very slow. Since the reactions carried out in thermocyclers are usually enzymatic, slow temperature transitions can lead to non-specific reactions and degradation of the enzyme concerned. In any event, slower temperature transitions lead to the reactions taking a longer time to perform.

The disadvantages mentioned above are important regardless of the type of reaction being carried out in the thermocyclers. Given the large throughput of samples generally required of a thermocycler for PCR reactions, however, the technical deficiencies in temperature transition rates and execution and imaging assume a greater significance. The use of large sample holders, sample blocks and thermal engines also inevitably makes the performance of such reactions more expensive than is necessary in principle. When such thermocyclers are employed for gene expression related QPCR studies, where the degree of accuracy in thermal control and detection is of paramount importance, the limitations of current apparatus are even more stark.

In EP 1045038 A, a thermocycler for rapid PCR is described, the thermal engine of which employs a small, low-profile, low thermal capacity sample block having an array of conical sample wells formed in its upper surface. Samples are introduced into the thermal engine by means of an ultrathin-walled plate which keys into the welled surface of the sample block. The ultrathin-walled plate described has a 6x6 arrangement of wells and is thus incompatible with the standard 96- well format used for microtitre plates. The sample block is typically formed from solid metal.

US6337435 is concerned with a thermal engine having a number of Peltier effect thermoelectric modules and wire heating elements embedded along the edges of the sample block assembly. The upper surface of the disclosed sample block contains an array of sample tubes having substantially parabolic inner cross-sections and designed to receive a tray of open-topped reaction vessels having a similar parabolic cross-section. An overhead resistance heating element is provided in order to avoid condensation of evaporating samples during themiocycling. A similar arrangement of sample block and sample vials is contemplated by WO98/43740. Both these documents are concerned with conventional (i.e. large) volume PCR thermocycling.

US5939312 describes a miniaturised, integrated multi-chamber thermocycler, the sample block of which is formed from an etched silicone slab. The base of the sample block is fitted with low conductivity projections to isolate the block from its surroundings and a number of heating elements are attached to the base of the block. The layout of the wells of the sample block is unlike that of a standard microtitre plate.

In EP 1157744, an automated integrated thermocycler is disclosed and has a machined aluminium sample block which has a plurality of wells in its upper surface for receiving sample vials. The sample vials intended to be used with this apparatus have a volume of 200 μl to 500 μl. In an attempt to overcome some of the limitations of using thermocycler apparatus of such a size, a complex zoned temperature control is employed.

WOO 1/35079 discloses a fluorimetric detection apparatus for use in conjunction with a thermocycler. The detection apparatus comprises a number of light emitting diodes (LEDs) having one-to-one registration with a number of samples in a standard multiple sample tray and a detection means, such as a charge-coupled device (CCD)-type camera, for recording the fluorescent output from the samples. The apparatus is designed to be used with a standard size and shape of sample block and sample holder.

GB2334581 concerns a microtitre plate for reflectometric interference spectroscopy and having a two part construction with a number of coatings of different refractive index in the region of the floors of the wells. EP 1266691 shows a two part microtitre plate defining a hollow chamber for the circulation of a temperature controlling fluid. The circulating fluid enters and exits the plate by means of tubing connected to an external bath. US6083763 describes an integrated apparatus for analysis of molecular arrays in multiwell plates. However, temperature control is not addressed.

The devices shown in the prior art are limited by the fact that they either employ the industry-standard 'large' size sample holders, resulting in oversized equipment and the thermal control and detection problems highlighted above, or they employ bespoke sample holders which cause incompatibility problems in relation to using the sample holders in other pieces of equipment. Even when the 'bespoke' sample holders of the prior art are used, their shape is generally such that excessive material is required to be used in constructing the sample holder in order to provide the required volume for the sample-receiving wells. This has necessitated, in the prior art apparatus, the sample blocks of the thermal engines to be similarly constructed from an excessive amount of material. Both material excesses add to the temperature control problems already mentioned. It should also be noted that the prior art apparatuses and sample holders are designed with either analysis or accurate temperature transition control in mind, but not both. It is an object of the present invention to provide thermocycler apparatus and parts therefor which ameliorate one or more of the limitations described above with reference to known apparatus.

Accordingly, one aspect of the present invention provides a. thermal engine for a thermocycler, the thermal engine comprising a first heating and cooling element, a sample holder receiver in thermal contact with the first heating and cooling element, and a second heating and cooling, element situated adjacent the sample holder receiver and removed therefrom so as to allow, in use, the placement of a sample holder between, and in thermal contact with, the first and second heating and cooling elements.

The use of a second heating and cooling element provides improvements in thermal transition rates and in accuracy of thermal control. As used herein, the term 'thermal contact' implies either physical contact between components or contact via an interposing, thermally conductive medium such that thermal transfer between the components is sufficiently tightly controlled to enable accurate use of the thermal- engine.

Preferably, at least one of the heating and cooling elements is electrically-controlled, preferably comprising a Peltier effect thermoelectric device, in which case the thermal engine is also provided with a heat sink. Alternatively or in addition, at least one of the heating and cooling elements comprises a heat pipe. Heat pipes comprise a sealed vessel containing a working fluid (for example, water) under a sub-atmospheric pressure (i.e. in order to lower the boiling point of the fluid). On heating the vessel in one region (eg. a first end), the working fluid is caused to evaporate and transfer to another region in the vessel (eg. a second end). The gaseous working fluid condenses in the vicinity of the second region of the vessel and thus transfers its thermal energy, both sensible and latent, to that second region. Heat pipes provide rapid transfer of thermal energy from a source to an intended destination and enable very close control of temperature at the destination. In a heating and cooling element for the present invention and which comprises a heat pipe, the destination of the thermal energy is the sample holder receiver; the source of thermal energy at the first region of the heat pipe may comprise a thermoelectric device or may comprise a stored energy source, eg. a water bath or ice bath. Heat pipes do not typically require ^' the presence of a separate heat sink (i.e. separate to that attached to the thermoelectric device in embodiments employing such an energy source) and provide a more^' energy efficient means of temperature control than simple thermoelectric devices - energy input is only required to 'top up' the thermal transfer to the destination and there is no requirement for long periods of power consumption as with a simple thermoelectric device such as a Peltier module. Because heat pipes can pump energy rapidly over large distances, the part of the heat pipe in direct thermal contact with the sample holder, or sample holder receiver, can be much smaller than^' conventional heating and cooling elements, with the active heating and cooling source situated some distance away. This provides advantages when combining the thermal engine with optics for concurrent imaging of the sample during thermal cycling as there is no heat sink for the optics to traverse through. A more complete description of heat pipe technology in general can be found in Peterson, G. P., An Introduction to Heat Pipes: Modelling, Testing and ■ Applications, John Wiley & Sons, Inc., 1994.

In most embodiments, the sample holder receiver will comprise a separate component to the first heating and cooling element, eg. it may comprise a sample block onto which the sample holder is mounted. However, in some embodiments, the sample holder receiver is a part of the first heating and cooling element itself, such that the sample holder is mounted directly onto the heating and cooling element.

It is preferred that the first and second heating and cooling elements are disposed, relative to each other, so as to be towards opposite sides of a sample holder placed into the thermal engine in use. Such an arrangement provides for particularly efficient thermal transfer to/from and control of the sample holder. In certain embodiments, the second heating and cooling element is positioned above the sample holder receiver when the thermal engine is in its normal operating orientation, the first heating and cooling element being positioned below the sample holder receiver. A sample holder placed into such a thermal engine is thus positioned above the sample holder receiver and below the second heating and cooling element. In preferred embodiments, the second heating and cooling element is situated so as to come into physical contact with a sample holder placed into the thermal engine. Alternatively, a thermally conductive interface plate may be positioned, in use, between the second heating and cooling element and the sample holder such that the interface plate comes into physical contact with the sample holder.

Thermally-conductive gaskets may be provided on the sample holder receiver, the second heating and cooling element and/or the interface plate. Such gaskets are formed of thermally-conductive compliant materials which may be selected from phase change materials (for example, T725 or T766 from Chromerics Inc.), preferably less than 1 mm thick phase change materials, carbon fibre-impregnated foamed plastics materials, silicone-impregnated foamed plastics material, metallic strips, conductive adhesive (e.g AR clad™ 8043 or AR clad™ 8044 from Adhesives Research, Inc)-coated metallic strips, thermally conductive greases (e.g Microfaze A4, a dry film thermal grease available from AOS Thermal Compounds of Eatontown, New Jersey), thermally conductive grease-coated mica, polyimide or ceramics, and combinations thereof. Combination gaskets are preferred and examples of such combinations include a thermally conductive paste surrounded by metal foil, thermally-conductive grease surrounded by metal foil, or a laminate of elastomeric binder (e.g silicon rubber)/thermally conductive filler applied to a carrier firm (e.g fϊbreglass, polyimide, polyester or aluminium film), such as that sold by Berquist as Sil-Pad. The gaskets help to ensure efficient heat transfer to and from the sample holder and samples.

The sample holder receiver may take the form of a fixed sample block or, alternatively, may comprise a heating and cooling stage onto which may be releasably mounted a variety of different format sample holders in the form of interchangeable sample blocks, as described in more detail below.

In an embodiment of the thermal engine of the invention, the first or second heating and cooling element is provided with one or more holes through which electromagnetic radiation may pass from samples held in a sample holder to a means for measuring at least one characteristic of the samples, the measuring means communicating with the holes by means of optical fibres and/or being positioned either towards the opposite side of the first heating and cooling element to the sample holder receiver or towards the opposite side of the second heating and cooling element to the sample holder receiver.

In such an embodiment, the thermal engine may be used as an integrated thermocycler and sample analyser. Thus, the thermal accuracy achievable by the use of the second heating and cooling element can be coupled with the convenience of analysis of the sample in situ in the thermal engine. Real-time QPCR analysis is also possible. Ideally, the holes in the first or second heating and cooling element are aligned with the wells of a welled sample holder placed into the thermal engine in use. From the point of view of improved thermal control, it is preferred that, in those embodiments employing thermoelectric heating and cooling elements, such as Peltier modules, the holes in the first or second heating and cooling element are of a diameter of 1mm or less.

Preferably, a source of incident electromagnetic radiation, such as UN and/or visible electromagnetic radiation, is provided such that, for example, fluorescence-based measurements may be conducted. The source preferably communicates with the holes in the first or second heating and cooling element by means of optical fibres and/or may be positioned adjacent a sample holder placed into the thermal engine in use or towards the opposite side of the sample holder to the measuring means. When the source is positioned other than towards the same side of the sample holder as the measuring means, the sample holder used in the thermal engine should be permissive to the incident radiation, for example as described below. If optical fibres are used, and/or if the source and measuring means are positioned towards the same side of the sample holder, the sample holder may be substantially opaque. The source may also be positioned towards the opposite side of the sample holder receiver to the measuring means. In embodiments in which the first heating and cooling element is situated below the sample holder receiver, the source or the measuring means may be positioned below the first heating and cooling element, the sample holder receiver and first heating and cooling element being provided, as necessary, with one or more holes, the holes being aligned with each other, and preferably with the wells of a welled sample holder placed into the thermal engine in use, so as to provide a passage for the incident or measured radiation from the source to the sample holder or from the sample holder to the measuring means, respectively. In embodiments in which the source is positioned towards the opposite side of the sample holder receiver to the measuring means, but not necessarily below the first heating and cooling element, the sample holder receiver is preferably provided with one or more holes to provide a passage for the incident radiation from the source to the sample holder. A source positioned adjacent the sample holder, with a measuring means positioned so as to take measurements of radiation substantially perpendicular to the direction of the incident beam of radiation, may usefully be employed for fluorimetric measurements.

When thermally-conductive gaskets are employed, these must either be provided with an appropriate arrangement of holes or must themselves be permissive to the incident and/or measured radiation. It will be appreciated that, in those instances in which a sample holder receiver which is itself substantially permissive to electromagnetic radiation is used, it is not necessary to provide holes in this part of the thermal engine.

Preferably, the means for measuring the characteristics of the samples comprises an optical device. More preferably, a CCD-type camera, capable of imaging a plurality of samples simultaneously either directly or indirectly utilising a mirror, lens or optical fibre-based light delivery system, is used. The measuring means may comprise a photo multiplier tube or CCD camera communicating with the holes in the first or second heating and cooling element or the sample holder receiver, as appropriate, by fibre optic cables. It is most preferred that the CCD-type camera is capable of imaging all the samples simultaneously. The source of incident electromagnetic radiation may comprise a quartz tungsten halogen lamp or LEDs such that, for example, fluorescence-based measurements may be conducted. The source of incident radiation may be connected directly or indirectly by utilising a mirror, lens or optical fibre-based light delivery system. In order to conduct analyses of samples containing multiple analytes (e.g multiple DNA targets for amplification by PCR), one or more dicliroic filters may be employed between the samples and the optical device(s) to allow distinction between the analytes. Alternatively, a rapid filter change system can be employed.

The use of an optical device allowing multi-sample simultaneous measurement of sample characteristics provides an even greater speed of sample throughput. In addition, the increased rate of data capture allows more accurate results to be obtained by virtue of the fact that a longer exposure of the camera to the samples can be comfortably tolerated. In a conventional apparatus, much time is wasted in switching the measuring means between samples. This waste of time is more significant when analysing multiple analytes.

Both the incident electromagnetic radiation and the measuring means may be provided from above the second heating and cooling element in those embodiments where the second heating and cooling element is provided with one or more holes, irrespective of whether or not the first element and/or the sample holder receiver are provided with holes. As will be described in more detail below, the use of a low volume sample holder according to a further aspect of this invention provides further advantages in terms of thermal control, the benefits of the second heating and cooling element being even greater in such a case. The ability to simultaneously measure a greater number of samples is also enhanced in such a case.

It will be appreciated that, in preferred embodiments in which the source and/or the measuring means communicate with the holes in the heating and cooling elements and/or sample holder receiver by means of optical fibres, the source and/or measuring means themselves may be positioned essentially anywhere, not excluding externally of the thermal engine. In a particularly preferred embodiment, the measuring means and the source are each capable of communicating with a given hole in the first or second heating and cooling element simultaneously, the optical fibre from the source and the optical fibre to the measuring means each passing through the same hole or each communicating with that hole by means of a junction fibre engaged with that hole. The junction fibre may be substantially Υ -shaped and is adapted for receiving both the fibre from the source (the excitation fibre) and the fibre to the measuring means (the detection fibre). In order to maximise the amount of electromagnetic radiation received from the sample by the measuring means, the junction fibre should preferably be arranged so as to substantially align the portion of the detection fibre engaged with the junction fibre along the axis between the sample and the hole in the first or second heating and cooling element.

According to a second aspect of the present invention, there is provided a thermal engine for a thermocycler, the thermal engine comprising a heating and cooling element and a sample holder receiver in thermal contact with the heating and cooling element, the sample holder receiver being provided with one or more holes through which electromagnetic radiation may pass, in use, from a source, communicating with the holes by means of optical fibres and/or placed adjacent the sample holder receiver, to a sample holder engaged, in use, with the sample holder receiver.

The thermal engine of the second aspect of the invention has the advantage that it allows incident radiation to be provided to a sample from, for example, below, the output radiation then being measurable from above. The path of sample matrix through which the radiation must pass is thus potentially reduced compared to conventional fluorescence-based systems. Furthermore, interference in the measured results due to the edges of the sample holder wells refracting and/or absorbing the radiation is reduced. In embodiments in which the heating and cooling element is situated below the sample holder receiver, the heating and cooling element is provided with one or more holes which are aligned with those provided in the sample holder receiver. The source of incident radiation may then be situated below the heating and cooling element. Preferably, the holes in the sample holder receiver and, where present, those in the heating and cooling element, are aligned with the wells of a welled sample holder placed into the thermal engine in use.

The features described above in relation to the first aspect of the present invention are also applicable to the thermal engine of the present aspect. The advantages of such features are as described above.

In a third aspect, the present invention provides a thermal engine for a thermocycler, the thermal engine comprising a heating and cooling element and a sample holder receiver in thermal contact with the heating and cooling element, characterised in that the heating and cooling element comprises a heat pipe.

The advantages of using a heat pipe for thermocycling applications are as described above. The invention also provides a thermocycler including a thermal engine according to any of the first, second or third aspects of the invention described above.

In a fourth, and related, aspect of the invention, there is provided a low volume sample holder for use in a thermocycler, the sample holder having a plurality of wells for holding samples, wherein the number of wells is a multiple of 96 and the spacing between the centres of neighbouring wells is approximately 4.5mm or 2.25mm, the wells being arranged in a substantially rectangular pattern of a multiple of 12 wells in length and a multiple of 8 wells in width.

The arrangement of wells ensures compatibility of the sample holders of this aspect of the invention with the standard microtitre plate format used by many related items of laboratory apparatus. In addition, however, the 4.5 mm maximum spacing ensures that the overall size of the sample holder is reduced by a factor of approximately 4. This makes significant improvements possible in both terms of thermal efficiency and control, and detection and imaging. The sample holders of this aspect of the invention thus confer additional advantages when used with the thermal engines and thermocyclers described above.

It is preferred that the surface area of the floor of each well is at least 50% of the area described by the mouth of each well.

The 'floor' of the wells refers to an interior surface of the wells, generally below the mouth of the wells when the sample holder is in a position for sample loading, (an 'upright' position) which is demarcated from, and forms an angle with, the other interior surfaces of the wells. The term 'low volume' means having an individual well volume of the order of the typical sample volume for PCR reactions. Preferably, the volume of each well is 100 μl or less, more preferably 1 to 60 μl and most preferably between 5 and 25 μl.

The floor of each well preferably extends in only one plane. Other floor configurations are possible, however, such as a shallow 'N'-shape or a shallow inverted dome shape. When the floor extends in only one plane, that plane is preferably parallel to the plane of the mouth. It will be clear that, under these conditions, the wells will provide a volume which is either cylindrical in shape (i.e. if the floor area is 100% of the area described by the mouth and if the mouth and floor are circular), cuboidal in shape (i.e. if the floor area is 100% of the area described by the mouth but the mouth and floor are rectangular) or a frustum (if the floor area is not 100% of the area described by the mouth). Depending on the shapes of the mouth and the floor, the frustum wells may be, for example, frusto-conical or frusto-pyramidal. When the wells have the shape of a frustum, it is preferred that the surface area of the floor is at least 70%, more preferably at least 90%ι of the area described by the mouth. A cylindrical shaped well is preferred.

The preferred designs of wells of the low volume sample holder of the present invention confer the advantage that the required volume of sample can be accommodated in wells of considerably lower depth than have been used hitherto. The relatively large floor area of each well allows adequate sample volume loading without the need for long, tapered well walls which inevitably lead to the use of excessive material in sample holder construction. Coupled with the fact that the volume of the wells of the sample holder of the present invention is appropriate to the volume of samples to be loaded into the wells, this results in the energy input required for thermocycling being reduced compared to that in prior art devices. The reduced size and mass of the sample holder also further facilitate improvements in thermal control. The reduced dimensions also allow for improvements in detection and imaging and allow the use, in a manner accurate enough for QPCR, of a CCD camera based detection system. This allows a much greater level of data capture per unit time, leading to faster and more accurate results. In the use of multiple fluorophores for the QPCR measurement of multiple DNA targets, this increase in acquisition speed is even more marked. In essence, only one change of filter is needed to simultaneously measure a given fluorophore in all the samples; in non-CCD based detection apparatus, filter changes have to be conducted every time the detector moves on to the next sample and again in order to measure each different fluorophore in that sample.

The spacing between the centres of each pair of neighbouring wells is preferably the same. Preferably, the spacing is approximately 4.5 mm and the number of wells is 96.

The number of wells in the sample holder of the present aspect of the invention may be increased to 384 (i.e. 16 x 24) or 1536 (i.e. 32 x 48). In the former case, the spacing is preferably approximately 4.5 mm or more preferably approximately 2.25 mm. When the number of wells is 1536, the spacing is preferably approximately 2.25 mm. The ability to employ such a large number of wells leads to improvements in throughput. In addition, the lower well spacings lend an increased thermal efficiency to the thermocycling process, the energy input from the heating and cooling element(s) being used to heat a greater sample volume relative to the volume of sample holder material.

The sample holder of this aspect of the invention preferably comprises a thermally-conductive biochemically-inert material which may be selected from liquid crystalline polymers, such as Cool Poly RS372™; thermally conductive polypropylene, such as Cool Poly RS032™; thermally conductive polycarbonate, such as Cool Poly RB019™; and thinly cast standard grade polypropylenes and polycarbonates. The Cool Poly™ polymers are products of Cool Polymers, Inc. of Warwick, Rhode Island. The thinly cast standard grade polypropylenes and polycarbonates preferably have base and wall sections less than or equal to 1 mm thick, more preferably with base sections less than 0.7 mm thick. The term 'cast' is intended to include manufacturing processes such as injection moulding and vacuum forming.

Preferably, the sample holder comprises a thermally conductive polymer having a low autofluorescence. Such a polymer may be selected from the following, all of which are available from Cool Polymers, Inc.: rs362, rsl51, rs228, rs392, rs032, E200, rs328, rb019, rsl43, rs264, rsl02, rsl59-2, rs415, rs224, rs263, rs367, rs416, rsl59, rs201, rsl78, rs277, rs426, rs501, rs395, rs343, rs441, rs035, rslOl, rs230 and rs396. The use of a low autofluorescence polymer in the sample holder's construction confers advantages in terms of accuracy of fluorescence based imaging and measurement of samples. Minimisation of extraneous fluorescence signals due to sample holder components allows the accurate use of a wider range of fluorophores.

In an embodiment of the sample holder of the present invention, the floors of the wells, and at least a region of the sample holder which abuts the floors of the wells, are substantially permissive to electromagnetic radiation such that electromagnetic radiation entering the said region may pass through the wells from the floors to the mouths.

In this embodiment, the need for excitation of samples from a source of electromagnetic radiation positioned above the samples is removed. Instead, the incident beam is transmitted through the body of the sample holder itself. This simplifies the apparatus required for detection/imaging and should also lead to a greater sensitivity of detection, the overall path of sample matrix through which incident and transmitted radiation have to pass being reduced. The entire sample holder may comprise material permissive to electromagnetic radiation or the sample holder may comprise an at least two-part assembly, a first part defining the walls of the wells and a second part, abutting the first part and forming the floors of the wells and providing a path for the electromagnetic radiation to the wells. The first part may, of course, if formed from a material permissive to electromagnetic radiation, also form the floors of the wells, the second part then forming a support for the first part. When the entire sample holder comprises electromagnetic radiation-permissive material, or when an at least two-part assembly is employed with the second part extending to the bottom of the sample holder when in an upright position, the incident electromagnetic radiation may be provided from below the sample holder. In addition, if the entire sample holder comprises the electromagnetic radiation-permissive material or if the second part of a two-part assembly sample holder extends to the lateral faces or corners of the holder, incident radiation may be provided from one or more of the lateral edges of the holder. Two sources of radiation at opposite sides of the holder are preferably employed in order to improve the uniformity of delivery of incident radiation to the samples, although a single source can be employed, preferably in conjunction with an oppositely positioned reflective surface in order to accomplish similar results.

The sample holder of this embodiment preferably comprises a thermally-conductive polycarbonate or polypropylene material. If an at least two-part assembly is employed, the second part may comprise a thin sheet of glass or polycarbonate and the first part may comprise one or more thermally-conductive, biochemically-inert materials such as those mentioned above.

The permittivity of the sample holder is preferably to radiation in at least the visible and/or ultraviolet wavelength ranges.

The sample holder of the present invention may be provided with a thermally-conductive gasket on its surface opposite that in which the mouths are formed (i.e. on its bottom surface when in an upright position). The thermally-conductive gasket comprises at least one compliant thermally-conductive material which may be selected from those mentioned above in relation to the thermal engine. The provision of a thermally conductive gasket on the bottom surface of the sample holder provides for better conduction of heat from the thermal engine to the sample holder. Minute imperfections in the surface of the sample holder and/or the sample block or heating and cooling stage result in air gaps at the interface. These air gaps generally rely on convection for thermal transfer, resulting in slower and less uniform heating of the sample holder and samples. The sample holder is preferably releasably engageable with the thermal engine and, in embodiments of the thermal engines which do not have a sample holder receiver having wells or other surface features related to the well layout of the sample holder, the versatility of the apparatus is considerably increased. The same thermal engine can be used with a variety of different sample holder layouts, the bottom surface of the sample holder not relying on keying into, for example, a welled sample block to ensure efficient thermal transfer. For these purposes, the bottom surface of the sample holder is preferably substantially flat. The sample holder and/or the sample holder receiver is preferably provided with one or more manually or electrically-actuated clamping mechanisms to ensure firm contact between the two during thermocycling.

The sample holder of the present invention may include a lid which is sealable to the holder over the mouths of the wells. The lid helps to reduce sample loss. The lid may be an integral part of the sample holder. The lid may also contain or constitute a lens to collimate electromagnetic radiation entering or leaving the wells.

In addition, the lid may be sealed over the mouths of the wells by means of an adhesive substance located on one or both of the lid and the sample holder. Alternatively the lid may be heat sealed. The lid may contain an optical film.

In a fifth aspect of the invention, there is provided a sample holder for use in a thermocycler, the sample holder comprising a thermally conductive material and having a plurality of wells for holding samples, each well having a mouth and a floor, the floors of the wells and at least a region of the sample holder which abuts the floors of the wells being substantially permissive to electromagnetic radiation such that electromagnetic radiation entering the said region may pass through the wells from the floors to the mouths. The advantages of sample holders according to this aspect of the invention are clear from what has been discussed above. Sample holders according to this aspect may also include any or all of the other features described in relation to the fourth aspect of the invention.

In a sixth aspect of the invention, there is provided a low volume sample holder for use in a thermocycler, the sample holder comprising a thermally-conductive, biochemically-inert material and having a plurality of wells for holding samples, the surface area of the floor of each well being at least 50% of the area described by the mouth of each well.

Sample holders of this aspect of the invention have the advantage that they can be efficiently used with thermocycling apparatus by accommodating samples of a size typical for PCR, and related, reactions in wells of the same order of size as the samples. The shape of the wells ensures that the required volume may be accommodated in a lower depth of well. Both these features contribute towards improvements in efficiency of thermal transfer to/from and control of the sample holder by the thermal engine, as discussed above. The volume of each well is preferably from 1 μl to 60 μl.

In a seventh aspect, the present invention provides a sample holder for use in a thermocycler, the sample holder comprising a thermally-conductive, biochemically-inert polymeric material having low autofluorescence.

The advantages of using low autofluorescence materials in sample holders for thermocycling applications are discussed above. It is preferred that the sample holder of this aspect of the invention has a plurality of low volume wells for holding samples, the volume of each well preferably being from 1 to 60 μl.

In an eighth aspect of the present invention, an integrated thermocycler and sample analyser is provided comprising: means for the releasable attachment of a low volume sample holder according to the fourth, fifth, sixth or seventh aspects of the invention; means for heating and cooling the sample holder when attached; and means for measuring at least one characteristic of samples held in the sample holder.

The integrated thermocycler and sample analyser (the "instrument") of the present invention is based around the low volume sample holders described above and accordingly has the advantage that it is of much lower size and mass than existing apparatus. The power requirements of the instrument are lower since the volumes of sample holder and samples which require heating and cooling are considerably reduced. Nevertheless, the sample throughout of the instrument is actually better than that of many existing devices. This is due in part to the fact that temperature transitions are more rapid. In addition, however, sample analysis is made more efficient since the data capture time necessary for each sample, and where a sample-by-sample analysis is performed, the lag time between samples, may be reduced. The integration of the functions also provides for real-time analysis of samples during a thermocycling run.

In preferred embodiments, the means for measuring the characteristics of the samples are those described above in relation to the thermal engine of the first aspect of the invention. A source of incident electromagnetic radiation may also be provided in equivalent manner to the preferred embodiments of the thermal engine of the first aspect of the invention. The advantages of these preferred features are as described in relation to that thermal engine.

The means for the releasable attachment of the low volume sample holder may comprise a retractable tray which is movable between a first position, substantially exterior of the instrument and in which the sample holder may be attached, and a second position proximal to the heating and cooling means.

The use of a retractable sample holder-loading mechanism allows the size of the instrument to be reduced further. In addition, it is more convenient to use, there being no need to actually detach any parts of the instrument to access the sample holder. It also allows easier loading and unloading by robots, facilitating continuous unmanned operation.

Other features of the thermal engines described above may be incorporated into the integrated thermocycler and sample analyser in order to achieve the advantages mentioned in connection with those features.

The means for attachment of the sample holder preferably does not have wells or other surface features related to the sample or well layout of the sample holder. As mentioned above, this increases the versatility of the instrument and removes the limitations imposed by having sample holders and sample blocks which key together, as employed in the prior art.

In a ninth aspect, the present invention provides an integrated thermocycler and sample analyser comprising: means for the attachment of a sample holder; means for heating and cooling the sample holder when attached; a source of incident electromagnetic radiation for excitation of samples held in the sample holder; and means for measuring at least one characteristic of samples excited by the incident radiation, the source and the measuring means being capable of each communicating with a given sample simultaneously by means of optical fibres.

As discussed above in connection with the thermal engine of the first aspect of the invention, the use of optical fibres between the source or measuring means and the samples allows improved versatility in the relative placement of these components. If necessary, the source and/or measuring means may thus be positioned externally of the thermocycler and sample analyser. The improved versatility allows for improvements in the reduction in size of the thermocycler and sample analyser. The thermocycler and sample analyser of this aspect may include any of the other features described above in relation to the thermal engines or the other integrated thermocycler and sample analyser of this invention and the advantages associated with those features may thereby be achieved.

Thus, the present invention achieves a number of advantages over the thermal engines, thermocyclers and related ancillary parts used hitherto. Substantially faster thermal ramp rates can be achieved, resulting in more specific reactions with a higher yield and reduced non-specific amplification. Thermal accuracy and uniformity may be improved, eliminating to a degree experimental error due to thermal variations. This makes sample-to-sample and batch-to-batch comparisons more informative and may increase the ability to detect smaller changes in expression level. Power requirements are reduced, resulting in lower running costs. The apparatus is also producible at reduced capital cost. The apparatus has a reduced size and thus takes up less laboratory space and is easier to transport, opening up the possibility of field-based experiments. In the use of the low volume sample holders, the well volume is more closely matched to the reaction sample volume. The sample loss during reactions is reduced and the resulting effects of concentration changes are lessened. Both these factors improve sample-to-sample comparisons. The ability to simultaneously measure/image an entire set of samples leads to improved accuracy through an increased uniformity of sample excitation and the opportunity for longer periods of data capture per sample. The ability to measure multiplex experiments is thereby enhanced. By way of comparison, a commercially available low volume QPCR thermocycler has a 32 sample batch size and images one sample at a time. The low volume sample holder and related apparatus of the present invention allow cycling of a greater number of samples at a time. In terms of imaging, the improvement is even greater since multiple samples can be imaged and analysed simultaneously.

The invention will now be described in more detail by way of example only and with reference to the appended drawings, of which:

Figure 1 shows an exploded perspective view of an integrated thermocycler and sample analyser according to the present invention;

Figure 2 shows a cross-sectional view of a low volume sample holder according to the present invention;

Figure 3 shows a cross-sectional schematic view of part of a thermal engine according to the present invention and having attached thereto a low volume sample holder.

Figure 4 shows a cross-sectional schematic view of another thermal engine according to the present invention and having attached thereto a sample holder, the thermal engine containing optical fibres for communication between the samples and optical devices; and

Figure 5 illustrates a number of possible alternative embodiments of the junction fibre illustrated in Figure 4.

The integrated thermocycler and sample analyser, generally indicated 11, has a thermal centre comprising a Peltier effect heating and cooling element 12, a fan-forced heat sink 13 and a heating and cooling stage 14. A sample holder 15 having 96 x 25 μl wells and an integral adhesive-sealed lid 16 fits into a drawer 17 which, under the control of the motor 18 facilitates the engagement of the sample holder 15 with the heating and cooling stage 14. Beneath the heating and cooling stage 14 is a source of UN and visible light (not shown) which provides excitation incident radiation to samples in the sample holder through a plurality of holes (not shown) in the heating and cooling stage 14.

Above the heating and cooling stage 14 is provided a printed circuit board (PCB)-mounted CCD module (not shown) which, via a lens 19, is able to image all 96 samples simultaneously. Above the PCB is a silicon rubber keymat 20 which provides a range of buttons for user control of the apparatus. The entire apparatus is housed within a two-part plastics moulding 20a, 20b.

Since the Peltier element 12 is situated to the side of the heating and cooling stage 14, there is no requirement, in the embodiment shown, for the Peltier element 12 to have holes through which incident radiation can pass. The apparatus is capable of performing QPCR analyses in real-time during an amplification run.

Figure 2 shows a low volume sample holder, generally indicated 21, having 96 (12 x 8) wells of 25 μl volume. The drawing is not to scale but, in practice, the sample holder 21 would be approximately 6 cm x 4 cm in plan. The wells 22 have diameters which would typically be 3.5 mm and the distance between adjacent well centres would typically be 4.5 mm. The upper part 23 of the sample holder is made from polypropylene whilst the lower part 24 consists of a thin plate of glass. For analysis of the contents of the wells 22 by fluorimetry, an incident beam of UV or visible light, indicated by the arrows I, is shone either from below or from the sides. The incident light is transmitted from the glass 24 into the polypropylene 23 and the wells 22. The light enters the sample wells 22 from the floors 25, undergoes an increase in wavelength on encountering sample molecules having fluorophores and can then be detected at the increased wavelength from a suitable position, e.g. above.

In Figure 3, a sample holder 31 is engaged, with the aid of resilient clamps 32, with a heating and cooling stage 33 having a flat upper surface. A thermally-conductive gasket 34 made of thermally-conductive grease sandwiched and contained by aluminium foil is interposed between the stage 33 and the holder 31. The heating and cooling stage 33 is fixed to a lower Peltier element 35 having a heat sink 36. The sample holder 31 is covered with a thin, thermally-conductive film lid 37 and has clamped upon it an upper Peltier element 38 having a heat sink 39. Each of the Peltier elements 35, 38, the heating and cooling stage 33 and the gasket 34 has a plurality of holes H which are aligned with sample holding wells 40 of the sample holder. Incident UN or visible light, indicated by the arrow I, is provided from below the Peltier element 35 and is able to pass through the aligned holes H, through the floors 41 of the wells 40 and into the samples contained thereby. The resulting UV absorbance signal, or fluorescent signal of increased wavelength, then passes through the holes H in the Peltier element 38 and is collected, via a lens 42, by a CCD type camera 43 which is capable of imaging a plurality of sample wells simultaneously.

Turning to Figure 4, a thermal engine, generally indicated 401, comprises a lower thermoelectric device 402 attached to a lower heatsink 403. A flat metal pressure plate 404 adjoins the upper surface of the thermoelectric device 402 and receives a sample holder 405 having 96 (12x8) 25μl wells 405a. The sample holder 405 is opaque and is formed from a low autofluorescence, thermally conductive polymer. A sealing film 405b is provided across the upper surface of the sample holder 405. In the upper part of the thermal engine 401, there is an upper thermoelectric device 406 and an upper heatsink 407. The lower surface of the thermoelectric device 406 is fitted with a flat metal pressure plate 408 for engagement with the sample holder 405 during thermocycling. Above the upper heatsink 407 is an array of 96 excitation optical fibres 409 and 96 detection optical fibres 410 (only one of each type of fibre shown). The fibres 409 and 410 are connected to a LED or tungsten halogen lamp 411 and a CCD camera 412, respectively. A lens 413 may be used in conjunction with the camera 412. The lamp 411 and the camera 412 may be positioned anywhere but are here represented above the upper heatsink 407 for simplicity. The upper heatsink 407 has a plurality of pin-like projections on its upper surface and between the bases of which are formed 96 holes 414 of 1mm diameter aligned with the wells 405a of the sample holder 405 and with the same number of holes (also of 1mm diameter) formed in both the thermoelectric device 406 and flat metal pressure plate 408. The aligned holes in the heatsink 407, thermoelectric device 406 and flat metal pressure plate 408 allow electromagnetic radiation to be transmitted to and from the sample wells 405a by the optical fibres 409 and 410, respectively.

The excitation fibre 409 and the corresponding detection fibre 410 for interrogation of the same well are each engageable with respective arms of a 'Y'-shaped junction fibre 415. The junction fibre 415 passes through the upper heat sink 407, thermoelectric device 406 and flat metal pressure plate 408 and terminates adjacent a sample well 405a. With reference to Figure 5, it can be seen that several different conformations of junction fibre can be used. However, it is preferable to have the junction fibre formed such that, when the detection fibre 410 is engaged with the junction fibre 415, the portion of the detection fibre proximal to the junction fibre is as close to vertical as possible and substantially directly above the centre of the sample well 405a. In such an arrangement, the amount of detectable radiation 501 captured by the detection fibre 410 is improved. The excitation fibre 409 can be engaged with the junction fibre at a variety of angles, provided that the junction fibre is arranged so as to transmit the excitation radiation 502 to the sample well 405a, as shown schematically in Figure 5.

Claims

1. A thermal engine for a thermocycler, the thermal engine comprising a first heating and cooling element, a sample holder receiver in thermal contact with the first heating and cooling element, and a second heating and cooling element situated adjacent the sample holder receiver and removed therefrom so as to allow, in use, the placement of a sample holder between, and in thermal contact with, the first and second heating and cooling elements.

2. A thermal engine according to claim 1 in which at least one of the heating and cooling elements is electrically-controlled.

3. A thermal engine according to claim 2 in which the electrically-controlled heating and cooling element comprises a Peltier effect thermoelectric device, the thermal engine also being provided with a heat sink.

4. A thermal engine according to any preceding claim in which at least one of the heating and cooling elements comprises a heat pipe.

5. A thermal engine according to any preceding claim in which the first and second heating and cooling elements are disposed, relative to each other, so as to be towards opposite sides of a sample holder placed into the thermal engine in use.

6. A thermal engine according to any preceding claim in which the second heating and cooling element is situated so as to come into physical contact with a sample holder placed into the thermal engine in use.

7. A thermal engine according to any of claims 1 to 5 in which a thermally conductive interface plate is positioned, in use, between the second heating and cooling element and the sample holder such that the interface plate comes into physical contact with the sample holder.

8. A thermal engine according to any preceding claim in which the sample holder receiver comprises a heating and cooling stage onto which may be releaseably mounted a variety of different format sample holders in the form of interchangeable sample blocks.

9. A thermal engine according to any preceding claim in which the first or second heating and cooling element is provided with one or more holes through which electromagnetic radiation may pass from samples held in a sample holder to a means for measuring at least one characteristic of the samples, the measuring means communicating with the holes by means of optical fibres and/or being positioned either towards the opposite side of the first heating and cooling element to the sample holder receiver or towards the opposite side of the second heating and cooling element to the sample holder receiver.

10. A thermal engine according to claim 9 in which a source of incident electromagnetic radiation is provided.

11. A thermal engine according to claim 10 in which the source communicates with the holes in the first or second heating and cooling element by means of optical fibres and/or is positioned adjacent a sample holder placed into the thermal engine in use or towards the opposite side of the sample holder to the measuring means.

12. A thermal engine according to claim 10 or claim 11 in which the source is positioned towards the opposite side of the sample holder receiver to the measuring means, the sample holder receiver being provided with one or more holes to provide a passage for the incident radiation from the source to the sample holder.

13. A thermal engine according to claim 12 in which the first heating and cooling element is situated below the sample holder receiver and is provided with one or more holes aligned with those of the sample holder receiver, the source or the measuring means being positioned below the first heating and cooling element and the aligned holes providing the passage for the incident or measured radiation from the source to the sample holder or from the sample holder to the measuring means, respectively.

14. A thermal engine according to any one of claims 9 to 13 wherein the holes are aligned with the wells of a welled sample holder placed into the thermal engine in use.

15. A thermal engine according to any of claims 9 to 14 wherein the measuring means and the source are each capable of communicating, by means of optical fibres, with a given hole in the first or second heating and cooling element simultaneously, the optical fibre from the source and the optical fibre to the measuring means each passing through the same hole or each communicating with that hole by means of a junction fibre engaged with that hole.

16. A thermal engine for a thermocycler, the thermal engine comprising a heating and cooling element and a sample holder receiver in thermal contact with the heating and cooling element, the sample holder receiver being provided with one or more holes through which electromagnetic radiation may pass, in use, from a source, communicating with the holes by means of optical fibres and/or placed adjacent the sample holder receiver, to a sample holder engaged, in use, with the sample holder receiver.

17. A thermal engine for a thermocycler, the thermal engine comprising a heating and cooling element and a sample holder receiver in thermal contact with the heating and cooling element, characterised in that the heating and cooling element comprises a heat pipe.

18. A thermocycler including a thermal engine according to any preceding claim.

19. A low volume sample holder for use in a thermocycler, the sample holder having a plurality of wells for holding samples, wherein the number of wells is a multiple of 96 and the spacing between the centres of neighbouring wells is approximately 4.5mm or 2.25mm, the wells being arranged in a substantially rectangular pattern of a multiple of 12 wells in length and a multiple of 8 wells in width.

20. A sample holder according to claim 19 in which the surface area of the floor of each well is at least 50% of the area described by the mouth of each well.

21. A sample holder according to claim 19 or claim 20 wherein the volume of each well is 100 μl or less.

22. A sample holder according to claim 21 wherein the volume of each well is from 1 to 60 μl.

23. A sample holder according to claim 22 wherein the volume of each well is between 5 and 25 μl.

24. A sample holder according to any of claims 19 to 23 wherein the floor of each well extends in only one plane.

25. A sample holder according to claim 24 wherein the plane of the floor is parallel to the plane of the mouth.

26. A sample holder according to claim 25 wherein the wells provide a volume which is cylindrical or cuboidal in shape.

27. A sample holder according to any of claims 19 to 26 wherein the spacing between the centres of each pair of neighbouring wells is the same.

28. A sample holder according to claim 27 wherein the spacing is approximately 4.5 mm and the number of wells is 96.

29. A sample holder according to any of claims 19 to 27 wherein the number of wells is 384 and the spacing is approximately 4.5 mm or approximately 2.25 mm.

30. A sample holder according to any of claims 19 to 27 wherein the number of wells is 1536 and the spacing is approximately 2.25 mm.

31. A sample holder according to any of claims 19 to 30 comprising a thermally-conductive biochemically-inert material selected from liquid crystalline polymers, thermally conductive polypropylene, thermally conductive polycarbonate, and thinly cast standard grade polypropylenes and polycarbonates.

32. A sample holder according to any of claims 19 to 31 comprising a thermally-conductive polymer having a low autofluorescence.

33. A sample holder according to claim 32 in which the polymer is selected from Cool Polymers, Inc. rs362, rsl51, rs228, rs392, rs032, E200, rs328, rb019, rsl43, rs264, rsl02, rsl59-2, rs415, rs224, rs263, rs367, rs416, rsl59, rs201, rsl78, rs277, rs426, rs501, rs395, rs343, rs441, rs035, rslOl, rs230 and rs396.

34. A sample holder according to any of claims 19 to 33 wherein the floors of the wells, and at least a region of the sample holder which abuts the floors of the wells, are substantially permissive to electromagnetic radiation such that radiation entering the said region may pass through the wells from the floors to the mouths.

35. A sample holder according to claim 34 wherein the entire sample holder comprises material permissive to electromagnetic radiation.

36. A sample holder according to claim 34 wherein the sample holder comprises an at least two-part assembly, a first part defining the walls of the wells and a second part, abutting the first part and forming the floors of the wells and providing a path for the electromagnetic radiation to the wells.

37. A sample holder according to claim 36 wherein the second part of the assembly comprises a thin sheet of glass or polycarbonate and the first part comprises one or more thermally-conductive, biochemically inert materials selected from liquid crystalline polymers, thermally conductive polypropylene, thermally conductive polycarbonate, and thinly cast standard grade polypropylenes and polycarbonates.

38. A sample holder according to any of claims 34 to 37 wherein the permittivity is to radiation in at least the visible and/or ultraviolet wavelength ranges.

39. A sample holder according to any of claims 19 to 38 fitted with a thermally-conductive gasket on its surface opposite that in which the mouths are formed.

40. A sample holder according to any of claims 19 to 39 wherein the bottom surface of the sample holder is substantially flat.

41. A sample holder according to any of claims 19 to 40 including a lid which is sealable to the holder over the mouths of the wells.

42. A sample holder according to claim 41 in which the lid is heat sealable or is sealable by means of an adhesive substance located on one or both of the lid and the sample holder.

43. A sample holder for use in a thermocycler, the sample holder comprising a thermally conductive material and having a plurality of wells for holding samples, each well having a mouth and a floor, the floors of the wells, and at least a region of the sample holder which abuts the floors of the wells, being substantially permissive to electromagnetic radiation such that electromagnetic radiation entering the said region may pass through the wells from the floors to the mouths.

44. A low volume sample holder for use in a thermocycler, the sample holder comprising a thermally-conductive, biochemically-inert material and having a plurality of wells for holding samples, the surface area of the floor of each well being at least 50% of the area described by the mouth of each well.

45. A sample holder according to claim 44 in which the volume of each well is from 1 to 60 μl.

46. A sample holder for use in a thermocycler, the sample holder comprising a thermally-conductive, biochemically-inert polymeric material having low autofluorescence.

47. A sample holder according to claim 46 having a plurality of wells for holding samples, the volume of each well being from 1 to 60 μl.

48. An integrated thermocycler and sample analyser comprising: means for the releasable attachment of a low volume sample holder according to any of claims 19 to 42, 44, 45 or 47; means for heating and cooling the sample holder when attached; and means for measuring at least one characteristic of samples held in the sample holder.

49. An integrated thermocycler and sample analyser according to claim 48 in which the means for measuring the characteristics of the samples comprises an optical device.

50. An integrated thermocycler and sample analyser according to claim 49 in which the means for measuring the characteristics of the samples comprises a CCD-type camera capable of imaging a plurality of samples simultaneously.

51. An integrated thermocycler and sample analyser according to claim 49 or claim 50 including a source of incident electromagnetic radiation.

52. An integrated thermocycler and sample analyser according to any of claims 49 to 51 having one or more dichroic filters between the samples and the optical device.

53. An integrated thermocycler and sample analyser according to any of claims 48 to 52 in which the means for the releasable attachment of the low volume sample holder comprises a retractable tray which is movable between a first position, substantially exterior of the instrument and in which the sample holder may be attached, and a second position proximal to the heating and cooling means.

54. An integrated thermocycler and sample analyser comprising: means for the attachment of a sample holder; means for heating and cooling the sample holder when attached; a source of incident electromagnetic radiation for excitation of samples held in the sample holder; and means for measuring at least one characteristic of samples excited by the incident radiation, the source and the measuring means being capable of each communicating with a given sample simultaneously by means of optical fibres.