WO2003059517A2

WO2003059517A2 - Sealing method for use with water-based thermal cyclers

Info

Publication number: WO2003059517A2
Application number: PCT/GB2003/000019
Authority: WO
Inventors: Susan Frances Kirby
Original assignee: Kbiosystems Limited
Priority date: 2002-01-04
Filing date: 2003-01-06
Publication date: 2003-07-24
Also published as: AU2003201635A1; GB0200157D0; WO2003059517A3; AU2003201635A8

Abstract

A method of applying a temporary seal to a reaction vessel for use in a water-based thermal cycler is provided. The temporary seal is achieved by placing a compression pad against an operative surface of the reaction vessels, and applying pressure to compress the pad against the operative surface of the vessel using another reaction vessel to apply the pressure. An apparatus for performing this method is also provided. The invention also provides a compressed vessel sandwich, comprising a first reaction vessel, a second reaction vessel, and a compression pad, arranged such that the operative surface of the first reaction vessel and the operative surface of the second reaction vessel are arranged face to face, are sealed, and are seperated by compression pad.

Description

SEALING METHOD FOR USE WITH WATER-BASED THERMAL CYCLERS

All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a method of sealing reaction vessels for chemical and/or biological reactions for use in water-based thermal cyclers. The invention particularly relates to a method and system of sealing reaction vessels prior to use in water-based thermal cyclers for Polymerase Chain Reaction (PCR) applications.

BACKGROUND ART

The PCR is one of the most powerful tools of modern molecular biology and is also used in diverse fields such as archaeology, forensic science, genotyping, clinical diagnostics and identity testing. It is a relatively simple in vitro technique that allows quick and reliable enzymatic amplification of any target DNA sequence, and effectively provides limitless supplies of the targeted DNA species. A typical PCR solution includes a sample of target DNA, a DNA polymerase enzyme, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer, magnesium and other optional additives. These components are mixed and then the reaction solution placed in a

'thermal cycler'. A thermal cycler is an automated instrument that controls the temperature of the reaction solution, varying the temperature over pre-programmed lengths of time. This thermal cycling allows the PCR to proceed. Each cycle comprises three steps (three different temperatures), and commonly between 30 and 40 cycles are used. There are many variations of the basic PCR methodology known in the art, and one important area for optimisation of the PCR is the method used for this temperature cycling.

Currently, bench-top Peltier effect thermal cyclers are commonly used for routine low and medium throughput laboratory PCR applications. Peltier effect cyclers comprise a heated block with recessions into which the appropriate reaction vessel is placed. This type of cycler may consist of a single block, the temperature of which is cycled, or may involve the transfer of samples between multiple blocks held at different temperatures. The heated blocks may also be used in conjunction with heated lids. However, Peltier effect thermal cyclers are not well suited to high throughput PCR applications due to their low capacity.

The ability to carry out large numbers of PCR reactions has been elegantly achieved by the use of hot water bath thermal cyclers (for example the 'Duncan' marketed by KBiosystems Ltd.). These are much larger instruments, consisting of a large 'basket', in which samples are placed, attached to an X, Z robotic arm assembly. Conventionally, these baskets are loaded with samples in 'microtitre plates' (reaction vessels each with 384 or 96 wells). The basket can be lowered into any one of three water tanks (held at different temperatures) for a designated time, with the immersion time and water temperature adjusted via computer software. The use of a basket allows a very large number of reaction vessels to be cycled in a single batch, thus allowing very high throughput PCR applications. As well as having increased capacity, these water-based thermal cyclers provide more uniform incubation temperatures (each sample within the reaction vessels is incubated in close proximity to the water), and have improved reaction yields relative to Peltier effect thermal cyclers. In addition, when using water-based thermal cyclers, the reaction vessel is constrained only by the size of the basket, whereas the reaction vessels used in Peltier effect thermal cyclers must be of the correct size and shape to fit within the recessions in the available heated blocks. Water-based thermal cyclers therefore have the advantage that they can be used to process large sample volumes or samples in unusually shaped reaction vessels, provided they fit within the basket. For these reasons, water-based thermal cyclers are often the preferred choice of thermal cycler in a given PCR application. However, there is currently one major disadvantage of this type of thermal cycler. Due to the fact that the reaction vessels are immersed in hot water tanks, the use of this thermal cycler requires the reaction vessels to be sealed before loading into the basket. Current technology for sealing microtitre plates for use in water-based thermal cyclers uses a welded lid to seal completely each individual well. A 'thermal weld' is achieved via the application of heat (approximately 180°C) and pressure resulting in the melting of a portion of the microtitre plate onto the sealing lid (the sealing lid consists of a film of thermal glue on a foil or polypropylene sheet). This current technology seals the microtitre plates extremely effectively.

Unfortunately, the use of a thermal weld to seal the plates means that the reaction solutions are not readily accessible for analysis after thermal cycling. The thermal weld seals are difficult to remove, and if removal is successful then resealing of the plate more than twice is not possible, with the result that the plates cannot be used in any subsequent processing steps. In fact, due to these major difficulties, the samples are usually recovered by puncturing the seal with a hypodermic needle and transferring them to fresh microtitre plates.

To compound these problems, in a number of cases it is desirable to go through the thermal cycling process a number of times. One example of this is the process used for Genotyping by Sequenom™ (Sequenom, San Diego, CA, USA). Their process comprises a PCR step, followed by elimination of residual PCR reactants (a simple enzymatic incubation), followed by a Single Base Chain Extension (SBCE, another thermal cycling step), finally followed by a desalting step. This necessitates the readdressing of the well four times, and hence the use of multiple microtitre plates and additional reagent handling steps.

A simple method for temporarily sealing reaction vessels suitable for use with water-based thermal cyclers would therefore be of great use, since it would remove a major disadvantage associated with the use of this type of thermal cycler.

Methods of temporarily sealing reaction vessels for use with thermal cyclers are known in the art. US Patent 5,721,136 describes the use of a multilayer composite material for sealing reaction vessels for use with Peltier effect heated block thermal cyclers. This method of sealing reaction vessels, marketed by MJ Research Inc., is currently used in many laboratories. However, the disclosed . .

multilayer material is not suitable for the level of pressure required when the samples are immersed in hot water, and hence is not suitable for high throughput water-based thermal cycling instruments. US Patent 6,153,426 describes a method for sealing reaction vessels via an adapted Peltier effect heated block thermal cycler which uses an electrochemical linear motor to ensure satisfactory sealing. This method is also not suitable for high throughput water-based thermal cyclers.

Furthermore, in the case of water-based thermal cyclers, it is the problem of water penetrating into the samples that is of priority, in contrast to the prior art wherein evaporation of liquid out of the sample is the most significant problem.

The present invention seeks to provide a method of temporarily sealing reaction vessels suitable for use in high throughput water-based thermal cyclers. The sealing method must be able to withstand the pressure exerted by the water in the tank in addition to any pressure resulting from the heating of the samples. It must also allow each of the samples within the reaction vessels to be incubated in close proximity to the water to ensure efficient and uniform heating of the samples. The sealing method should also allow re-addressing of the samples numerous times without the need for sample transfer to further reaction vessels, and also allow re-use of the reaction vessels for further thermal cycling. The sealing method should preferably also allow the use of various sizes and shapes of reaction vessels and be amenable to use with existing high throughput automated thermal cycling instruments. The current invention provides a solution to these requirements.

DISCLOSURE OF THE INVENTION The invention provides a method of applying a temporary seal to a reaction vessel for use in a water- based thermal cycler, wherein said seal is achieved by placing a compression pad against an operative surface of the reaction vessel, and applying pressure to compress the pad against the operative surface of the vessel using another reaction vessel to apply the pressure.

Preferably, the two reaction vessels are arranged with their operative surfaces face to face, a pad is placed between the vessels, and pressure is applied to cause a seal to be formed with the operative surface of each vessel.

The reaction vessels used with the invention are preferably microtitre plates e.g. having 96, 384 or 1536 wells. These plates are standard in the art and are usually made of a plastic e.g. polypropylene.

Preferably, an impervious sheet of material (e.g. a plastics material such as a compressible film, or a metal foil) is placed between the pad and each operative surface. This sheet preferably has an adhesive on its face designed to abut the operative surface of the vessel, or another form of adhesion may be used to join the sheet and the vessel e.g. a removable heat seal. The opposite face of the sheet is preferably non-adherent e.g. for abutting the compression pad. The impervious sheet may be applied to the face of the reaction vessel prior to contact with the compression pad, or it may be temporarily positioned on or attached to the compression pad and then positioned on and adhered to _ _

the reaction vessel. The sheet will typically not itself form a water-tight seal on the vessel until it is compressed against the vessel face by the compression pad.

The compression pad may be formed from any one or more of a variety of suitable materials. The material will be sufficiently conformable that it enables a water-tight seal to be created upon application of a compressive force, but sufficiently non-conformable that it is not permanently damaged by the compressive force e.g. so it can be re-used. Preferably, the compression pad is formed from a sheet of silicone, but materials such as silicone foam may also be used in addition to or in place of silicone sheets, particularly where the faces of the reaction vessels are not smooth.

The compression pad may be of different thicknesses for different applications. Preferably, the thickness is between 0.75mm and 2.75 mm. More preferably, the thickness is between 1mm and 2mm. If the compression pad is too thin it will become difficult to handle, and may tear easily. If the compression pad is too thick it may disrupt temperature distribution within the modified loading basket and would also be needlessly expensive to manufacture.

A typical compression pad will be a 2 mm thick silicone pad sheet. The invention also provides a compressed vessel sandwich, comprising a first reaction vessel, a second reaction vessel, and a compression pad, arranged such that the operative surface of the first reaction vessel and the operative surface of the second reaction vessel are (i) arranged face to face, (ii) separated by the compression pad, and (iii) sealed. Sealing may be achieved by the contact made between an operative surface and the compression pad e.g. with one face sealing the first operative surface and the opposite face sealing the second operative surface, or it may be achieved by contact with a material which abuts an operative surface and is situated between the compression pad and the abutted surface. The compressed vessel sandwich is suitable for use in nucleic acid amplification, and the reaction vessels will typically contain nucleic acid amplification reagents (e.g. PCR reagents) such as a polymerase, a nucleic acid template, and dNTP^s. The invention also provides a compressible vessel sandwich, comprising a first reaction vessel, a second reaction vessel, and a compression pad, arranged such that the operative surface of the first reaction vessel and the operative surface of the second reaction vessel are (i) arranged face to face and (ii) separated by the compression pad. The operative surfaces may be sealed by compressing the compressible vessel sandwich such that they are squeezed together against the compression pad. The invention also provides a method as described above, wherein the method is achieved using a plate holder comprising multiple loading layers separated by guide rods and compression springs, and wherein at least one reaction vessel and a pad (e.g. in the form of a compressible vessel sandwich) are placed on at least one (preferably each) loading layer, and pressure is applied to compress the loading layers towards a central area of the holder to compress each pad against an operative surface of its associated reaction vessel e.g. to form a compressed vessel sandwich as described above. The pressure may be applied by turning locking twist catches associated with each guide rod to produce sufficient compressive force to temporarily seal each reaction vessel held within the modified plate holder.

Preferably the loading layers comprise sheet material in which holes of an appropriate size and shape are formed, and onto which supports of an appropriate size and shape are provided to support reaction vessels.

In use, each loading layer preferably holds a plurality of compressed vessel sandwiches, preferably in a layer one sandwich thick.

In an alternative embodiment, the method is achieved using a compression compartment adapted for the application of a compressive force to one or more compressible vessel sandwiches positioned therein, wherein the compressive force is sufficient to temporarily seal the reaction vessels within the compressible vessel sandwiches held therein (i.e. to form a compressed vessel sandwich). Further details of compression compartments of the invention are given below.

The invention also provides apparatus for forming a seal between a reaction vessel and a pressure pad for use in a water-based thermal cycler, said apparatus including a holder for supporting at least two reaction vessels therein and means for applying pressure thereto, wherein the applied pressure is sufficient to form a temporary seal between a face of the compression pad and one or more openings in a surface of one of the reaction vessels within the holder, said pressure being applied to the pad by another reaction vessel within the holder. Preferably, the holder is designed to support at least two reaction vessels with their operative surfaces facing one another, with a compression pad located between the operative surfaces, whereby a single compression pad forms a seal with each operative surface when the pressure is applied.

Preferably, the holder comprises a plurality of vertically spaced loading trays each designed to support at least one reaction vessel. Preferably also, each support tray incorporates a plurality of holes therein, each one being provided to support a reaction vessel.

Preferably, a support is provided on both an upper and lower surface of each tray surrounding each hole, and each tray is supported in the holder by a plurality of horizontally spaced rods, and spaced from its adjacent tray by a plurality of coil springs, each of which is mounted on one of the rods. The means for applying pressure may comprise a locking twist catch provided on one end of each rod to move the loading trays towards a central region of the holder, against the bias of the springs, the arrangement being such that the supports surrounding each hole bear against the peripheries of the reaction vessels to squeeze each pair of reaction vessels into sealing engagement with the pad therebetween, to form a seal with the pad. _ _

Preferably, a plastics sheet overlies each face of the pad to form a temporary seal with the vessels.

The invention also provides an apparatus for forming a seal between a reaction vessel and a compression pad for use in a water-based thermal cycler, wherein the apparatus comprises (a) at least one compression compartment for holding at least two reaction vessels therein and (b) means for applying compressive pressure during use to vessels located within the compartment, and wherein the pressure is (a) sufficient to form a temporary seal between a face of the compression pad and one or more openings in a surface of one of the reaction vessels within the compartment, and (b) applied to the pad by another reaction vessel within the compartment.

Preferably, the compression compartment is designed to support at least two reaction vessels with their operative surfaces facing one another, with a compression pad located between the operative surfaces, whereby a single compression pad forms a seal with each operative surface when the pressure is applied. Thus reaction vessels and compression pads (compressible vessel sandwiches) may be positioned within the compartment, and pressure can be applied to compress pads against vessels' operative surfaces to form a temporary seal (i.e. to form a compressed vessel sandwich). The compression compartment is typically cuboidal. It will be made of a rigid and lightweight material e.g. a metal such as aluminium.

Compression of compressible vessel sandwiches situated within the compartment can be achieved in various ways. For example, the vessel sandwiches positioned within the compartment may be compressed using a clamping mechanism, an arrangement of hydraulic or pneumatic pistons perpendicular to the plane of the reaction vessels, or solenoid- or electrical motors-based compressors. In a preferred embodiment, the compartment includes a screw mechanism which can be tightened to compress compressible vessel sandwiches situated within the compartment. This can conveniently be achieved by locating a screw within a wall of the compartment such that, when it is tightened, it protrudes further through the wall into the compartment, with the protruding end thereby applying compressive force. The extending end of the screw may optionally be attached to a plate to spread its pressure evenly across reaction vessels. The compressing mechanism may include some form of limiting the amount of compression, to prevent the reaction vessels being crushed and damaged. The same effect can be achieved by the use of an appropriately calibrated torque wrench to tighten the screw. Screws can be located in opposite walls of the compartment such that pressure can be applied from either or both ends. Preferably, however, one screw is used on a single wall of the compartment.

The compression compartment can preferably accommodate a plurality of compressible vessel sandwiches. These will typically be stacked within the compartment, and the compartment may include flanges to facilitate the arrangement of vessels within it. Furthermore, a plurality of compression compartments (e.g. three) may be arranged adjacently in a unitary construction (a 'compression basket') to allow maximal load capacity during thermal cycling. Reaction vessels (within compressible vessel sandwiches) within the compression compartment may be situated between compression plates. These plates are preferably made of a rigid material which prevents warping of the reaction vessel during compression and which facilitates the application of an essentially uniform compressive force on the compression pads situated between reaction vessels. The plates may fit around the reaction vessels, or they may be adapted to fit within and support vessels (e.g. to fit within the base of a microtitre plate). The plates may thus have one or more flanges to hold vessels in place. During use, therefore, the compartment may contain a stack of compressed vessel sandwiches, with compression plates between sandwiches (preferably between each sandwich). It may be preferred to include a compression plate at either or both ends of a stack of sandwiches such that compressive force is directly applied to a plate rather than to a reaction vessel.

Different compression plates may be used for different reaction vessels e.g. one type of compression plate for a 96-well microtitre plate, and another type for a 384-well microtitre plate.

The compression plates are preferably perforated such that, during use of the compression compartment, water can contact reaction vessels to ensure efficient heating e.g. during thermal cycling. The perforations also ensure that, when the compartment is removed from a water bath, the water drains quickly. They also lighten the apparatus for easier handling.

For the same reasons, at least one of the walls of the compression compartment will be perforated. At least one of the walls will be open to allow insertion of reaction vessels, and others may also be open.

The apparatus may have a handle to facilitate its loading and unloading into a cycler, either by manual or robotic manipulation.

Other arrangements for holders and compartments will be apparent. For instance, sandwiches could be stacked between compression plates which have a slightly larger area than the reaction vessels. The plates will thus expose a margin around the sandwiches, and bolts can be placed through holes in the corners of the plates. On tightening the bolts, the plates will squeeze the sandwiches together. The invention also provides an apparatus of the invention, wherein the apparatus contains at least one compressible vessel sandwich. Similarly, it provides an apparatus of the invention, wherein the apparatus contains at least one compressed vessel sandwich.

The invention also provides a compressible vessel sandwich or a compressed vessel sandwich, wherein reaction vessels within the sandwich contain nucleic acid amplification reagents (e.g. PCR reagents) such as a polymerase, a nucleic acid template, and dNTPs.

The invention also provides a water-based thermal cycler, characterised in that a compressed vessel sandwich and/or an apparatus of the invention is situated within the cycler. The sandwich and/or apparatus is preferably submerged within water.

Creation of compressible and/or compressed vessel sandwiches may be automated. _ _

The invention also provides a compressed vessel sandwich of the invention for use in nucleic acid amplification. It also provides an apparatus of the invention for use in nucleic acid amplification. More generally, the invention can be used in any reaction which involves thermal transfer, and is particularly suited for thermal cycling techniques. The invention also provides a method of performing a nucleic acid amplification reaction which requires thermal cycling (e.g. PCR), wherein the reaction is performed within reaction vessels of a compressed vessel sandwich. The sandwich may be located within an apparatus of the invention.

The invention also provides a kit comprising an apparatus of the invention which includes a screw mechanism for applying compressive pressure, and a tool for tightening the screw (e.g. a torque wrench). The kit may also comprise one or more microtitre plates and/or compression pads.

The invention relates to the sealing of reaction vessels. Reaction vessels typically comprise a number of separate reaction wells, and it will be appreciated that, although it may be preferred to seal each well within a vessel, the invention can be performed equally well by sealing only a fraction of the wells. For 96-welI microtitre plates (8x12), for instance, a compression pad might cover only a 8x10 fraction of the surface, leaving two rows of 8 wells open on each plate in a pair. Similarly, facing microtitre plates might not be wholly aligned, with a one row offset causing one row in each plate in a pair to remain open. The reaction vessels are still sealed in these two instances, although some wells within the vessels are not.

BRIEF DESCRIPTION OF DRAWINGS Figure 1 is a partly schematic side view showing a method of sealing a compression pad between a pair of opposed microtitre plates which have previously been covered with a thin adhesive-backed plastics sheet, to form a 'compression sandwich'.

Figure 2 is a schematic representation of a plate holder for a plurality of pairs of microtitre plates, with only one compression sandwich shown in position therein. Figure 3 is a schematic representation of a compression basket comprising three adjacent compression compartments, in which only one compartment is loaded with compression cassettes, and Figure 4 is a photograph of the Figure 3 compression basket.

Figures 5 and 6 show an alternative compression basket.

Figure 7 shows a pair of compression plates and a compression pad. Figure 8 shows 384-well microtitre plates after temporary sealing.

MODES FOR CARRYING OUT THE INVENTION

Preferred embodiments of the invention are now described by way of example. . .

First embodiment

Figure 1 shows two microtitre plates 101, 103 with each being provided in known manner with a plurality of wells 105 therein, containing PCR mixtures 107. In accordance with the method of the invention, each plate 101 , 103 is covered with a thin plastics sheet 109. One surface of each plastics sheet is coated with an adhesive substance. The adhesive substance should be of any type such that it adheres to the microtitre plate, but only in such a manner that the sheet can subsequently easily be removed from the plate when required. This thin plastics sheet prevents contamination of the PCR mixtures, and also allows safer handling of the loaded microtitre plates. However, it is not itself sufficient to create a water-tight seal. The two covered microtitre plates are orientated face to face and then placed in contact with a compression pad 1 1 1, such that the entire surface of each microtitre plate containing the wells 105 is covered by the compression pad. It will thus be appreciated that the two plastics sheets 109 also prevent contamination of the compression pad, allowing its re-use. The compression pad is a silicon sheet of 1 mm thickness and of equal area to the upper surface of the microtitre plates. The surface of each plastics sheet in contact with the compression pad is not coated with an adhesive substance, and thus they do not adhere to the compression pad. The compression sandwich of Figure 1 can be assembled either horizontally or vertically. During tilting of the microtitre plates 101 , 103, the PCR mixtures 107 at the base of each of the wells 105 will remain in position due to surface tension.

The compression sandwiches can be assembled in situ on the loading layer or in the compression compartment or pre-assembled, then transferred onto the loading layer.

Figure 2 shows a plate holder 1 13 onto which only one compression sandwich of Figure 1 has been placed. The plate holder comprises a series of spaced loading layers 1 15, each comprising a loading tray 1 17 in which three holes 1 19 are formed. As shown, rectangular holes have been provided, and around the perimeter of these rectangular holes, rectangular supports are formed on both the upper and lower surface of the trays 1 17. These rectangular supports are of the appropriate size to support an inside ridge found on the base of commonly-used microtitre plates. The rectangular supports ensure that the compression sandwiches are held in the correct positions on the loading layer 1 15 to enable subsequent efficient temporary sealing of the microtitre plates. Thus, each loading layer of the plate holder can hold numerous compression sandwiches, with each layer being one sandwich thick. The holes 1 19 formed in the loading trays 1 17 allow the water to come into close proximity to the samples, once the plate holder 1 13 is immersed in a water-based water thermal cycler.

The loading trays 1 17 of the plate holder 1 13 are held apart by a series of springs 121 supported on horizontally spaced, vertically extending guide rods 123, which pass through each loading tray 1 17 perpendicular to the plane of the tray. The springs 121 are stainless aluminium compression springs positioned around each rod 123. This arrangement of guide rods 123 and springs 121 ensures that the loading layers 1 15 are held at a suitable distance apart to allow loading of compression sandwiches, and also contributes to the mechanism of seal formation. _ _

After loading of the compression sandwiches onto the respective rectangular supports of the upper surface of each loading tray 117, locking twist catches 125 positioned at an upper end of each rod 123 are manually turned, requiring an input of force by the operator. Turning of the locking twist catches 125 causes the movement of the loading layers 1 15 towards one another such an upper face of each compression sandwich of one loading layer 115 comes into contact with a support on the lower face of the adjacent loading layer 1 15. The rectangular supports provided on the lower faces of the loading layers ensure that the compression sandwich is correctly positioned. The movement of the trays 117 caused by turning of the locking twist catches 125 is such that the springs between each tray 117 are significantly compressed. The springs therefore exert a compensating compressive force on the loading trays, and particularly, the compression sandwiches held between them. This compressive force is sufficient to cause the formation of a temporary water-tight seal around the edge of the microtitre plates due to compression of the compression pads 111 against the upper surfaces of the microtitre plates 101, 103. The force exerted by the compression pads onto the central area of the microtitre plates ensures that the plates do not warp and thus threaten the efficiency of the seals. The regular arrangement of the guide rods 123 and compression springs 121 between the loading trays ensures that the compressive pressure is evenly spread across the compression sandwiches of each loading layer and between the compression sandwiches of the numerous loading layers.

Once the temporary seal has been applied by engaging the locking twist catches, the modified plate holder is placed in a basket of a water-based thermal cycler and processed as normal. In combination, the 'face to face' orientation of the microtitre plates within the modified plate holder, and the holes formed in each loading layer, ensure that samples at the base of each well of the microtitre plates are in an optimal position for heating, ensuring uniform and efficient thermal cycling of the samples.

After thermal cycling, the locking twist catches 125 are released, and the springs 121 are able to extend, forcing the loading trays 1 17 apart, and concomitantly this may break the temporary seals. In addition to breaking the seals, when the loading trays 117 are forced apart by releasing the locking twist catches 125, access to the microtitre plates 101, 103 is again possible, allowing them to be easily removed from the plate holder 1 13 and analysed, or prepared for further thermal cycling.

In alternative arrangements of this first embodiment of the invention, the compression pads may be replaced by a plurality of 'O-rings' or gaskets of a suitable material. In embodiments, the compression pad may be sufficiently large to be used in a plurality of compression sandwiches or cassettes or to cover multiple reaction vessels, or even all the reaction vessels in one loading layer of the plate holder.

Also, the number of loading layers, the size and shape of the holes formed therein, and the supports formed thereon, may be varied. This variation will allow various types of reaction vessels to be temporarily sealed for water-based thermal cycling. The number, positioning and types of guide rods _ _

and springs utilised for the modified plate holder may also be varied to allow optimisation of the disclosed sealing method for use with varied reaction vessels.

A further variation of this embodiment of the invention provides for the use of alternative means for the application of a compressive force to the compression sandwiches within the plate holder. For example, an arrangement of hydraulic or pneumatic pistons perpendicular to the loading layers, which when actuated cause a suitable degree of compression of the compression sandwiches, could be used, or perhaps even solenoids or electrical motors.

Second embodiment

Figures 3 and 4 show an alternative embodiment of the invention. Apparatus 200 is designed for direct mounting on the support arm of a thermal cycler and consists of three cuboidal compression compartments 210 and a handle 202. Two side walls 212 of each compartment 210 are open except for a flange 214 on one wall. The other two side walls 216 are thin perforated aluminium plates, to one of which flange 214 is attached. The end walls 218 are open except for aluminium bushes 264, 265 in the centre of each. Bush 264 is threaded and includes a tensioning screw 260. The end of the screw 260 mates to an aluminium plate 265 of the same size as end wall 218.

In Figure 3, the top compartment 210 contains eight 384-well microtitre plates 220, arranged as four adjacent pairs. Each pair is made up of facing microtitre plates 220 separated by a 2mm thick silicone sheet compression pad 230 having the same area as microtitre plates 220. Each microtitre plate 220 is placed over a metal compression plate 240 (Figure 7) to provide rigidity. Each compression plate 240 is the same size as end wall 218 and plate 265, and has feet 244 to create space for water flow between adjacent plates 240 when stacked.

Thus the top compartment 210 contains four adjacent compression cassettes 250, each cassette consisting of two outer compression plates 240, with each plate accommodating a microtitre plate 220, and a middle silicone sheet 230. An optional foil sheet 270 is located between plate 220 and sheet 230.

Cassettes 250 can be assembled prior to positioning within the compartment 210, or they may be assembled by sequential positioning of components within the compartment 210. After four cassettes 250 are in place, screw 260 is tightened using a torque wrench and plate 265 thus moves further into the compartment 210, thereby squeezing the faces of microtitre plates 220 onto sheets 230 to form a seal. The same procedure is used until all compartments 210 are full, and apparatus 200 is then used in a thermal cycler. After use, screw 260 is released and microtitre plates 220 are removed (Figure 8).

It is not necessary, of course, to fill all compartments 210 prior to use, and a compartment 210 may be left empty. If a compartment 210 cannot be filled (e.g. fewer than eight microtitre plates 220 are needed) then there are several alternative ways of proceeding. Empty microtitre plates 220 can be used to assemble cassettes 250, but this is wasteful. The microtitre plates 220 can be omitted, giving _ .

thinner cassettes 250, thereby requiring additional protrusion of screw 260. Finally, "dummy" cassettes can be used e.g. formed of appropriately-dimensioned aluminium. The full capacity of the Figure 3 apparatus 200 is eighteen 96-well plates (6 per compartment) or twenty-four 384-well plates (8 per compartment), or combinations between these two extremes (e.g. one compartment contains 96-well plates and the other two contain 384-well plates). Suitable dummies can also be used where an odd number of microtitre plates 220 are to be used, so that a cassette 250 includes only once microtitre plate 220. Thus the face of a reaction vessel does not absolutely have to be sealed by using the operative surface of another reaction vessel to apply the pressure.

Each compartment 210 is attached to the next by studs and nuts 290. Further compartments 210 can thus be added in a modular fashion, although this will necessarily require a thermal cycler with a larger capacity.

Third embodiment

Figures 5 and 6 show an apparatus 300 which is similar to apparatus 200, but which is intended for use in a larger thermal cycler. Three compression compartments are arranged horizontally, and the apparatus 300 includes drawer runners 380. Apparatus 300 can therefore be inserted and removed like a drawer from an appropriately modified basket cage, hence the position of handles 302. The basket cage can then be used in conjunction with conventional water-based thermal cyclers as usual.

Automation

One or more of the steps of the sealing method of the invention could be automated. For example, the assembly of the compression sandwiches or compression cassettes, their transfer into the plate holder 113 or compression compartment 210 and the engagement of the locking twist catches 125 or screw 260 are steps that are all amenable to automation. Such automation is highly desirable for the use of the sealing method of the invention for high-throughput thermal cycling applications.

The invention thus provides a method of forming a temporary seal that prevents the movement of liquid between the water-based thermal cycler and the wells in microtitre plates (or other reaction vessels), thus allowing the microtitre plates to be safely processed in a water-based thermal cycler.

It will, of course, be appreciated that the present invention has been described above only by way of example. Many other embodiments falling within the scope of the accompanying claims will be apparent to those skilled in the art, and modifications of the examples may be made whilst remaining within the scope and spirit of the invention.

Claims

_ .

1. A reaction apparatus for use in a reaction which involves thermal transfer, comprising a first reaction vessel, a second reaction vessel, and a compression pad, arranged such that the operative surface of the first reaction vessel and the operative surface of the second reaction vessel (i) are arranged face to face, (ii) are separated by the compression pad, and (iii) either (a) are sealed, or

(b) can be sealed by squeezing the vessels together.

2. The reaction apparatus of claim 1, wherein the reaction vessels contain a polymerase, a nucleic acid template, and dNTP^s for nucleic acid amplification.

3. The reaction apparatus of any preceding claim, wherein sealing of a reaction vessel is achieved by the contact made between an operative surface of the vessel and the compression pad.

4. The reaction apparatus of claim 1 or claim 2, wherein sealing of a reaction vessel is achieved by contact with an impervious sheet of material which abuts an operative surface of the vessel and is situated between the compression pad and the abutted surface.

5. The reaction apparatus of claim 4, the sheet has an adhesive on the face which abuts the operative surface of the vessel.

6. The reaction apparatus of any preceding claim, wherein the compression pad is between 0.75mm and 2.75mm in thickness.

7. The reaction apparatus of claim 6, wherein compression pad is between 1mm and 2mm in thickness. 8. The reaction apparatus of any preceding claim, wherein the compression pad is formed from a sheet of silicone.

9. The reaction apparatus of any preceding claim, wherein the reaction vessels are microtitre plates.

10. A reaction apparatus substantially as hereinbefore described with reference to the accompanying drawings. 1 1. A method of applying a temporary seal to a reaction vessel for use in a water-based thermal cycler, wherein said seal is achieved by placing a compression pad against an operative surface of the reaction vessel, and applying pressure to compress the pad against the operative surface of the vessel using another reaction vessel to apply the pressure.

12. A method according to claim 11, wherein two reaction vessels are arranged with their operative surfaces face to face, a pad is placed between the vessels, and pressure is applied to cause a seal to be formed with the operative surface of each vessel.

13. A method according to claim 1 1 or 12, wherein an impervious sheet of material is placed between the pad and each operative surface. _ .

14. A method according to claim 13 wherein the sheet has an adhesive on its face designed to abut the operative surface of the vessel.

15. A method according to any one of claims 1 1 to 14, wherein the compression pad is formed from a sheet of silicone. 16. The method according to any one of claims 1 1 to 15, wherein the compression pad is between 0.75mm and 2.75mm in thickness.

17. The method of claim 16, wherein the compression pad is between 1mm and 2mm in thickness.

18. A method according to any one claims 1 1 to 17, wherein said method is achieved by using a plate holder comprising multiple loading layers separated by guide rods and compression springs, and wherein at least one reaction vessel and a pad are placed on each loading layer, and pressure is applied to compress the loading layers towards a central area of the holder to compress each pad against an operative surface of its associated reaction vessel.

19. A method according to claim 18, wherein pressure is applied by turning locking twist catches associated with each guide rod to produce sufficient compressive force to temporarily seal each reaction vessel held within the modified plate holder.

20. A method according to claim 18 or claim 19, wherein said loading layers comprise sheet material in which holes of an appropriate size and shape are formed, and onto which supports of an appropriate size and shape are provided to support reaction vessels.

21. A method according to any one of claims 1 1 to 20, wherein said reaction vessel is a microtitre plate.

22. A method of applying a temporary liquid-tight seal to a reaction vessel substantially as hereinbefore described with reference to the accompanying drawings. 3. Apparatus for forming a seal between a reaction vessel and a compression pad for use in a water- based thermal cycler, said apparatus including a holder for supporting at least two reaction vessels therein and means for applying pressure thereto, wherein the applied pressure is sufficient to form a temporary seal between a face of the compression pad and one or more openings in a surface of one of the reaction vessels within the holder, said pressure being applied to the pad by another reaction vessel within the holder. 4. Apparatus according to claim 23, wherein the holder is designed to support at least two reaction vessels with their operative surfaces facing one another, with a compression pad located between the operative surfaces, whereby a single compression pad forms a seal with each operative surface when the pressure is applied. 5. Apparatus according to claim 23 or 24, wherein the holder comprises a plurality of vertically spaced loading trays each designed to support at least one reaction vessel.

26. Apparatus according to claim 25, wherein each support tray incorporates a plurality of holes therein, each one being provided to support a reaction vessel.

27. Apparatus according to claim 26, wherein a support is provided on both an upper and lower surface of each tray surrounding each hole, and wherein each tray is supported in the holder by a plurality of horizontally spaced rods, and spaced from its adjacent tray by a plurality of coil springs, each of which is mounted on one of the rods.

28. Apparatus according to claim 23, wherein the means for applying pressure comprises a locking twist catch provided on one end of each rod to move the loading trays towards a central region of the holder, against the bias of the springs, the arrangement being such that the supports surrounding each hole bear against the peripheries of the reaction vessels to squeeze each pair of reaction vessels into sealing engagement with the pad therebetween, to form a seal with the pad.

29. Apparatus according to claim 23, wherein the holder is compressed using a clamping mechanism, an arrangement of hydraulic or pneumatic pistons perpendicular to the plane of the reaction vessels, or solenoid- or electrical motors-based compressors. 30. Apparatus according to claim 23, wherein the holder is compressed using a screw mechanism comprising a screw located within a wall of the holder such that, when it is tightened, it protrudes further through the wall, with the protruding end thereby applying compressive force.

31. Apparatus according to any one of claims 23 to 30, wherein reaction vessels are situated within compression plates in the holder. 32. Apparatus according to claim 31, wherein the plates are perforated.

33. Apparatus according to any one of claims 23 to 32, wherein the holder is cuboidal.

34. Apparatus according to any one of claims 23 to 33, wherein a plastics sheet overlies each face of the pad to form a temporary seal with the vessels.

35. Apparatus according to any one of claims 23 to 34, wherein at least one of the walls of the holder is perforated.

36. Apparatus according to any one of claims 23 to 35, wherein reaction vessels are microtitre plates.

37. The reaction apparatus of claim 1 , for use in nucleic acid amplification.

38. The apparatus of any one of claims 23 to 36, for use in nucleic acid amplification.

39. A method of performing a nucleic acid amplification reaction which requires thermal cycling, wherein the reaction is performed a sealed reaction apparatus of claim 1.

40. A kit comprising an apparatus of claim 30, and a tool for tightening the screw.

41. Apparatus for forming a seal between a reaction vessel and a compression pad, substantially as hereinbefore described with reference to the accompanying drawings.