GB2432667A

GB2432667A - Apparatus and method for the separation of material from biological samples

Info

Publication number: GB2432667A
Application number: GB0518073A
Authority: GB
Inventors: Osborn Jones
Original assignee: GWERNAFALAU GYFYNGEDIG
Current assignee: GWERNAFALAU GYFYNGEDIG
Priority date: 2005-09-06
Filing date: 2005-09-06
Publication date: 2007-05-30
Also published as: WO2007028987A3; EP1938108A2; GB0518073D0; WO2007028987A2

Abstract

The invention relates to a closure member 1 and a vessel having a chamber 7 which form a sample separation device. The closure member has apertures 2 which can be positioned above exit ports 3 having an opening 10 with an extension 11 into the body of the vessel. Running along the length of the base of each chamber is a channel 8 which can receive balls for disrupting in particular samples including nucleic acid.

Description

Apparatus and Method for the Separation of Material from Biological

Samples The present invention relates to apparatus and a method of separating material from a biological sample, the sample is in particular but not exclusively, biological cells, with the material that is separated being a nucleic acid.

The separation and extraction of nucleic acids such as DNA or RNA from cells is well known. Manual extraction, where a technician uses a hand held pipette holder, is one way of extracting nucleic acids. Such a method may involve using microplates having an array of open wells or tubes, which can receive a sample. A lysis buffer is added to each well using a disposable pipette tip. Multiple samples can be treated using a pipette holder having a series of pipettes arranged in a linear array on the holder. A biological cell sample containing nucleic acid is then added to the wells using a disposable pipette tip. The sample is mixed with a solid substrate such as a number of magnetic beads that can bind with the nucleic acid that has been released from the cells and the sample is incubated. The nucleic acid and solid substrate complex is then attracted using a magnetic field to confine the complex within an area of the extraction apparatus so that it can be removed from extraneous contaminants such as haemoglobin and other proteins. Another disposable pipette is used for each sample, to separate buffer and the contaminants from the resulting nucleic acid/solid substrate complex. To ensure a substantially pure complex, the complex is washed a number of times, usually four or more times, using a fresh disposable pipette tip each time and for each sample. There may even be the step of transferring the complex to a further microplate so that further washing may occur to remove contaminants. As each microplate remains open during the process, there is the risk of aerosols being generated, when the sample is agitated by material being sucked in and out of the pipette, which, if pathogens or hazardous reagents are being used, could endanger the health of the technician and can pollute the environment. Also, as the action of washing and transferring samples relies on hand operation, there is the risk of a technician incurring Repetitive Strain Injury (RSI) as a result of the multiple repetitive actions of operating a button on the pipette tip holder so that a sample can be picked up and released. The button is usually pushed by the thumb and it may be operated up to fifty times during a single washing of the sample.

Automated separation and extraction methods follow the steps of manual separation and extraction, with the microplates being held in an accessible platform such as a tray or carousel or a chamber. Automation can increase the risk of the generation of aerosols, especially if using open trays and there is the technical difficulty of alignment of the pipette tips with the wells by a machine. Also, unlike a human operator, during an automated operation, it is not so easy to visually check that the method is being carried out properly for example, when the nucleic acid/solid complex sample is being re-suspended, the human eye is the best judge of when to start this re-suspension procedure. The best point to carry this out is when the complex has been successfully attracted to the orifice of a pipette tip and the point at which this occurs can best be judged by the human eye. When using a machine, the re-suspension may start when the sample is not in proximity to the pipette tip, which could result in a sample where the nucleic acid is not properly re-suspended. Also, there is the risk that a pipette tip may become blocked, which may not be detected when using an automated technique, while if a human operator is carrying out the procedure, then is will be visually apparent when a tip is blocked.

To date there is no commercially available automated equipment that can provide a consistently re-suspended sample.

Microplates can have eight or twelve rows of open wells or tubes, and fresh disposable plastic pipette tips are used at each stage of the processing of the sample, this means that there is a great deal of wastage of plastic material. Also, as the plastic components are simply disposed of once used, there is the likelihood of environmental contamination occurring by reagents or nucleic acid material that remains on the disposed components. The more components that are disposed of then there is the greater risk of contamination occurring to the environment.

The present invention seeks to overcome the problems associated with the prior art by providing apparatus that minimises the number of components used, so there is reduced waste material, in particular plastics waste material. The invention also seeks to provide apparatus and a method of efficiently separating and extracting nucleic acid from biological samples, with minimal damage to the nucleic acid by providing a system that shears the nucleic acid from a magnetic bead-nucleic acid complex without causing the resultant long strands of nucleic acid to incur damage and which also provides for consistent re-suspension of material.

According to a first aspect of the present invention, there is provided a closure member arranged to seat on a vessel formed of a chamber that can receive a sample from which material is to be separated, the closure member having an inlet aperture for introducing one or more reagents into the chamber, the closure member also having an elongate exit port which extends downwardly into the chamber such that material that is to be extracted from the chamber can be removed without the need to remove the closure member.

It is preferred that the material to be extracted is waste reagents or alternatively, the material may be the sample itself, in particular, once washed.

It is envisaged that the closure member can be push fitted onto a vessel, or alternatively, a click-lock mechanism may be used to join the closure member with the open mouth of the vessel. In certain arrangements, the closure member and vessel can be sealed together, for example by heat welding.

In a preferred arrangement the inlet aperture is angled such that reagent introduced is directed against a sidewall of the vessel. By having this arrangement, there is the avoidance of splash back of the reagent(s), which could occur if reagents are introduced such that they fall directly into a volume of liquid contained at the base of the vessel.

Alternatively, a lower wall forming the base of the vessel may have a stepped profile against which fluid or reagents entering the chamber may be directed to avoid the risk of splash back.

It is preferred that the inlet aperture is provided as an opening that can receive a pipette tip. However, a -5..

seal arrangement may be provided over the opening that has to be broken by the pipette tip before material can be introduced into the vessel. This maintains the sterility of the vessel prior to use.

It is envisaged that the inlet aperture may receive a pipette tip that is inserted in the aperture In a preferred arrangement, the outlet port is an elongate aperture. This means that the aperture has a length that is longer than its width, with the length extending in the axis of the length of the chamber.

It is preferred that the seal is a plastic, rubber or metal material that is either pierce able or which has an area of weakness allowing the seal to be broken.

It is also envisaged that the inlet aperture may include a valve system, whereby material is introduced into a reservoir by the pipette and then the material is slowly released into the vessel by way of the valve mechanism which controls the flow of material flowing through the inlet aperture or port, into the vessel.

This will allow for controlled introduction of material into the vessel.

The inlet port may have a lid, which can be opened so that a sample can be introduced into the chamber and closed during incubation of the sample to reduce the risk of air-born contamination.

Preferably, the exit port extends to be in proximity to a lower wall of the elongate chamber, said lower wall forming the base of the chamber. The exit port, which is advantageously provided as a pipette tip extends to approximately 0.5mm -4mm above the base of the chamber.

It is envisaged that the exit port extends into the vessel such that the outlet at the end of the exit port is substantially directed to the centre of the lower wall of the vessel. Alternatively, the exit port may be to one or other side of the centre point of the lower wall.

It is preferred that the part of the exit port that is in communication with the closure member is provided with a seal, which forms a seal between the exit port and the closure member. A preferred type of seal would be a ring seal. However, it is envisaged that the seal may be provided by way of a close tolerance moulding and having an integral moulding keeps manufacturing costs to a minimum.

Further, associated with the upper portion of the exit port, is a non-return valve. The valve may be provided by a ball, such as a steel ball, for example, a 3mm steel ball. The valve operates to control the flow of material from the vessel through the exit port.

It is envisaged that the exit port includes a filter to prevent contaminants from being extracted from the chamber with the material from the sample. Ideally the filter is towards an upper portion of the exit port and may be located in the part of the exit port, which is contact with the body of the closure member. The filter acts to prevent contaminants being drawn through the exit port, which again, reduces the risk of the sample obtained and the environment from being contaminated.

This is especially important where sample from pathogenic microorganisms are being handled.

Preferably the filter is a hydrophilic filter to absorb any waste fluid passing through the exit port so there is a reduction is the risk of contamination.

The exit port preferably is in communication with a vacuum source to extract the sample material from the chamber.

It is preferred that a tube extends from the upper portion of the exit port is connected to the vacuum source. The exit of the tube is in proximity to the filter and the vacuum maintains pressure on the filter such that substantially no material caught on the filter from the sample can return to the chamber. The tube may include a viewing window so that the sample or waste that is being extracted can be observed as it exits the vessel.

It is envisaged that the waste material is dispensed into a waste receptacle in communication with the vacuum source.

It is preferred that the material from the sample is a nucleic acid material.

The nucleic acid may be DNA or RNA or any forms of such material for example mRNA.

In a preferred arrangement the closure member is formed of a metal material such a stainless steel or aluminium. By having a metal rather than a plastics material the closure member can be sterilised, for example by autoclaving, thereby reducing further the production of contaminated waste material. Also by having a closure member that can be re-used, this reduces the costs of sampling material.

Further, the exit port may also include a closure that can be sealed when the closure member is not in use.

Such an arrangement would be advantageous where the lid and vessel have been sterilised and placed together.

Having closures or lids on the ports would keep the chamber sterile until required for use.

It is further envisaged that the exit port is formed integrally with the closure member.

According to a second aspect of the invention there is provided a vessel having a base and upwardly extending walls to form a chamber for receiving a sample from which material is to be separated, the base including a channel extending along the base of the vessel, with two of said upwardly extending walls forming side walls for the channel and a further two of said side walls forming end walls of the channel, the channel including at least one ball that can travel along the channel to agitate material in the vessel.

It is preferred that the vessel is deeper than the width of the vessel.

Preferably, the ends of the channel are curvilinear and extend to meet the end walls of the vessel.

It is envisaged that the base of the vessel is inclined so that that sample material or material separated from the sample can collect at the bottom of said incline. This provides for ease of extraction of material from the vessel, for example by using a closure member having an exit port according to a first aspect of the invention.

In an alternative embodiment the base of the container includes recesses for the collection of sample material.

It is preferred that the recess is between the part of the channel in proximity to an aperture at the end of an exit port extending into the vessel.

It is envisaged that the channel includes at least one ball the can travel along the length of the channel.

Preferably, the channel includes two or more balls.

The curved ends of the channel, which are preferably or similar dimensions to the periphery for example correspond to the circumferential dimensions of the ball or balls, allows for maximum travel of the ball(s) along the channel.

In a preferred arrangement the two or more balls are positioned in the channel either side of an aperture for the exit port.

It is envisaged that there is a small or narrow tolerance between the periphery of the ball(s) and the sidewalls forming the channel. Ideally there is a difference of 0.5mm between the width of the channel track and the diameter of the ball. This will mean that there is a clearance of 0.25mm either side of the b all in the channel. By having this narrow tolerance between the balls and the side walls, shear forces are produced which break up nucleic acid, which is bound to magnetic beads.

The action of the ball or balls running along the narrow groove forming the channel provides a shearing action as the nucleic acid is pushed through the narrow gap between the ball(s) and the sidewalls and the base of the vessel. The balls are caused to move by movement of the vessel within a carrier. Also there is break up of the nucleic acid as the ball hits the curved ends of the channel. A further contributory factor is that the operation may be carried out at an elevated temperature, preferably 50-65 degrees centigrade. All these factors interact to re-suspend material so that a homogenous suspension is obtained. Poor suspension, which the current invention seeks to avoid, is when what can be seen as a "floating globule" occurs.

Preferably the balls are formed of stainless steel for example 316 non-magnetic stainless steel.

It is preferred that the material from the sample is nucleic acid material.

It is envisaged that either the sidewall or a base of the vessel includes a profile, which may be a gradual slope, in proximity to the inlet aperture such that reagents entering the vessel hit the profiled surface of the vessel rather than the material in the base of the vessel and this reduces splash back of material in the vessel.

Preferably, the vessel is adapted to be used with a closure member according to the invention.

According to a third aspect of the invention there is provided a sample separation device the device comprising a closure member and a vessel formed of a chamber that can receive a sample from which material is to be separated, the closure member being adapted to seat on the vessel and having an inlet aperture for introducing one or more reagents into the chamber, the closure member also having an exit port which extends downwardly into the chamber such that material extracted from the chamber without the need to remove the closure member with the vessel, the chamber having a base and upwardly extending walls to form the chamber, the base including a channel extending along the base of the vessel, with two of said upwardly extending walls forming side walls for the channel and a further two of said side walls forming end walls of the channel, the channel including at least two balls one of the balls being on one side of the point at which the exit port extends into the vessel, the other of said balls being on the other side of the point at which the exit port open into the vessel, the balls being free to travel along the channel on either side of the exit port, independently of one another.

Preferably, the curved ends of the channel, which are preferably of similar dimensions to the periphery of the ball or balls allows for maximum travel of the ball(s) along the channel.

It is envisaged that there is a small tolerance between the periphery of the ball(s) and the sidewalls forming the channel. Ideally there is a difference of 0.5mm between the width of the channel track and the diameter of the ball. This will mean that there is a clearance of 0.25mm either side of the ball in the channel. Nucleic acids include not only DNA and/or RNA but also messenger or recombinant DNA can be sheared from a magnetic bead complex formed using the balls.

Preferably the balls are formed of stainless steel.

It is preferred that the lower wall of the vessel forming the base includes a recess, preferably in proximity to the opening for the exit port. The recess allows for reagents or nucleic acid that has been separated from the magnetic bead complex to accumulate so that such material can be easily and cleanly withdrawn from the vessel through the exit port.

It is envisaged that the exit port extends into the vessel such that the outlet at the end of the exit port is substantially directed to the centre of the lower wall of the vessel.

It is envisaged that the closure member can be push fitted onto a vessel, or alternatively, a click-lock mechanism may be used or indeed the closure member and vessel can be sealed together.

In a preferred arrangement the inlet aperture is angled such that reagent introduced is directed against a sidewall of the vessel.

Alternatively, a lower wall forming the base of the vessel may have a profile, which could be a step or a slope, against which fluid or reagents entering the chamber may be directed to avoid the risk of splash back.

It is preferred that the inlet aperture is provided as an opening that can receive a pipette tip and a seal arrangement may be provided over the opening. It is preferred that the seal is a plastic, rubber or metal material that is either pierceable or which has an area of weakness allowing the seal to be broken.

Preferably, the exit port extends to be in proximity to a lower wall of the elongate chamber, said lower wall forming the base of the chamber.

It is envisaged that where the exit port is in sealed communication with the closure member. A preferred type of seal would be a ring seal although the closure member may be moulded such that that no seal is needed.

Further, associated with the seal is a non-return valve. The valve may be a ball valve.

It is envisaged that the exit port includes a filter to prevent contaminants from being extracted from the chamber with the material from the sample.

Preferably the filter is a hydrophilic filter to absorb and wick away any waste fluid passing through the exit port.

The exit port preferably is connected to a vacuum source to extract the material such as reagents or sample from the chamber. Having a vacuum maintains pressure on the filter such that substantially no material caught on the filter from the sample can return to the chamber.

In a preferred arrangement the vessel and/or closure member is formed of a metal material such a stainless steel or aluminium to allow for sterilization for re-use.

The inlet port and/or the exit port may have lids that can be sealed when the closure member is not in use so that the inside of the vessel can be maintained in a sterile condition.

According to a fourth aspect of the invention there is provided a method for separating material from a sample, wherein material is introduced into a chamber in which material is to be separated, the material is mixed with magnetic beads so allow material in the sample and the beads to complex, and placed in a magnetic field to attract the complex to a specific area in the vessel, balls are caused to travel along the channel there being a small tolerance between the walls of the channel and the balls to shear material in the complex from the beads.

The complex is preferably incubated before being subjected to treatment with a magnet. When under the influence of a magnet, the magnetic bead complex can be held in position while extraneous material such as haemoglobin and other proteins are removed into a waste container. The complex can then be washed with buffers and the sample may be monitored for re-suspension.

Material such as waste or re-suspended material is removed at appropriate stages from the vessel by way of an exit port through which the material is extracted from the vessel. The fourth aspect is carried out using equipment as described in previous aspects of the invention.

The invention is intended to cover not only all individually described aspects as described but also combinations of the aspects that have been discussed.

An embodiment of the invention will now be described by way of example only with reference to the accompanying figures in which: Figure la: shows a plan view of a closure member according to a first aspect of the invention; Figure ib: shows a perspective view of the closure member of Figure la; Figure 2: shows a vessel having a channel that can be used with a closure member as shown in Figure la and lb; Figure 3: shows the closure member of Figure la or Figure lb and the vessel of Figure 2 in combination; Figure 4: shows an upper end of an exit port for the closure member, with a non-return valve and a filter; Figure 5: shows a further aspect of the invention including a vessel having balls within the channel at the base of a vessel; Figure 6: shows the base of the vessel of Figure 5; and Figure 7: shows the vessel and lid of the first and second aspects of the invention supported in an automated carousel for harvesting nucleic acid.

As shown in Figure 1, the closure member according to a first aspect of the invention comprises a main body, generally shown as 1 in Figure la. The closure member has a series of four apertures 2 running along the length of the closure member 1. Although four apertures are shown, more than four or less than four apertures for example two, can be used depending upon how many vessels are to be associated with the closure member. There is a series of four exit ports 3 which are parallel to the inlet ports 2. The exit ports are substantially central of the closure member with the inlet ports being to one side of the exit ports. Each of the exit ports has a ring seal 4 to seat the respective exit port within the body of the closure member and within the exit port there is a valve 14. As can be seen from Figure lb, the closure member has a flange or extension 5 which extends from underneath the closure member and this forms a seat which can sit within the mouth of a vessel as shown in Figure 2. The vessel comprises a chamber 7 having a mouth 6 which can receive the extension 5 of the closure member. The vessel may include a number of mouths which can be lined up with extensions 5 for a closure member which extends underneath a set of apertures which comprise an inlet aperture 2 and an exit port 3 in side-by-side relationship. The vessel may have a lip 9 on one or both longitudinal sides which allow for the vessel to be seated within a carrier, for example a carousel for a series of vessels. Running along the length of the base of each chamber is a channel 8.

Figure 3 shows in more detail a vessel having an inlet port 2 and in side-by-side relationship an exit port 3. The exit port 3 includes an opening 10 which narrows to a tube like member 11 which extends into the body of the vessel. The tube like member can be provided as an integral pipette tip. The exit aperture of the tube 11 ends in proximity to the channel 8 at the base of the vessel. As shown in Figure 3, the tube 11 extends to a point that is substantially equidistant from the two sides of the vessel. In a preferred arrangement, the exit aperture of the tube is approximately 4mm from the bottom of the channel although this distance can vary for example between 1mm and 6mm.

Figure 4 shows the upper portion of the exit port in more detail.

The exit port 4 has a sealing ring which seals around the upper portion of the exit port which is in communication with closure member 1. The exit port extends downwardly into the body of the closure member forming a first chamber 10. The end walls of the chamber narrow to form an aperture which is narrower than the chamber 10 and in the neck of the aperture, there sits a valve member 14 which is shown in this case as a ball valve. The ball valve is positioned above an exit tube which can include a filter member 15 beneath the ball valve. The ball valve 14 controls the flow of fluid which is taken upwardly through the tube 11 and into chamber 10. The filter 15 allows for the capture of any contaminated material in the sample to reduce the risk of environmental contamination and also to ensure that the sample that is extracted from the vessel is substantially pure.

Turning to Figure 5, the closure member of the first aspect of the invention can be used with a vessel of a second aspect of the invention. The closure member 1 in this case is shown with the exit port being substantially central of the width of the closure member. There is a vacuum tube 16 which is connected via a connection member 17 to the top of the closure member 1. The vacuum tube 16 extends into chamber 10 which is positioned above valve 14. The valve 14 sits above the entrance to tube 11 which extends downwardly into the body of the vessel.

The exit aperture 12 of the tube, which is cut at an angle to the length of the tube is in proximity to but does not come into total communication with the base of chamber 8. Having a cut away portion of the end of the tube 11 provides a space between the end of the tube and the bottom of the chamber where material that has been extracted from a complex containing DNA, for example DNA strands can accumulate. Either side of the tube 11 can be found balls 8 which can travel between the side walls of the vessel and the tube 11.

As shown in Figure 6, the base of the vessel may include a sloped area with the profile of the lower wall of the vessel falling away to the lowest point being underneath the exit port 12 of the tip 11. This allows for a globule of extracted DNA 19 to accumulate in proximity to the tip so that the DNA can collect underneath the tip 11 so it can be extracted or reagents can be removed from the DNA as cleanly as possible.

According to a first aspect of the invention during operation in an automatic procedure as shown in Figure 7, blocks of closure members may be associated with corresponding vessels and seated in chambers 21 within a rotating carousel 22. The combined closure member and vessel is shown as 20 in Figure 7. During operation in, for example, an automated system, into each inlet

aperture, 200/21 of lysis buffer is dispensed, together with 2OOil of whole blood. The blocks containing the blood and lysis buffer are positioned on the carousel as shown in Figure 7 and each block is identified with the correct sample number. The carousel is programmed to gently shake the blood and lysis buffer for a period at room temperature, this period is usually five minutes and this is to release DNA from white blood cells. The carousel may include means to gradually heat the sample to achieve optimum incubation conditions. A timer may also be included to time the period that incubation occurs. Further, the carousel may also include means to either heat up the reagents to stop a reaction or alternatively, cooling may occur to terminate the reaction, depending on the sample and/or reagents being used.

Magnetic beads are added together with a binding buffer and the carousel is again programmed to gently shake the blood and lysis buffer for a further five minutes at room temperature.

The carousel is positioned above an array of magnets so that DNA that has been complexed with magnetic beads is attracted to for example the bottom corner of a chamber. Supernatant is removed using a vacuum connected to the exit port and then wash buffer is added to a chamber using the inlet aperture which contains more lysis buffer. The DNA bead complex is mixed vigorously to completely break up the complex into an even constituency.

A mixture for example ethanol or isopropynol is added to recombine the DNA onto the magnetic beads. The

vessel is then put within the magnetic field and

supernatant is again removed.

Washing is repeated four times with decreasing amounts of lysis buffer and increasing amounts of alcohol at each stage. The final wash involves removing last traces of alcohol by adding a final tris-HCL buffer wash at pH 8.5. Again, supernatant is removed using a vacuum connected to the exit port. It is possible to clean samples by including protein digestion enzyme to remove all traces of protein from DNA and heating the sample can accelerate the digestion process up to a point where the sample is cleaned and the protein digestion enzyme can then be deactivated by heat treatment so that further enzymatic reactions are not compromised.

In this first aspect of the invention, the DNA/bead complex is broken up and re-suspended by the shearing action of DNA passing through the tip of the exit port.

In the second aspect of the invention, the process of inclusion of magnetic beads and washing occurs as described in the first aspect of the invention. However, rather than using shearing action of material passing through a pipette tip to break up the nucleic acid/magnetic bead complex, the complex is broken up using balls travelling along a channel in the vessel in which the complex is contained. The shearing action of removing DNA from the complex can be achieved by way of using one or more stainless steel balls which run along the narrow elongated horizontal trough as shown in Figures 5 and 6. The elongated trough is approximately 30mm long with a width of 6.5mm. The diameter of the ball running within the trough is 6mm. The ball material is preferably, non-magnetic 316 stainless steel. The action of the ball running along the narrow groove which forms the trough contributes to shearing action as nucleic acid is pushed through the narrow gap between the ball wall and bottom of the trough. The balls are caused to move along the trough as a result of movement of the carousel or by manual movement, causing the balls to travel back and forth along the channel. Also, as the operation is carried out at a temperature of roughly 50 C to 65 C, this elevated temperature as well as the impact of the ball hitting the end of the trough contributes to the shearing action and release of the nucleic acid from the complex. The ball can be caused to move back and forth at six movements per second to cause shearing and the distance travelled back and forth is approximately 13.6mm. The radial acceleration of the ball moving within the channel is 2.04m/second2. It is envisaged that the balls can move as a result of movement of the vessel in the carrier for example the balls are caused to move in a sideways back and forth movement as the vessels are shaken. Also, due to friction some of the DNA complex can stick to the balls can be caused to be broken up as the ball impacts against the sides of the vessel, the action of the chamber in which the ball is being shaken back and forth can result in the beads and DNA complex being broken up as the balls pass the DNA and beads complex thereby releasing DNA from the complex so that it can then be washed and extracted from the vessel.

Therefore it can be seen that the invention provides not only an improved method or introducing reagents into a vessel, but also an improved way of breaking up nucleic acid/carrier complexes.

The invention covers not only individual embodiments as discussed but combinations of embodiments as well. It is to be understood that modifications and variations of the present invention will become apparent to those skilled in the art and it is intended that all such modifications will be included within the scope of the present invention.

Claims

Claims 1. A closure member arranged to seat on a vessel formed of a

chamber that is adapted to receive a sample from which material is to be separated, the closure member having an inlet aperture for introducing one or more reagents into the chamber, the closure member also having an elongate exit port which extends downwardly into the chamber such that material that is to be extracted from the chamber can be removed without the need to remove the closure member.

2. A closure member as claimed in claim 1, wherein the material to be extracted is selected from waste reagents or the sample itself.

3. A closure member as claimed in claims 1 or 2, wherein the inlet aperture is angled such that when :.:::. 20 the closure member is seated on a vessel a reagent S...

introduced into the vessel is directed against a sidewall of the vessel. S. * S * *5*

4. A closure member as claimed in any preceding claim, * , 25 wherein the inlet aperture is provided as an opening ::::: that can receive a pipette tip. S...

5. A closure device as claimed in claim 4, wherein a seal arrangement is provided over the opening.

6. A closure member as claimed in claim, wherein the outlet port is an elongate aperture.

7. A closure member as claimed in claim 5, wherein the seal is a plastic, rubber or metal material that is either pierce-able or which has an area of weakness allowing the seal to be broken.

8. A closure member as claimed in any preceding claim, wherein the inlet aperture comprises a valve system for controlling the flow of material thought the inlet aperture.

9. A closure device as claimed in any preceding claim, wherein the exit port extends to be in proximity to a lower wall of the elongate chamber, said lower wall forming the base of the chamber.

10. A closure member as claimed in any preceding claim, wherein the part of the exit port that is in communication with the closure member is provided with a seal, which forms a seal between the exit port and the closure member. S... * *

SSSS

11. A closure member as claimed in claim 10, wherein the seal is a ring seal. S..

* 25 12. A closure member as claimed in any preceding claim, wherein the exit port includes a non-return valve.

* S S. S..

13. A closure member as claimed in any preceding claim, wherein the exit port includes a filter to prevent contaminants being extracted from the chamber.

14. A closure member as claimed in claim 13, wherein the filter is a hydrophilic filter.

15. A closure member as claimed in any preceding claim, wherein when the closure member is seated on the vessel, the exit port is in communication with a vacuum source to extract material from the chamber of the vessel.

16. A closure member as claimed in claim 15, wherein a tube extends from the upper portion of the exit port to the vacuum source.

17. A closure member as claimed in any preceding claim, wherein the material separated from the sample is nucleic acid material.

18. A closure member as claimed in any preceding claim, wherein the closure member is formed of a metal material such as stainless steel or aluminium.

19. A vessel having a base and upwardly extending walls to form a chamber for receiving a sample from which material is to be separated, the base including a S...

channel extending along the base of the vessel, with : .. two of said upwardly extending walls forming side walls for the channel and a further two of said side * 25 walls forming end walls of the channel, the channel including at least one ball that can travel along the channel to agitate material in the vessel.

20. A vessel as claimed in claim 19, wherein the vessel is deeper that the width of the vessel.

21. A vessel as claimed in claim 19 or 20, wherein the ends of the channel are curvilinear and extend to meet the end walls of the vessel.

22. A vessel as claimed in any of claims 19 to 21, wherein the base of the vessel is inclined so that sample material or material separated from the sample can collect at the bottom of said incline.

23. A vessel as claimed in any of claims 19 to 22, wherein the base of the container contains recesses for the collection of sample material.

24. A vessel as claimed in claim 23, wherein the recess is between the part of the channel in proximity to an aperture at the end of an exit port extending into the vessel.

25. A vessel as claimed in any of claims 19 to 24, wherein the channel includes at least one ball that can travel along the length of the channel.

26. A vessel as claimed in claim 25, wherein the channel *.S.

* * includes two or more balls. em..

27. A vessel as claimed in claim 26, wherein the two or more balls are positioned in the channel either side of an aperture for the exit port.

28. A vessel as claimed in any of claims 25 to 27, wherein the balls are formed of stainless steel.

29. A vessel as claimed in any of claims 19 to 28 wherein the material separated from the sample is nucleic acid material.

30. A vessel as claimed in any of claims 19 to 29, in combination with the closure member of any of claims 1 to 18.

31. A sample separation device the device comprising a closure member and a vessel formed of a chamber that can receive a sample from which material is to be separated, the closure member being adapted to seat on the vessel and having an inlet aperture for introducing one or more reagents into the chamber, the closure member also having an exit port which extends downwardly into the chamber such that material can be extracted from the chamber without the need to remove the closure member with the vessel, the chamber having a base and upwardly extending walls to form the chamber, the base including a channel extending along the base of the vessel, with two of said upwardly extending walls forming side walls for the channel and a further two : .". 20 of said side walls forming end walls of the channel, the channel including at least two balls one of the S...

balls being on one side of the point at which the exit port extends into the vessel, the other of said balls being on the other side of the point at which : * 25 the exit port open into the vessel, the balls being free to travel along the channel on either side of S.... the exit port, independently of one another.

32. A sample separation device as claimed in claim 31, wherein the ends of the channel are curvilinear and extend to meet the end walls of the vessel.

33. A sample separation device as claimed in claim 32, wherein the curved ends of the channel allow for maximum travel of the ball or balls along the channel.

34. A sample separation device as claimed in any of claims 31 to 33, wherein the balls are formed of stainless steel.

35. A sample separation device as claimed in any of claims 31 to 34, wherein the lower wall of the base includes a recess in proximity to the opening of the exit port.

36. A sample separation device as claimed in any of claims 31 to 35, wherein the exit port extends into the vessel such that the outlet at the end of the exit port is substantially directed to the centre of the lower wall of the vessel.

37. A sample separation device as claimed in any of claims 31 to 36, wherein the inlet aperture is angled such that reagent introduced is directed against a sidewall of the vessel.

38. A sample separation device as claimed in any of * 25 claims 31 to 37, wherein the inlet aperture is ::::: provided as an opening that can receive a pipette tip.

39. A sample separation device as claimed in claim 38, wherein a seal arrangement may be provided over the opening.

40. A sample separation device as claimed in claim 39, wherein the seal is a plastic, rubber of metal material.

41. A sample separation device as claimed in claim 39 or 40, wherein the seal is pierce-able or has an area of weakness allowing the seal to be broken.

42. A sample separation device as claimed in any of claims 31 to 42, wherein the exit port is in sealed communication with the closure member.

43. A sample separation device as claimed in any of claims 31 to 42, wherein the exit port includes a filter.

44. A sample separation device as claimed in claim 43, wherein the filter is a hydrophilic filter.

45. A sample separation device as claimed in any of claims 31 to 44, wherein the exit port is connected to a vacuum source to extract material and/or : ... reagents from the chamber.

46. A sample separation device as claimed in any of ::: claims 31 to 45, wherein the vessel and/or closure member is formed of a metal material such as stainless steel or aluminium to allow for sterilization for re-use.

47. A method for separating material from a sample using a sample separation device according to any of claims 31 to 46, wherein material is introduced into a chamber in which material is to be separated, the material is mixed with magnetic beads so allow material in the sample and the beads to complex, and placed in a magnetic field to attract the complex to a specific area in the vessel, balls are caused to travel along the channel there being a small tolerance between the walls of the channel and the balls to shear material in the complex from the beads.

48. A method for separating material from a sample as claimed in claim 47, wherein the complex is incubated before being subjected to treatment with a magnet.

49. A closure member substantially as herein described with reference to the accompanying drawings.

50. A vessel substantially as herein described with reference to the accompanying drawings. * S. * . * *..

51. A sample separation device substantially as herein described with reference to the accompanying drawings.

* 25 52. A method for separating material from a sample :::::: substantially as herein described with reference to the accompanying drawings.