EP1766360A1

EP1766360A1 - Method and device for taking and dispensing a sample

Info

Publication number: EP1766360A1
Application number: EP05756321A
Authority: EP
Inventors: Matti Korpela; Sanna PIETILÄ; Pekka Mattsson
Original assignee: Bio Nobile Oy
Current assignee: Bio Nobile Oy
Priority date: 2004-06-18
Filing date: 2005-06-17
Publication date: 2007-03-28
Also published as: WO2005124310A1; FI20045231A0

Abstract

The object of the invention is a method and a device for taking and dispensing a sample, such as a biological sample, for purification. A device according to the invention includes an oblong hollow unit, that contains a first end (1100') and an opposite, second end (1200'), that contains a sample space (900') separated by a flexible membrane (800') as well as a tip (500'), which is designed to be a picking tip for taking a sample. There is a magnetic rod inside the device (1000').

Description

METHOD AND DEVICE FOR TAKING AND DISPENSING A SAMPLE

FIELD OF THE INVENTION The object of the invention is a method for taking and dispensing a biological sample for purification.

Another object of the invention is a device for dispensing a sample, such as a biological sample. The device includes an oblong hollow unit, that contains a first end and an opposite second end, that includes a sample space and a tip, which is designed to be a picking tip for taking a sample.

BACKGROUND OF THE INVENTION

Complex biological samples, such as animal tissue, plant, eukaryotic and bacterial cell homogenates, reaction solutions and electrophoresis gels, such as agarose, low melting agarose and acrylamide, obtained by PCR and other amplification methods are common starting materials in purification processes of nucleic acids, proteins and peptides. A large variety of pre-treatment methods have been described in the litterature for the kind of sample materials described above, depending on whether DNA is desired to be purified, for example, from plant tissue, agarose gel or whole blood. A common feature for the methods is that the sample volume may not exceed the maximum limit of the purification method in use. Exceeding the maximum limit may lead to a loss of the whole sample.

There is a variety of purification methods in use, for example, commercially available purification kits based on the use of a spin column, chromatography column, filter or particles. Pre-treatment of the sample, for example, homogenisation, weighings, aliquoting and storing and the actual purification process have so far been performed apart from each other. One remarkable reason for this is that the purification methods that are most generally in use (gravitation columns, filters, spin columns) are limited to the use of centrifugated sample materials. If a sample containing untreated, for example, particulate material accidentally comes into the column, this in most cases results in the stoppage of the column and the loss of the sample. Scientists need to take care of the pre-treatment of the sample material manually and check carefully the amount of sample that may be applied to the actual purification process.

At present, the most commonly used way to isolate DNA from an agarose gel after gel electrophoresis is to cut the desired piece of gel by means of a scalpel out of the gel. The procedure is laborious and demands precision. The cut piece of gel has to be weighed separately in order to be able to calculate the needed amount of the reagents for purifying the DNA from the piece of gel. The commonly used purification kits determine the amount of the reagents to be used in a direct relation to the weight of the cut gel. The amount of the needed reagents has to be calculated separately for each piece of gel. A maximum limit has also been set for the weight of the piece of gel and this may not be exceeded. Generally taken, cutting the gels is laborious and troublesome, but in the prior art it is an obligatory procedure in the laboratories of molecular biology.

Ethidium bromide is traditionally used in the DNA/RNA gels for visualisation of the nucleic acid zones by means of UV light. UV radiation causes rapid mutations in the nucleic acids and therefore the detachment of the pieces of gel has to happen as quickly as possible.

In the futher treatment, the piece of gel is typically transferred to an Eppendorf tube. The tubes need to be weighed separately in order to determine the weight of the piece of gel. The scalpels that are used need to be sterile and free of DNase. If RNA is desired to be isolated from the gel, the requirements concerning the cleanness of the equipment in use get considerably more strict, as RNase needs to be eliminated from each equipment.

Also electroelution and DEAE paper may be used for isolating nucleic acids from gels. Most of the methods in use, such as dispersing the gel by passing it through a filter, dialysis, the use of chaotropes and the so called "freeze-squeeze" methods, require that the gel is cut with a scalpel before the actual purification of the nucleic acids. Many of the methods have not become very popular among scientists either because of their laboriousness or slowness.

Cut pipette tips have been used to cut a piece of gel out of the gel (J. M. Thimson and M. M. Compton, Biotechniques 24, 1998, 942). The greatest problem in using pipette tips is how to remove the piece of gel from the pipette tips. The gels also break easily and transferring the pieces of gel entirely to a tube is almost impossible. For this reason, the isolated pieces of gel need to be weighed even in this case in order to determine, how much of gel was managed to get stored up. In practice, using pipette tips is almost as laborious as cutting the piece of gel by means of a scalpel out of the gel.

A device for taking liquid samples is described in the US Patent 3768978. The method according to the invention is also of considerable use in the treatment of such samples that are viscous or contain particulate material. Pipetting such samples is very problematic due to the risk of stoppage in the pipette tips.

By means of the method according to the invention, dispensing, picking, transfer, isolation and purification of biological samples and various gels may be simplified and facilitated.

BRIEF DESCRIPTION OF THE INVENTION

The objective of the invention is to develop a method and a device to realise the method in such a manner, that the above mentioned problems can be solved. The objective of the invention is achieved with the method and the device characterised in the text of the enclosed independent claims. The preferred embodiments of the invention are the object of the dependent claims.

The greatest advantages of the device according to the invention are that it enables a fast and easy taking and releasing of a biological sample. Additionally, the device may be autoclaved or baked in an oven several times, when the need arises, and thus render it sterile as well as free of DNase and RNase.

The great advantage of the method is that it may be used to speed up and secure a successful treatment of a sample from various especially difficult biological samples and different gels. The method is also suited for collecting colonnies from a growth plate, tube or other corresponding growth surfaces. The speed of the method according to the invention reduces for example the negative effects of UV light on the DNA fragments to be isolated from the gel. The method described in the invention enhances also clearly the treatment of viscous samples and samples containing particulate material. The method and the device according to the invention is well suited for different samples and the method is easily automated.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is now described further together with the preferred embodiments, referring to the enclosed drawing, where

Fig. 1 illustrates the essential unit of the device according to the invention and the way, how it can be used, when a piece of gel is desired to be removed manually from a gel, Fig. 2 presents sideways the first embodiment of the essential unit of the device according to the invention, Fig. 3 presents a view of the unit of Fig. 2 along the cutting line 11-11, Fig. 4 presents sideways another embodiment of the essential unit of the device according to the invention,

Fig. 5 presents a view of the unit of Fig. 4 along the cutting line IV-IV,

Fig. 6 presents sideways a third embodiment of the essential unit of the device according to the invention, Fig. 7 presents a view of the unit of Fig. 6 along the cutting line VI-VI, Fig. 8 presents sideways a fourth embodiment of the essential unit of the device according to the invention, Fig. 9 presents a view of the unit of Fig. 8 along the cutting line VIII-VIII, Fig. 10 presents the preferred embodiment of a device according to the invention and illustrates its use, when a sample is desired to be taken manually or in an automated manner from the gel and released,

Fig. 1 1 illustrates the use of the device in Fig. 10, when a liquid sample is desired to be taken and given further, and Fig. 12 presents another preferred embodiment of the device according to the invention and illustrates its use, when a sample is desired to be taken manually or in an automated manner from the gel and released

Fig. 13 presents an agarose gel, where DNA fragments purified by means of the method according to the invention have been run.

DETAILED DESCRIPTION OF A DEVICE ACCORDING TO THE INVENTION Figure 1 presents an essential part, marked generally with the index number 1 , belonging to the device according to the invention and its use for taking the sample 3 manually out of the gel 2. Unit 1 , which is an oblong hollow unit, includes the open upper part 11 and the bottom 12, which includes the open tip 5. Within a distance from the tip 5 there is the flexible, transparent membrane 8, which sets limits for the sample space 9. The membrane is loose.

In the picture farthest left in Figure 1,unit 1 is presented sideways within a distance from the gel 2. In the picture second farthest left in the Figure, the view seen from above inside the unit 1 in the direction of the arrow A is presented. It is seen, that the cross-section of the unit 1 is circular and that a part of the gel 2 together with its sample 3 can be seen through the membrane 8 of the unit. Thus, the sample may be optically observed. When a piece of gel 4 containing the sample 3 is desired to be taken out of the gel 2, the unit 1 is pushed in the direction of the arrow A in such a manner, that the unit penetrates the gel 2 to the position presented in the middle picture, whereby the sample space 9 situated in the tip 5 of the unit contains the piece of gel 4. The tip 5 of the unit 1 may not be very thick at the very end, that is at the lower edge of the tip, i.e. the tip may not be very thick or blunt, because this would make the penetration of the tip into the gel 2 more difficult. Depending on the size of the unit and the material that it consists of, the thickness of the tip is in the range of 0.02 - 2 mm and 3 mm at the most, which is mentioned also further on. The tip 5 may be sharp, straight or rounded.

After the unit 1 is pushed into the gel 2, it is lifted up, whereby the piece of gel 4 stays inside the tip 5, where the sample space 9, inside, cf. the second picture farthest right. Before the unit 1 is lifted up from the gel 2, it may be moved slightly around its longitudinal axis (for example, 40 - 90 degrees), when the need arises, in such a manner, that detachment of the piece of gel 4 from the gel 2 becomes easier. This requires that the cross-section of the tip 9 is circular, which is not required in all of the embodiments of the device. The picture farthest right illustrates that the piece of gel 4 is easily detached from the unit 1 by squeezing with fingers, that is, with minor force at that end of the unit, which is near to the tip, whereby the piece of gel is released as whole.

When the unit 1 together with its membrane is prepared of elastomer material, which is recommended, the said squeezing for detaching the piece of gel 4 is especially easy. The elastomer material yields resiliently while squeezing it and it restores to its former state, when the outer force focused on it is withdrawn. As an example of an especially recommended elastomer material is silicone rubber. Silicone rubber may be heated up to a temperature of +200 °C and kept in that temperature for long periods without causing any damage to it. Heating to a high temperature is important, when given biological materials are desired to be destroyed. Another advantage of silicone rubber is that it is very inert, that is, it practically does not react with other materials. Furthermore, it does not release components, that might inhibit the desired reactions. The unit 1 prepared of silicone rubber or other elastomer material may be used multiple times.

Instead of flexible material, the unit may be prepared of a material that is plastically, i.e. permanently, deformed, when an outer force is focused on it, whereupon the device is disposable. In manual use the unit needs to be easily deformed, that is, in consequence of a quite minor outer force, when it is squeezed. In consequence of what has been said, the wall thickness of the plastically deformable material has to be sufficiently small, preferably 0.02 - 2.0 mm, and more preferably 0.5 - 1.0 mm. The material may be aluminium, because of its quite low yield strength and plastic deformation happens with a minor force.

When the unit 1 and its tip 5 have been prepared of elastomer material, the recommended thickness of the material is 0.2 - 2 mm. The thickness of the material at the end of the tip cannot in practice exceed about 3 mm, as if the material is too thick, it breaks the gel 2 and cannot penetrate it evenly.

The unit 1 together with its tip 5 is, for reasons related to the technical manufacture, most preferably prepared to one single integrated unit.

In practice, the length of the unit 1 needs to be 10 - 300 mm and preferably 20 - 200 mm and more preferably 20 - 100mm. The shape of the cross-section of the unit 1 at the tip 5 may vary depending on the scope of application of the device. If the cross-section of the unit 1 at the tip 5 is cylindrical (cf. Figures 1 , 6 - 9), the inner diameter of the cross-section at the tip is 2 - 15 mm. If the cross-section of the tip 5 deviates from a cylindrical shape (cf. Figures 3 and 5), the tip includes a long diameter (L in Figure 3), which is 5 - 20 mm and a short diameter (I in Figure 3), which is at least 1 mm.

In the units of Figures 2 and 3, a corresponding index numbering has been used as for the corresponding parts in Figure 1. The cross-section of units 1 ' at the tip 5' in Figures 2 and 3 is rectangular. The length of the long diameter, L, is 5 - 30 mm and the length of the short diameter, I, is at least 1 mm. The short diameter, l, is preferably 0.5 x the long diameter L, at the most. Due to the fact that the unit includes the long diameter, which is manifold in its size compared to the short diameter, the volume of the piece of gel remains small, when the gel contains oblong sample zones.

In the units of Figures 4 and 5 a corresponding index numbering has been used as for the corresponding parts in Figure 1. The unit of Figures 4 and 5 corresponds to the unit of Figures 2 and 3, except that the tip 5" of the unit 1 " is bevelled. The bevelling forms, for example, an angle of 20 - 70 degrees, preferably 30 - 60 degrees, with respect to the longitudinal direction of the unit 1 ". Due to the bevelled tip 5" the tip may, when a gel having a very soft consistence is being cut (for example, moved around in the gel), function like a spoon upon leaning and detaching the unit from the gel by lifting. When the unit 1" is lifted out of the gel, that particular part of the tip 5", which reaches farthest, that is, the part 5a", is kept the lowest. In the units of Figures 6 and 7, a corresponding index numbering has been used as for the corresponding parts in Figure 1. The unit V" of Figures 6 and 7 has a circular cross-section. In addition, a shoulder 6'" is formed on the outer surface of the unit. The purpose of the shoulder 6'" is to form a support for the unit, when it is stored, but also to form a support, when the unit is handled, for example, in connection with taking a sample. The unit may be situated in an autoclavable storage, out of which the unit may be easily picked, e.g. for gel isolation. In this way one can make sure that the lower surface of the tip of the unit, which is in contact with the gel and the nucleic acids, is not contaminated. Further, at the shoulder 6", the inner space of the unit 1'" may receive a bar-like unit, cf. Figures 10 and 12. A shoulder 7'" is also formed at the lower end 12'" and the outer surface of the unit. The purpose of the shoulder T" is to form a support for the unit 1 '", when it is stored in its holder (not shown), including a hole for the unit. The diameter of the said hole is smaller than the greatest outer diameter of the shoulder 7'".

It is thinkable that either one of the shoulders 6'" and 7'", or both of the shoulders, is replaced by shaping the unit as a wedge, however in such a manner, that there would not be a wedge-shaped structure at the tip.

In the embodiment of Figures 8 and 9, a corresponding index numbering has been used as for the corresponding parts in Figure 1. The unit 1"" in Figures 8 and 9 deviates from the unit in Figures 6 and 7 with the distinction that the tip 5"" is bevelled. The purpose of the bevelled tip 5"" is partly the same as in the embodiment of Figures 4 and 5. In addition, due to the fact that the cross-section of the unit 1 "" in Figures 8 and 9 is circular, the unit may be rolled, when it is placed inside the gel. Upon rolling the lower part 5a"" of the tip 5"" cuts the bottom of the gel and the bevelled edge of the tip 5"" cuts a piece of gel out of the gel, which piece of gel may be lifted out of the gel by leqaning the unit 1 "" and lifting, that is, by using it like a spoon as is described in the embodiment of Figure 4 and 5. The unit in Figures 8 and 9 may include a shoulder at its lower end like in Figures 6 and 7, although this is not drawn. The lower edge of Figure 6 may be bevelled like in Figure 8.

It should be noted that in the embodiments of Figures 6 - 9 the circular cross-section is easily changed to an ellipse by pushing the unit lightly at the end, where the tip is situated. The advantage of the elliptic shape compared to the circular shape is, that an oblong sample can be picked in such a manner, that it is surrounded by a smaller amount of gel. The amount of chemicals solubilising the gel and needed for the purification of the sample may, respectively, be reduced and the melting time of the gel is shortened. Some preferred embodiments according to the invention may completely lack the membrane described in Figures 1 - 9.

A preferred embodiment of the device according to the invention, generally marked with the index number 100, is presented in Figure 10 for taking a sample 300 manually or in an automated manner, for example, out of the agarose gel 200. Figure 10 illustrates also the use of the device 100. The device 100 farthest left in the Figure is presented sideways from a distance to the gel 200 and the sample 300. The cross-section of the device 100 is preferably circular, but it may also deviate from a circular shape, whereupon it preferably includes an oblong hole at the tip, for example, such a hole that is presented above referring to Figure 4.

In the same way as the units in Figures 1 - 9, the device 100 of Figure 10 includes a flexible membrane 800, that sets limits to the sample space 900. The membrane 800 is prepared of silicone rubber or some other flexible material. Due to the fact that the device 100 includes a flexible membrane 800, the flexible tip 500 of the device does not need to consist of flexible material. Also due to the flexible membrane, the upper end of the device does not need to be, but it may be, open at the top. The device 100 includes a bar-like organ 1000 consisting of magnetic material adapted inside of it, which organ is adapted to be moved in the longitudinal direction of the device. The bar-like organ 1000 is one part of a tool, which is adapted to be supported by the conical portion on the inner surface of the upper end 1100 of the device 100. When the upper end 1100 of the device 100 consists of flexible material, the said tool is easily and well attached to the device 100. The tip 500 of the device 100 may, as distinct from Figure 10, be bevelled like in Figures 4 and 8.

In the farthest left picture in Figure 10 the bar-like organ 1000 is adapted to push the flexible membrane 800 in such a manner, that the sample space 900 is diminished. The second picture from the left illustrates pushing the device 100 into the gel 200, while the bar-like organ 1000 is in a position, where it stretches the flexible membrane 800. Upon pushing the device 100 downwards the bar-like organ 1000 is at the same time let to move upwards in such a manner, that a vacuum is formed inside the sample space 900 and the tension, that the bar-like organ is focusing on the membrane 800, is decreased. When the tip 500 of the device 100 is pushed to the bottom of the gel 200, the bar-like organ 1000 is typically in a position, where it does not stretch the flexible membrane 800 and there is a vacuum inside the sample space 900, which vacuum helps to keep the piece of gel 400 in the sample space, when the device 100 is lifted out of the gel 200, cf. the upper picture to the right. When the piece of gel 400 is desired to be removed from the device 100, the bar-like organ 1000 is pushed towards the flexible membrane 800, see the lower picture to the right.

Figure 11 illustrates the use of the device 100 in Figure 10 for taking a liquid sample. The use corresponds to what is presented in connection with Figure 10. Due to the vacuum, the liquid sample stays inside the device 100, although the device is lifted from the container, where the sample is taken, cf. the middle picture. When the sample is desired to be released from the device, the bar-like organ 1000 is pushed downwards. The tip of the device may then be soaked in the liquid, which is illustrated in the picture farthest right, or not. If the release of the sample is performed by keeping the tip of the device soaked in the liquid, the bar-like organ 1000 is kept in the said down-pushed position and the device is lifted up from the liquid.

Due to the fact that the bar-like organ consists of magnetic material or may be magnetised by other means, for example by means of electricity, magnetic particles may gather around the flexible membrane 800. This is very useful in the treatment of samples.

Figure 12 presents another recommended realisation of the invention, where liquid samples or samples of gel can be taken either manually or in an automated manner. The device 100' in Figure 12 includes a bar-like organ 1000' in the same way as the device in Figure 10.

The device 100' in Figure 12 deviates from the device in Figure 10 in that way, that the end 1200' of the device together with its tip 500' form an own unit, which may be detached from the rest of the device, see the picture farthest left. Due to the detachable end, the device may be used in multiple ways. The bar-like organ 1000' includes a magnet 1100' formed at its lower end, while the bar-like organ consists in other parts of non-ferromagnetic material. The magnet 1300' is a transversely magnetised permanent magnet, whereby magnetic particles may gather on its vertical edges around the flexible membrane 800'. The magnet may be magnetised along the longitudinal axis or transversely against the longitudinal axis ox in another way. The advantage of a transverse magnetisation is that a large number of magnetic particles may gather compared to a situation, where the magnet is vertically magnetised. It is thinkable that instead of a permanent magnet the device includes an electric magnet. The size and the shape of the magnets may vary case by case.

The index number 1400' refers to a sleeve-like part, which surrounds the bar-like organ 1000'. The sleeve-like part 1400' consists of ferromagnetic material. When the magnet 1300' is completely inside the sleeve-like part 1400', no magnetic field of any kind is caused outside of the sleeve-like part. The flexible membrane 800' separates the sample space 900' from the magnet 1300'.

The tip 500' of the device may, as distinct from Figure 12, be bevelled like in Figures 4 and 8.

When a sample is taken from the gel 200', the device 100' is first put to the position presented in the picture second farthest left, in which position the end 1200' is attached to the other parts of the device and the device is above the gel 200' and the sample 300' in it. The end may preferably consist of non-flexible material. The bar-like organ 1000' is pushed to a down position in such a manner, that the magnet 1300' stretches the flexible membrane 800' to diminish the sample space 900'. In the said position the magnet 1300' projects from the sleeve-like part 1400'.

Thereafter the device 100' is pushed into the gel 200' by moving the bar-like organ 1000' upwards at the same time. When the tip 500' of the device 100' is pushed to the bottom of the gel 200', the bar-like organ 1000' is completely inside the sleeve-like part 1400' and there is a vacuum inside the sample space 900', see the third picture from the right. Due to the vacuum, the piece of gel 400' together with its sample 300' stays inside the device 100', while the device is lifted up from the gel 200", see the second picture farthest right.

The piece of gel 400' is released from the device 100' by pushing the bar-like organ 1000' downwards, whereupon the flexible membrane is stretched, the sample space 900' is diminished and the piece of gel falls out of the device, for example, to a vessel, cf. the picture farthest right.

By means of the purification method based on the transfer of magnetic particles complex biological samples may be in many cases processed without a need for centrifugation. Magnetism is not a common property of biological material, whereby the use of magnetic particles is an excellent separation technology in combination with these materials. A new way of dispensing samples, magnetic particle technology and different purification chemistries may be combined in the present invention, thus, the method considerably simplifies and facilitates the current methods. Magnetic particle refers to a material, that contains a core, that consists of paramagnetic or superparamagnetic material and a polymer surrounding the core. A coating may be prepared on the outer surface of the polymer, which coating is able to form a complex together with the material of interest (to be purified or isolated). The polymer may be, for example, polystyrene, cellulose, agarose or silica. Para- or superparamagnetic particles do not have a magnetic field, but they form a magnetic dipole in the presence of an outer magnetic field. A magnetic particle as used herein refers also to particles containing ferromagnetic material. Magnetic particles may have different sizes and preferably they have a diameter of 0.5 - 100 μm. Magnetic particles may also be a combination of non-magnetic and magnetic particles.

It is clear for a man skilled in the art that the device according to the invention may, within the claims enclosed herein, differ in multiple ways from the details and design described in the examples above.

DESCRIPTION OF THE METHOD ACCORDING TO THE INVENTION

The invention describes a method and a device used in the method for dispensing a sample into an appropriate vessel, where the cells, bacteria, viruses, DNA, RNA, mRNA, protein or peptide in the sample may be further purified. For example, agarose gel, acrylamide gel, tissue homogenate, whole blood, plant homogenate or growth medium can be used as sample material. By means of the method and the device described in the invention, for example, nucleic acids, proteins or peptides may be especially efficiently isolated and transferred from the gel.

The method according to the invention is suitable for dispensing and transferring various complex biological samples: tissue, feces, whole blood, buffy coat, serum, saliva, cultured cells, plants, sperm, gels, yeasts, molds, fungi, gram-negative bacteria, gram positive bacteria and foodstuff.

By means of the methods and the device described in the invention, for example, the following components may be isolated and purified from complex biological samples: nucleic acids (DNA, RNA, mRNA), proteins, peptides, polysaccharides, lipids, viruses, bacteria, parasites, yeasts, eucaryotic cells and cell organelles.

The method described in the invention may be used immediately together with purification methods, that have been described earlier, such as, for example, methods based on the use of silica and chaotropes, in the purification of nucleic acids (Vogelstein B. and Gillespie D., (1979) Proc. Natl. Acad. Sci. USA 76, 615-619; Smith H.O., (1980) Methods. Enzymol. 65, 371-380; Yang R.C.-A. et al., (1980) Methods Enzymol., 65, 176-182; Chen C.W. and Thomas CA. Jr., (1980) Anal. Biochem. 101, 339-341 ; Marko M.A., et al., (1982) Anal. Biochem. 121, 382-387; Bowtell D.D.L., (1987) Anal. Biochem. 162, 463-465; EP 0 389 063; Kristensen T., (1987) Nucleic Acids Res. 15, 5507-5516; Zimmermann J., (1989) Methods Mol. Cell Biol. 1, 29-34; Boom R., et al., (1990) J. Clin. Microbiol. 28, 495-503; Yamada O. et al. (1990) J. Virol. Meth. 27, 203-210; Zeillinger R. et al., (1993) Biotechniques 14, 202-203). The now described method is a considerable improvement to the earlier described purification methods. By means of the method described in the invention, for example, weighing of the gels and the tubes may be completely ignored and duration of the purification may be considerably decreased. The pieces of gel that have been picked from the same gel by means of the device described in the invention, are very similar in their size. Because of this, the user does not need to determine the weight of the cut piece of gel in a time-consuming weighing step in order to calculate the right amount of reagents for the purification process. By means of the method and the device described in the invention the purification process may be considerably simplified and speeded up.

The method described above is suitable for use together with different gel types, for example, normal agarose gels, low melting agarose gels and acrylamide gels.

In the preferred embodiment of the invention, the DNA fragment to be purified is isolated from an agarose gel by means of the method and the device according to the invention and transferred to the vessel, where the purification is performed by means of the magnetic silica and the chaotrope. The use of chaotropes in melting gels and together with silica material brings about the attachment of DNA to the silica material.

The present method for taking and dispensing a biological sample suits especially well to be combined with a purification method, that is based on the transfer of magnetic particles from one vessel to another, as for example it is described in the publication US Patent 6468810. Combining the use of the method and the device described in the invention to the technology based on the transfer of magnetic particles is a very functional combination. There are no filters in the magnetic particle technology (e.g. spin columns) or chromatography columns, that would clog up because of impurities in the sample. Thus, particulate biological material (for example, residual cell membranes) and other disturbing "rubbish" does not cause any major problems in a purification method based on the magnetic particle technology. Technology based on the transfer of magnetic particles is a considerably faster method than the conventional magnetic particle technology (International Patent Publication WO 2004035217). Either conventional magnetic particle technology (a magnet outside of the vessel) or spin column technology make it possible to dispense the reagents beforehand for the purification process. The device described in the invention may also be used together with the conventional magnetic particle and spin column technology, whereby the methods are considerably speeded up. A particular picking tip is used in the present method, which tip has properties that make the handling of the sample easy and secure. The tip may in one embodiment be prepared of elastomer material, such as, for example, silicone rubber. Other possible elastomer raw materials are silicone rubber, natural rubber, polyurethane, fluoroelastomers, polychloroprene, chlorosulfonated polyethylene, nitrile elastomers and butyl elastomers. Silicone rubber is a very appropriate material because of its flexibility and good heat stability. The material may be baked in +160 - +200 °C for long periods of time and thereby destroy both RNases and DNases in the picking tip. Tips that are prepared of silicone rubber do not require a toxic treatment in order to remove RNases and the tips may be used several times. Silicone does not either release significant amounts of heavy metals or other, for example, components inhibiting the PCR.

Especially in the manual embodiment of the invention, even other alternatives for the material come into question: metals (for example, aluminium), polystyrene, polypropylene, polycarbonate, polysulfone, nylon, polyethylene, polyvinylechloride, polyvinylfluoride or other suitable material. The prerequisites for the material of the tip are as follows: one has to be able to shape the tip with fingers, that is, the material needs to be flexible in a way that is opposite to stiff material, that has no ability to change its shape. The material does not necessarily need to restore to its original state, but it may even stay in a squeezed state. When using this kind of materials, that do not restore to their original state, one needs to be much more careful. Many of the conventional materials do not endure temperatures high enough (> +160 °C) to destroy RNases simply by heating. A large number of plastic materials do not even endure autoclaving, that is needed to sterilise materials and destroy DNases.

The device according to the presented invention may be appropriately coated with different coatings (e.g. teflon), which introduce preferred properties depending on the intended use.

EXAMPLE 1. ISOLATION OF DNA FROM THE GEL

The device described in the invention may be used as a picking tip after gel electroforesis to detach the desired piece of gel (containing, for example, DNA, RNA or mRNA) out of the gel. The device is suitable for normal agarose and low melting agarose gels, TAE and TBE buffer gels as well as gels of various thickness.

After taking the device out of its storage, it is brought in an upright position above the gel in such a manner that the bottom of the tip faces the gel. Through the tip it is possible to target, for example, a DNA zone precisely in the middle of the inner diameter of the tip. The tip is pushed through the gel in a vertical position. Depending on the strength of the gel the tip may be turned around 45 - 90 degrees, when the need arises, folded downwards and both the tip and the piece of gel may be lifted up from the gel.

By means of the picking tip, a DNA fragment broader or longer than the inner diameter of the tip may be picked either by releasing the first piece of gel to a tube and taking the second piece of gel only after this, or by taking two or more pieces of gel one after another by means of the picking tip. By means of the picking tip, pieces of the same size are obtained, whereby the amount of the chaotrope to be used is derived directly from the number of pieces of gel. There is no limitation for the size of the piece of gel in purification, because the purification may be performed in different vessel formats.

Detaching the piece of gel comes off by pushing the picking tip with fingers slightly above the piece of gel inside of it. In a preferred embodiment a finger can be placed as a "plug" on the upper part of the device, whereby pushing the upper part of the tip together with fingers some pressure is formed inside the tip, which pressure drives the piece of gel out of the tip.

In the automated version, the detaching of the gel is performed by means of a specific bar and/or overpressure. The piece of gel comes unbroken out of the tip and is ready for further treatment. When dealing with very "soft" gels, it may be necessary to lean the tip to a horizontal position in order to make sure that the gel is kept entirely inside the tip during the lifting step.

Picking the piece of gel (incl. taking the piece of gel out of the geland removing it from the device) by means of the device described in the invention is about three times quicker than when cutting by means of a scalpel, thus, the damage caused by UV light decreases. When all the steps needed before the actual purification procedure are taken into consideration (picking from the gel, weighings, calculations), the now developed method is considerably quicker: 1 minute vs. 10 minutes (per 5 pieces of gel).

EXAMPLE 2. VARIATION IN THE SIZE OF THE PIECES OF GEL

The variability of the pieces of gel is minimised by means of the method descibed in Example 1 and weighing the tubes and the pieces of gel before further treatment is avoided. Because of the homogeneity of the pieces of gel picked by means of the method described herein, there is no need to calculate separately, piece by piece, the reagents used in the actual purification. In this experiment, pieces cut by means of a scalpel and by means of the device according to the invention were weighed and compared to each other. The pieces of gel were taken from a 1% low melting agarose gel (0.7 mm).

Table I presents the reproducibility of the pieces of gel as a function of the weight compared to the pieces of gel cut with a scalpel.

TABLE 1 Sample Weight of the piece of gel 3 vol chaotr. μi Cutting device (mg) 1 83 249 2 82 246 3 86 258 4 81 243 5 78 234 6 86 258 Picking tip 7 84 252 8 87 261 9 96 288 10 95 285

Mean 86 ± 6 257 + 17

Variation coefficient CV% 6,7 11 130 390 12 236 708 13 216 648 14 231 693 15 187 561 16 152 456 Scalpel 17 214 642 18 205 615 19 192 579 20 207 621

Mean 207 ± 25 591 + 101

Variation coefficient CV% 17

It is concluded as a result of the experiment that the variation in the weights of the pieces of gel (1 -10) picked by means of the device according to the invention is minor and therefore a predestined amount of the needed chaotrope, that has to present in a given volume in relation to the piece of gel, can be used while using the picking tip according to the invention. A sufficient amount of chaotrope for all the samples (1 - 10) picked by the device according to the invention is 300 μl. Respectively, this experiment shows that pieces of gel (1 1 - 20) cut by means of a scalpel, even by an experienced man skilled in the art, vary so dramatically, that adding the same amount of chaotrope in further treatment might cause great problems.

EXAMPLE 3. PURIFICATION METHOD FOR DNA When using the method described in the invention in connection with magnetic particle technology and especially with such a technology, where magnetic particles are transferred from a vessel to another, a very fast and efficient purification is achieved. The above mentioned facts together bring about the rapidity: a simple deattachment of the piece of gel, homogenious pieces of gel, standard amounts of reagents and a transfer method for magnetic particles render pre-dispensing of the all the needed solutions possible already while running gel electroforesis.

In this Example, DNA was picked from a gel by means of the device according to the invention and a further purification was performed by means of silica magnetic particles and the Pick-Pen 8-M™ (Bio-Nobile Oy, Finland) magnetic tool on a 96 well plate. By means of the Pick-Pen 8-M™, 8 samples may be treated simultaneously, i.e. the silica magnetic particles are transferred from one row to another (1 - 12) while the purification procedure proceeds.

The hBtk insert (1980 bp) included in the pBAT4 plasmid was amplified by means of PCR and the product was pipetted on an agarose gel (1% (w/v) low melting agarose gel in TEA buffer). The SybrGreen I dye was added to the samples in order to detect DNA.

During electroforesis, the solutions needed for the purification were pipetted as follows: To Eppendorf tube 1 was added 400 μl (3 vol) NaCIO solution

To the row 1 (wells A1-H1) of the well plate were added 20 μl silica magnetic particles (Chemicell, SiMAG/K-MP/1 , 200 mg/ml, 20% isoprop.)

To row 2 (wells A2-H2) of the well plate was added 750 μl wash buffer, which was A3-B3: 70% EtOH. A2-B2: 70% EtOH. C2-D2: 10% PEG, 1 M NaCI, 53% EtOH, E2-F2: 50 mM Tris-HCI (pH 7.2), 1 mM EDTA, 70% EtOH, or G2-H2: 20 mM Tris-CI (pH 7.5), 1mM EDTA, 100 mM NaCI, 58 % EtOH. To row 3 (wells A3-H3) of the well plate was added 750 μl wash buffer, which was A3-B3: 70% EtOH. C3-D3: 10% PEG, 1 M NaCI, 53% EtOH, E3-F3: 50 mM Tris-HCI (pH 7.2), 1 mM EDTA, 70% EtOH, or G3-H3: 20 mM Tris-CI (pH 7.5), 1mM EDTA, 100 mM NaCI, 58% EtOH. To row 4 (A4-H4) of the well plate was added 750 μl sterile water

To row 5 of the well plate (wells A5-H5) was added 100 μl TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA)

After electrophoresis the DNA fragments, 1980 bp in their size, were cut out of the gel by means of the device according to the invention. As shown in Example 2, all pieces of gel taken from a 1% agarose gel by means of the picking tip had weight sufficiently close to one another, so that a separate weighing step was not necessary.

The DNA fragments were picked from the gel by means of the picking tip and transferred to Eppendorf tube 1. The tube was incubated by mixing it now and then in a heat block at 55 °C, until the agarose had completely melted. The melted mixture of agarose and DNA was pipetted to row 1 of a 96 well plate (wells A1-H1).

The silica magnetic particles and the mixture of agarose and DNA were incubated for 5 minutes on a plate shaker at room temperature. The silica magnetic particles were collected from row 1 of the well plate (A1-H1 ) by means of the Pick-Pen 8-M™ magnetic tool and released to row 2 of the well plate (A2-H2). Silica magnetic particles (and bound DNA) were washed in wells A2-H2 for about 10 seconds by mixing the solution by means of the Pick- Pen 8-M™ magnetic tool.

The silica magnetic particles were collected by means of the Pick-Pen 8-M™ from the wells of row 2 of the well plate and transferred to the wells of row 3 (A3-H3). The treatment performed in row 2 was repeated in the wells of row 3.

The silica magnetic particles were collected by means of the Pick-Pen 8-M™ from row 3 of the well plate and transferred to row 4 (wells A4-H4). The collected particles were flushed for about 5 seconds without releasing the particles to the solution. Thereafter, Pick-Pen 8- M™ and the particles were transferred to the wells of row 5 (wells A5-H5) and the particles were released from the tip of Pick-Pen 8-M™ to the elution solution. The particles were incubated in the elution solution for 5 minutes in a plate shaker at room temperature and thereafter the particles were collected by means of Pick-Pen 8-M™ from the solution. Pure DNA is in row 5 (wells A5-H5).

Gel electroforesis (1% agarose gel in TAE buffer) was performed with the purified samples. A size standard (λ DNA - H/ndlll, Haelll - Digest Marker) was run on the gel together with the samples in order to estimate the size of the DNA fragment. The fragments were visualised with a fluorescent dye by using Dark Reader device and photographed.

Figure 13 shows an agarose gel with purified DNA fragments and the effect of different wash buffers on the purification result (well 1 : 70% ethanol was used as wash buffer, well 2: 10% PEG, 1 M NaCI, 53% EtOH, well 3: 70% EtOH, 50 mM Tris-HCI (pH 7.2), 1 mM EDTA, well 4: 20 mM Tris-HCI (pH 7.5), 1mM EDTA, 100 mM NaCI, 58 % EtOH. Well M: λ DNA - H/ndlll, Haelll - Digest Marker. Based on the size standard, the purified sample had the right size, the sample had not been broken and there was no contamination of RNA detected.

As chaotrope, instead of a natriumperchlorate (NaCIO₄) solution, for example, sodium iodide (Nal), potassium iodide (Kl), sodium chloroacetate (NaCIAc), guanidine isothiocyanate (GuHSCN) or guanidine hydrochloride (GuHCl) may be used. The concentration of the chaotrope needs to be at least 6 molar, when using 3 volumes of solution in relation to the piece of gel in order to achieve a final chaotrope concentration of 4.5 molar or above.

Alternatively the particles may be pre-added to the Eppendorf tube together with the chaotrope, whereby DNA is bound to the particles in proportion as it is released from the agarose, while it melts. It is good for the particles to be in suspension in order to maximise the binding of the sample. By combining the melting and the sample binding step the purification may be speeded up. The mixture of chaotrope and magnetic particles may also be pre-heated, whereby the melting of the agarose and, thus, the binding of the sample is accelerated. The use of warmth while binding the sample to the particles and also while eluting it from the particles increases the yield somewhat, so that it is also useful.

It is obvious for a man skilled in the art that, as the art develops, the leading idea of the invention may be realised in many different ways. Thus, the invention and its embodiments are not limited to the Examples described above, but they may vary within the claims.

Claims

1. A device for taking and releasing, such as dispensing, a sample, such as a biological sample, which device includes an oblong hollow unit, that contains a first end (11 , 11 ', 11 ", 11'", 11"", 1100, 1100') and an opposite second end (12, 12', 12", 12'", 12"", 1200, 1200'), that includes a wall section with a sample space (9, 9', 9", 9'", 9"", 900, 900') and a tip (5, 5', 5", 5'", 5"", 500, 500'), which is designed to be a picking tip for taking a sample, characterised in,

- that the above mentioned opposite second end (12, 12', 12", 12'", 12"", 1200, 1200') contains a flexible membrane (8, 8', 8", 8'", 8"", 800, 800'), which outlines the sample space (9, 9', 9", 9'", 9"", 900, 900') and which is adapted to yield by changing its shape, when an external force is focused to it, in order to push the sample out of the device, and that a magnetic, bar-like organ (1000, 1000') is fit inside the oblong hollow unit, which organ is adapted to be moved along the length of the oblong unit in order to obtain the above mentioned external force.

2. A device according to claim 1, characterised in, that the flexible membrane consists of elastomer material.

3. A device according to claim 2, characterised in, that the flexible material consists of silicone rubber.

4. A device according to claim 1, characterised in, that the said opposite second end (12, 12', 12", 12'", 12"", 1200, 1200^*) is longer than the first end (11, 11', 11", 11'",

11 "", 1100, 1100') in its diameter.

5. A device according to claim 1, characterised in, that there is a shoulder (6'", 6"", 600, 600') in the first end.

6. A device according to claim 1, characterised in, that the tip (5", 5'") is bevelled.

7. A device according to claim 1, characterised in, that the length of the oblong unit is 10 - 300 mm and that its wall thickness in the said second end (12, 12', 12", 12"', 12"", 1200, 1200') is 0.02 -3 mm.

8. A device according to one of the previous claims, characterised in, that the cross-section of the device (1 '", 1 "") by the tip is cylindrical and that the diameter of the cross-section at the tip is 2 - 15 mm.

9. A device according to one of the claims 1-8, characterised in, that the cross- section of the tip of the device (1'", 1"") deviates from a cylindrical shape, whereby the tip contains a long diameter (L) and a short diameter (I), the long diameter being 5-20 mm and the short diameter being at least 1 mm.

10. A device according to claim 1, characterised in, that the oblong unit surrounds the bar-like organ (1000, 1000') essentially without clearance in such a manner, that the bar-like organ and the oblong unit form an arrangement, which is like a combination of a piston and a cylinder.

11. A device according to claim 1, characterised in, that the bar-like organ (1000, 1000') is adapted to stretch the flexible membrane (800, 800'), when it moves towards the tip of the oblong unit (500, 500').

12. A device according to claim 1, characterised in, that the flexible membrane (800') separates the sample space (900') from the magnet (1300') and that the magnet is adapted to be moved inside the oblong unit towards the tip in such a manner, that it stretches the flexible membrane (800').

13. A device according to claim 12, characterised in, that the bar-like organ (1000') contains a non-ferromagnetic part, that the magnet (1300') is transversely magnetised and that it is attached to the non-ferromagnetic part.

14. A device according to claim 13, characterised in, that the bar-like organ (1000') is surrounded by the ferromagnetic, sleeve-like part (1400') and that the magnet of the bar- like organ (1300') is adapted to be moved from the first position, where it is inside the sleeve-like part, to a second position, where it projects from the sleeve-like part.

15. A device according to claim 1 , characterised in, that its second end (1200') together with its tip (50') is a unit that is detachable from the first end (1100').

16. A method for taking and dispensing a biological sample from a gel for purification, characterised in, that a device is transferred above the gel, which device contains an oblong hollow unit, that includes a first end (11, 11', 11", 11'", 11"", 1100, 1100') and an opposite second end (12, 12', 12", 12"', 12"", 1200, 1200'), that includes a sample space (9, 9', 9", 9'", 9"", 900, 900') and an open tip (5, 5', 5", 5'", 5"", 500, 500') for taking a sample, - the hollow unit is pushed into the gel in such a manner, that the piece of gel containing the sample to be picked is getting transferred to the sample space (9, 9', 9", 9'", 9"", 900, 900'), the hollow unit is detached from the gel together with the piece of gel, the device is transferred to the sample treatment vessel, and - the piece of gel is removed from the hollow unit by focusing an external force to the vicinity of the tip of the device in order to push the piece of gel out of the device.

17. A method according to claim 16, characterised in, that the biological sample is DNA, RNA, mRNA, a fragment of nucleic acid, protein or peptide separated by means of gel electrophoresis.

18. A method according to claim 16 or 17, characterised in, that the gel consists of agarose, low melting agarose or acrylamide.

19. A method according to claim 18, characterised in, that the piece of agarose gel containing the DNA fragment is treated with a chaotrope: Nal, Kl, NaCIO₄, NaCIAc, GuHSCN or GuHCI.

20. A method according to one of the claims 16-19, characterised in, that the hollow unit of the device consists of elastomer material, whereby the said external force is focused to the wall of the hollow unit.

21. A method according to claim 20, characterised in, that the elastomer material consists of rubber silicone.

22. A method according to claim 16, characterised in, that the device contains a bar-like organ containing a magnet adapted inside an oblong hollow unit, which organ is adapted to be moved along the length of the hollow unit and that the sample contains magnetic particles.

23. A method for purifying a biological sample from a gel, characterised in, that the sample is taken from the gel by means of a method according to one of the claims 16-22 and a further treatment is performed by means of a method based on the use of magnetic particles.

24. A method according to claim 22 or 23, characterised in, that the magnetic particles are silica magnetic particles.

25. A method according to claim 23, characterised in, that the reagents needed for the purification are added or they are pre-added to the vessels, where the biological sample is transferred.

26. A method according to claim 23, characterised in, that the magnetic particles are washed with ethanol or a buffer containing ethanol.

27. A method according to claim 26, characterised in, that ethanol is washed away from the particles by bringing the particles into water or a dilute buffer for 1 - 60 seconds without releasing the particles to the solution.

28. A method according to claim 27, characterised in, that the magnetic particles and the bound nucleic acid is finally brought into water or a dilute buffer and the magnetic particles are released to the solution for the duration of the elution step.

29. A method according to claim 28, characterised in, that the magnetic particles may, when the need arises, be removed from the solution.