DE3717211C2 - - Google Patents

Description

The invention relates to a device for the separation and purification of molecules from a Solution by adsorbing the molecules onto one in the Device arranged matrix and the use of this device.

For sample preparation in chemical, biochemical and molecular biology-oriented laboratories Chromatography materials of all kinds long used in a proven manner. Often fall there for the samples on a micro scale, especially in the bio chemical and molecular biological, but also in ana lytic-chemical laboratory. The chromatographi materials are also used for the extraction of be agreed components from the samples. The Procedure to be referred to as solid phase extraction You can also use it to clean and separate Use substance mixtures in solution. So far were used in addition to the chromatography columns, especially in high pressure liquid chromatography, also Kartu disposable syringes or small plastic chromatographs phy pillars, each with the desired chromato graphic material are used.

These aids that are used in the low pressure range find, are disadvantageously characterized in that not with established laboratory equipment are compatible. Another disadvantage of this aid tel is that it is only one direction of flow of the the molecules to be separated and cleaned Allow solution. So you have to go with the procedure according to the prior art, the sample is first placed in a Storage vessel, for example a disposable syringe, brin conditions to them by the flux, in particular Cartridges or extraction columns.  

This is why these tools are used in practice mostly when you get the unwanted products wants to adsorb. Otherwise you have to use the storage container, for example, remove the disposable syringe with a the adsorbed substance eluting solution again send and then push through the tool, to get the products you want. By the Ab take and put on a storage vessel each but a risk of contamination for the employee who Environment and the sample itself. The danger is just now in the medical-technical field, if in particular working with infectious material, or in biochemical-molecular biological laboratories, if especially handling radioactive substances.

In practice there is a considerable need for one Device that allows a simple Process for the purification and separation of molecules to carry out a solution.

The object of the invention is therefore a device to provide, which enables

• - extract molecules from a solution,
• - No preferred extraction or elution direction to demand,
• - a separation and re-addition of a supply to avoid vessel
• - the separation and cleaning operations in one closed system,
• - the risk of contamination for personnel, the environment and Avoid sample and
• - At the same time to facilitate the overall working method tern.

The object is achieved by a pre direction according to claim 1. Describe the subclaims preferred embodiments of the invention Contraption.

DE-A 32 11 309 relates to a device for carrying out chromatographic separations in cylindrical Hollow bodies run off. With this so-called Column chromatography is there for a reasonable separation performance required the flow rate of the To keep eluent as constant as possible. Through the pointed end of the device according to the invention however, this is not possible. Furthermore, it is an essential requirement, the dead volume in the Keep the pillar as small as possible. No way In the event, however, it is possible to use only 5 to 50% of the chromatography column with the matrix intended for separation to fill. Furthermore, it is in column chromatography not known to connect pipettes to the columns.

Beythien / Diemair, laboratory book der Lebensmittelchemiker, 8th edition (1963), pp. 62-64, describes column chromatography (column filtration) (see Fig. 14, page 64). That made for DE-A 32 11 309 applies mutatis mutandis, since it is also necessary for separation here is constant flow conditions of the eluent to guarantee.

The catalog "Technical specialties, apparatus, instruments and preparations for laboratory and operation ", Fa. Reichelt Chemietechnik GmbH & Co. (1979), pp. 91-93 a large number of chromatographic accessories can be removed, however, without exception for use in column chromatography. The items offered there can the disadvantages inherent in the prior art do not eliminate.  

DE-A 32 11 309 also relates to a method with which Mixtures of substances can be broken down into their components. As with all column chromatography procedures, be it HPLC, FPLC, MPLC or simple column chromatography - and only these procedures are affected here (see page 5, description of Figure 1) - is the sample abandoned on the column head, and without changing the The mixture flows through in the direction of flow with separation the pillar. However, in the method according to the invention a reversal of the direction of flow of the solvent indispensable in order to at all the one wanted according to the invention Purpose, namely simple separation of a certain one To achieve component from a mixture.

Figs. 1 and 2 schematically show the construction of a preferred embodiment of the device according to the invention before. The matrix 2 is delimited by two devices 3 a, 3 b, which simultaneously ensure that the matrix 2 is fixed in the selected position. The devices 3 a, 3 b are preferably clamped to the wall of the hollow body 1 . However, in addition to fastening by tension, fastening by gluing the devices in place can also be achieved. Both methods lead to a sealing effect between the devices 3 a, 3 b and the wall of the hollow body 1 . Preferably, there is a free space between the lower device 3 b and the narrower opening 4 b, this free space should be small compared to the total volume of the hollow body. The opening 4 a of the two openings 4 a and 4 b forming the base of the truncated cone is designed such that it fits on a commercially available pipette. A preferred embodiment of the device according to the invention is that the hollow body 1 is designed in the form of a pipette tip that is commercially available in specialist shops. The matrix 2 of the device according to the invention preferably consists of a porous chromatography material, in particular based on silica gel, aluminum oxide, titanium dioxide, hydroxylapatite, dextran, agarose, acrylamide, polystyrene, polyvinyl alcohol or other organic polymers, derivatives or from copolymers of the above-mentioned carrier materials. The material forming the matrix 2 can also be a surface-modified, porous chromatography material based on silica gel, aluminum oxide, titanium dioxide, hydroxyapatite, dextran, agarose, acrylamide, polystyrene, polyvinyl alcohol or other organic polymers, derivatives or from copolymers of the above-mentioned carrier materials.

The choice of the appropriate chromatography material depends on the particular molecules to be separated and purified and is known to the person skilled in the art on the basis of the rules in chromatography. For example, a polar molecule, if it is to be adsorbed on the matrix 2 , is only adsorbed on the surface of the matrix 2 if corresponding interactions are present, which are also, for example, due to polar groups on the surface of the matrix 2 , but with the opposite Polarity. For example, ion exchange materials can be used as matrix 2 of the device according to the invention for the separation and purification of polar molecules. Further preferred configurations of the device according to the invention can contain a reversed-phase and / or an affinity chromatography material as matrix 2 . The pore size of the porous chromatography materials is preferably 20 to 1000 nm, and the grain size of the materials in particular 10 to 2000 microns, preferably 75 microns to 125 microns.

The devices 3 a, 3 b fixing the matrix 2 are preferably porous frits with pore sizes of 10 μm to 1 mm, preferably 70 μm to 2000 μm.

The porous frits can be made of plastics, in particular made of Teflon (PTFE), polyethylene, polypropylene, polystyrene, Polyurethane, etc. exist. But also inorganic Materials such as glass and / or sintered metal are suitable materials for the production of porous Fries.

The advantage of the device according to the invention lies in the practical handling and the type of packing of the matrix 2 , which allows the solution containing the molecules to flow in the back and forth direction. If the frustoconical hollow body 1 is formed, for example, as a pipette tip, the piquette tip is immersed in the sample, the sample is sucked up through the matrix 2 in the pipette tip and then pressed out. This achieves a higher efficiency of adsorption, since the sample passes through the chromatography material twice. Since you can work with a closed system, contamination of the sample or danger to the environment is avoided. The device according to the invention facilitates the overall method of operation which is to be used in the separation and purification of molecules. In particular, with the aid of the device according to the invention, biopolymers, in particular nucleic acids and proteins, can also be fractionated quickly, easily and safely.

The device according to the invention is suitable for use in a separation and purification process for Molecules, preferably biopolymers such as proteins and / or Nucleic acids, especially from other cell components.

The device according to the invention can advantageously be used in a method for separating molecules. The molecules containing, to be separated and to be purified can be conveyed through the matrix 2 by means of a pipette. If the pipette tip is to be used as a column replacement in conventional processes, the pipette tip can also be connected to a disposable syringe using a silicone tube.

The device according to the invention for the separation and purification, in particular of biopolymers, such as nucleic acids and proteins, preferably contains 2 ion exchange chromatography materials as matrix, as are proposed in the German patent application P 36 39 949.3. It is silica gel-based chromatography material modified on the surface with anion exchange groups. For example, if the matrix 2 is in a pipette tip, the nucleic acid is sucked through the matrix 2 with the pipette. The long-chain nucleic acids are adsorbed using buffers of low ionic strength. If you then want to utilize the short-chain nucleic acids, you push the solution back through the matrix 2 and catch it. Then further procedural steps can follow. However, if you are interested in analyzing the long-chain nucleic acids, you can elute the long-chain nucleic acids again after a few washing steps by elution with buffers with a high salt concentration (high ionic strength) and process them accordingly.

Fig. 3 shows an elution profile dung at USAGE of anion exchange materials for the USAGE dung of the device according to the invention can be obtained. It can be seen that when using the material proposed for the separation of nucleic acids in German patent application P 36 39 949.3, an elution profile is obtained by which nucleic acids with increasing chain length become eluent with increasing ions. Double-stranded DNA with base pairs <500 is only obtained with an ionic strength of 1.3 M sodium chloride, whereas single-stranded DNA of phage M 13 is eluted with 1.1 M sodium chloride. The elution peaks are so sharp that even "baseline" separations are possible. With an average ionic strength between 0.5 and 1 M sodium chloride, nucleic acids such as tRNA, 5 RNA and mRNA elute. At low ionic strengths of 0.1 to 0.5, polysaccharides, proteins, metabolites, dyes, individual nucleotides, proteins such as BSA (bovine serum albumin) and smaller nucleotides such as, for example, a nucleotide decamer, which is used as the "left", are eluted .

One method variant is to use an affinity adsorbent as matrix 2 , as is proposed, for example, in German patent application P 36 27 063. The procedure is carried out analogously to the procedure described above.

The use of the device according to the invention in Separation process ensures the following advantages, especially in molecular biological operations:

• - Implementation of DNA preparations in so-called "mini creps",
• - Purification of mRNA, rRNA, viral RNA and RNA train scripts,
• - Cleaning samples after nick translation, end label or oligonucleotide-induced label tion (oligolabeling) can be obtained,
• - Removal of the DNA linker in cloning experiments ten,
• - Purification of nucleic acids after gel extraction as well
• - Fast cleaning of nucleic acids after digestion with restriction enzymes, phosphatase treatments, Polymerase reactions etc. instead of a time consuming one phenol treatment.

When using the device according to the invention available purity in routine preparations is at least least with cesium chloride density gradient centri fugation obtained samples comparable. The isolated Nucleic acids are free of proteins, polysacchari the and other cell metabolites and show none Inhibition of enzymes in enzymatic reactions. The device according to the invention can be used for insulation and purification of nucleic acids from oligonucleotides down to plasmids are used, the time expenditure on fractions compared to other processes is restricted.

The use of the device according to the invention in Different methods are used in the following examples explained in more detail. The inventive Device in a preferred embodiment, the Pipette tip used.

example 1

A 70 µm polyethylene frit is placed in a 1 ml pipette tip with 4 mm diameter, thickness 1.6 mm and clamped by pressing. Then approx. 70 mg of the chromatography material proposed in the German patent application P 36 39 949, filled dry. The chromatography material is used with a like Sealed frit with a diameter of 5.7 mm and the frit by pressing on her Fixed space.  

The same procedure is followed for a 200 µl pipette tip zen or 5000 µl pipette tips.

Example 2

General handling of those prepared in Example 1 The pipette tip happens as follows:

First the pipette tip is filled with 100 µl egg equilibrated nes suitable adsorption buffer. At a sample volume to be processed from 100 to 200 µl is sufficient, the sample through the chro about five times to press matography material. But should be a size re amount of liquid to be processed, as in for example after gel elution, the sample can by means of a silicone tube that holds the pipette tip and connects a disposable syringe through the matrix be pulled through. It is then sufficient that the Sample only passes the chromatography material twice. The flow rate of the solution should not be be higher than 1 ml per minute.

As an equilibration and adsorption buffer, the Buffer A can be used.

Buffer A:
400 mM sodium chloride
15% ethanol
50 mM MOPS (3-N-morpholinopropanesulfonic acid)
1 mM EDTA (ethylenediaminetetraacetate)
pH 7.0

Analogous to the adsorption process described above performed the washing steps. Serve as a wash buffer in the case of nucleic acid preparations, typically the buffers B, C and D.

Buffer B:
750 mM sodium chloride
15% ethanol
50 mM MOPS
1mM EDTA
pH 7.0

Buffer C:
1000 mM sodium chloride
15% ethanol
50 mM MOPS
1mM EDTA
pH 7.0

Buffer D:
1000 mM sodium chloride
50 mM MOPS
pH 7.0

An Eppendorf vessel is filled with 1 ml of washing buffer and rinses 150 µl three times through the chromatography material, to remove contaminants. This step will repeated.

The adsorbed nucleic acids are eluted as in both previous steps either with a Eppendorf or Gilson pipette or with a disposable syringe, taking care that the pipettes tip with a silicone tube with the disposable syringe is connected. It just becomes the elution puff fer E or F

Buffer E:
1100 mM sodium chloride
15% ethanol
4 M urea
50 mM MOPS
1mM EDTA
pH 7.0

Buffer F:
1500 mM sodium chloride
15% ethanol
50 mM MOPS
1mM EDTA
pH 7.50

sucked or pressed through the pipette tip. This Step is repeated once. The eluates are ver agreed and the nucleic acid precipitated from it.  

Example 3

A pipette tip, manufactured according to Example 1, is hydrated with 50 mM sodium phosphate buffer and equilibrated by sucking 750 μl buffer several times through the pipette. A sample containing 20 µg plasmid pBR322 DNA in 500 µl 0.5 M sodium chloride and 50 mM sodium phosphate, pH 7 , is adsorbed onto the chromatography material by sucking it up five times and squeezing it out. Unbound constituents and impurities are washed out five times by sucking up and squeezing out 1 ml of fresh 0.5 M sodium chloride and 50 mM sodium phosphate, pH 7. The plasmid DNA is then eluted with 1.5 M sodium chloride and 50 mM sodium phosphate, pH 7, by sucking up and expressing 1 ml of the elution buffer three times.

Example 4

A plasmid DNA sample is in a so-called mini preparation typically by following the steps won:

The bacterial cells are incubated overnight and on harvested by centrifugation the next day. The pellet is dissolved in 85 µl of an ice-cold solution of 50 mM Tris HCl, pH 7.4, with 2 mg / ml lysozyme (freshly prepared) disrupted and incubated on ice for 10 minutes. After that 20 µl of a 0.5 M EDTA solution are added and incubated for a further 10 minutes. After adding 4 µl 2% Triton X ′ 100 is incubated on ice for a white tere hour. The sample is placed in a laboratory centrifuge 30 Centrifuged at maximum speed for minutes, and 100 µl of cell lysate freed from cell debris into another microcentrifuge tube (so-called Eppendorf tube) transferred, after which 1 volume of 2 M sodium chloride,  100 mM MOPS, pH 7, and 5 ul isoamyl alcohol added will. A pipette tip according to the invention manufactured according to Example 1, is equi with 100 ul buffer C. librated. Then adsorb 100 µl of an E. coli Plasmid DNA sample on the chromatography material by the sample is pipetted up and down five times. After Adsorption is carried out with a total of 2 to 5 ml of buffer C washed, whereupon the plasmid DNA using 300 ul of Buffer F is eluted. Then you drop the DNS with 300 µl isopropanol, let stand for 15 minutes and zen the sample is then centrifuged in a laboratory centrifuge For 30 minutes.

Example 5

Isolation of mRNA, rRNA and viral RNA from Roh extracts happens as follows:

The RNA extract is precipitated with ethanol and this is obtained tene pellet in TE buffer (10 mM Tris / 1 mM EDTA, pH 7.5) resuspended. One sets the adsorption conditions by adding 0.1 part by volume (based on the right suspended solution) 5 M sodium chloride and 0.2 vol portion 250 mM MOPS to pH 7.0. The pipette tip is equilibrated with 1 µl buffer A. After that the Sample by pipetting up and down five times on the Chromatography material adsorbed. You wash with puf fer A to eliminate contaminants. The elution is done with 300 µl of buffer C. The RNS is through Add 2.5 parts by volume of ethanol. According to Ste Leave at -20 ° C, 30 minutes, in one step village centrifuge for a total of 30 minutes at maximum speed centrifuged number. The pellet is pre-processed work carefully washed with ethanol. The sample should have a nucleic acid content of at most 15 µg ha ben.  

Example 6

Purification of mRNA to synthesize the corresponding cDNA is carried out as described in Example 5.

Example 7

Triphosphates not used in labeling reactions such as nick translations, end marks and oligonucleo tid-dependent labeling (oligolabeling) of DNA are removed as follows:

The labeling reaction of the DNA is carried out by adding 2 µl 0.5 M EDTA per 20 µl sample volume ended, and man sets the adsorption conditions by adding 1 Volume part of the buffer C. The total salt end con concentration should be about 500 mM. The pipettes tip according to the invention is with 100 ul buffer B equilibrated. The sample is raised five times conditions and expressions on the chromatographic material adsor beer and washed with a total of 10 ml of buffer B, to separate the unreacted nucleotides. The Flow rate of the wash buffer should preferably about 5 ml per minute. The highlighted DNS is then with a total of 300 µl of the buffer F elu iert. The sample can be used directly, if with the buffer intended for hybridization is diluted at least 1:20. Otherwise the Sample precipitated and dissolved in TE buffer.

Example 8

Removing a DNS linker from a DNS with more as about 400 base pairs in a cloning experiment is carried out as follows:  

The sample to be cleaned is mixed with 1 volume of the puf fers C diluted so that the final concentration of salt is about 500 mM. The pipettes according to the invention tip is equilibrated with 100 µl of buffer A. The Sample is adsorbed as in the previous examples beer. The pipette tip according to the invention is included Buffer B flushed to remove contaminants. The DNA is desorbed and eluted with a total of 300 µl of buffer F. The DNA is then isolated by isopropanol precipitation (addition of 1 part by volume) and leaves on ice for 15 minutes, after which there is a zen centrifuge the sample in a centrifuge for the duration of 30 minutes. Then you wash carefully with 70% ethanol.

Example 9

The quick cleaning of a DNA sample after enzymatic Modification (for example by phosphatase, restriction endonuclease, Polymerases etc. as well as cofactors) can be achieved according to the following procedure:

The underlying reaction volume is 50 µl and contains no more than 100 mM sodium chloride. 5 µl 0.5 M EDTA, pH 8.0, are added to the reaction volume added to cancel the modification reaction. A Volume part of buffer C is added to the adsorp conditions with a salt concentration tion of about 500 mM. The pipettes according to the invention tip is equilibrated, the sample is adsorbed and then eluted as described in Example 8. The further treatment of the sample is done as in Example 8 described.  

Example 10

The device according to the invention can also be used for this to agarose and acrylamide impurities efficient separation. This is especially necessary This is the case if gel elution of the sample has taken place ben. When using the Ver the DNA is concentrated at the same time, even if the starting volume is a few ml. The DNS has a size of <400 base pairs and can be combined in one Amounts of 5 ng to 15 µg are available. The sample preparation device and the equilibration of the Pi according to the invention pette tip is as described in Example 8 by guided. Then you transfer the sample to a disposable syringe and pushes it twice through the fiction moderate pipette tip. The flow speed should be speed are around 250 µl / min. The invention Pipette tip is then covered with 5 ml of buffer B washed at a flow rate of 5 ml / min. The further treatment of the DNA sample happens as described in Example 8.

Claims (11)

1. A device for the separation and purification of molecules from a solution by adsorption of the molecules on a matrix, characterized in that the matrix ( 2 ) in a hollow body ( 1 ) ( 4 a, 4 b) open at both ends ( 4 a, 4 b), a cylindrical hollow body adjoins the opening ( 4 a) forming the base of the truncated cone, between two devices ( 3 a, 3 b) which are permeable to the solution, the matrix ( 2 ) and the devices ( 3 a, 3 b) together fill 5 to 50% of the hollow body volume and the opening ( 4 a) of the frustoconical hollow body ( 1 ) forming the base of the truncated cone can be connected to a pipette. 2. Device according to claim 1, characterized in that the hollow body ( 1 ) is a pipette tip. 3. Apparatus according to claim 1 or 2, characterized in that the devices ( 3 a, 3 b) fix the Ma trix ( 2 ) in the hollow body ( 1 ). 4. Device according to one of claims 1 to 3, characterized in that between the narrower opening ( 4 b) and the lower device ( 3 b) is a free space. 5. Device according to one of claims 1 to 4, characterized characterized in that the matrix of porous chromatogra Phi material based on silica gel, aluminum oxide, titanium dioxide, hydroxylapatite, dextran, agarose, Acrylamide, polystyrene, polyvinyl alcohol or others organic polymers, derivatives or from copolymers of the above-mentioned carrier materials. 6. Device according to one of claims 1 to 5, characterized characterized in that the matrix is a superficially modi  Chromatography material made from silica gel, alumi nium dioxide, titanium dioxide, hydroxylapatite, dextran, aga rose, acrylamide, polystyrene, polyvinyl alcohol or an their organic polymers, derivatives or from copoly meren of the above-mentioned carrier materials. 7. The device according to claim 6, characterized in that the superficially modified chromatography mate rial an ion exchanger, a reversed phase and / or is an affinity chromatography material. 8. Device according to one of claims 1 to 7, characterized characterized in that the pore size of the porous chroma tography materials 20 to 1000 nm and the grain size the materials 10 to 2000 microns, preferably 75 to 125 µm is. 9. Device according to one of claims 1 to 8, characterized in that the matrix fixing Einrich lines ( 3 a, 3 b) are porous frits with pore sizes of 10 microns to 1 mm, preferably 70 to 200 microns. 10. Use of the device according to one of claims 1 to 9 for the separation and purification of molecules, preferably Biopolymers. 11. Use of the device according to claim 10 for Tren Cleaning and purification of proteins and / or nucleic acid ren.