DE3717211A1 - DEVICE AND METHOD FOR SEPARATING AND CLEANING MOLECULES - Google Patents

Abstract

In a device for separating and cleaning molecules by adsorption of the molecules on a matrix (2), the solution is forced through the device. The matrix (2) is arranged, between two devices (3a, 3b) permeable to the solution, in a hollow frustum-shaped body (1) open at both ends (4a, 4b), the wider opening (4a) being connected, if necessary, to a cylindrical hollow body. The matrix (2) and the devices (3a, 3b) together occupy 5 to 50% of the volume of the hollow body and the wider opening (4a) of the hollow frustum-shaped body (1) can be connected to a pipette. The use of the device in a process for separating and cleaning molecules on a matrix is also described.

Description

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

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 an on device, which is characterized in that the Ma trix 2 in a at both ends 4 a , 4 b open, frustoconical hollow body 1 , to which a cylindrical hollow body connects to the further opening 4 a, if necessary , is arranged between two devices 3 a , 3 b which are permeable to the solution, the matrix 2 and the devices 3 a , 3 b together filling up 5 to 50% of the hollow body volume and the further opening 4 a of the frustoconical hollow body 1 being connectable to a pipette is.

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 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 . There is preferably 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 further opening 4 a of the two openings 4 a and 4 b is designed so that they pass on a standard 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 commercially available from specialist dealers (Brand, Gilson, Eppendorf). 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, hydroxyapatite, dextran, agarose, acrylamide, polystyrene, polyvinyl alcohol or other organic polymers, derivatives or 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 Be 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, for example also by polar groups on the surface of the matrix 2 , but with 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 refinements 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, Po lystyrene, polyurethane, etc. exist. But also anorga African 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.

In a particular embodiment, the method according to the invention is carried out with a hollow body 1 configured as a pipette tip. The molecules containing, to be separated and purified molecules can then 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 hose.

The inventive method is suitable to NEN tren in particular biopolymers such as nucleic acids and proteins and purify by 2 Ionenaustau scherchromatographiematerialien as a matrix, as proposed in the interpreting rule Patent Application P 36 39 949.3 who are to be used. It is silica gel-based chromatography material modified on the surface with anion exchange groups. If, for example, 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 the analysis of the long-chain nucleic acids, after a few washing steps you can elute the long-chain nucleic acids again 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, while 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 elute nucleic acids such as tRNA, 5S RNA and mRNS. At low ionic strengths from 0.1 to 0.5 polysaccharides, proteins, metabolites, Dyes, individual nucleotides, proteins such as BSA (bo vine serum albumin) and smaller nucleotides as in for example a nucleotide decamer that acts as a "left" is used, elutes.

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 method according to the invention ensures the fol 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 device according to the invention is suitable for ver in a separation and purification process for Molecules, preferably biopolymers such as proteins and / or nucleic acids, especially from other cells share.

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

example 1

In a 1 ml pipette tip, suitable for Gilson Pipet one becomes a 70 µm polyethylene frit with a 4 mm diameter knife, thickness 1.6 mm, inserted and pressed clamped. Then approx. 70 mg of the Chromatogra phiematerials, proposed in the German patent message P 36 39 949, filled in dry. The Chromato graphic material is made with a as described above Frit with 5.7 mm diameter closed and the frit fixed in place by pressing.  

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 harvested the next day by centrifugation. The pellet is disrupted in 85 ul of an ice-cold solution of 50 mM Tris-HCl, pH 7.4 with 2 mg / ml lysozyme (freshly prepared) and incubated on ice for 10 minutes. Then 20 µl of a 0.5 M EDTA solution are added and incubated for a further 10 minutes. After adding 4 µl of 2% Triton X '100, incubate on ice for a further hour. The sample is centrifuged in a laboratory centrifuge for 30 minutes at maximum speed and 100 μl of the cell lysate freed from cell debris are transferred to another Eppendorf tube, after which 1 volume of 2 M sodium chloride, 100 mM MOPS, pH 7 and 5 μl Isoamyl alcohol can be added. A pipette tip according to the invention from Gilson, Brand, Eppendorf, manufactured according to Example 1, is equi-librated with 100 μl buffer C. Then 100 .mu.l of an E. coli plasmid DNA sample is adsorbed on the chromatography material by pipetting the sample up and down five times. After adsorption, washing is carried out with a total of 2 to 5 ml of buffer C, whereupon the plasmid DNA is eluted using 300 μl of buffer F. The DNA is then precipitated with 300 μl of isopropanol, left to stand for 15 minutes and the sample is then centrifuged in an Eppendorf 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 portions of 250 mM MOPS, 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 centrifugation of the sample in an Eppendorf centrifuge for followed by 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 (14)

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 ), to which, if necessary, at the further opening ( 4 a ) a cylindrical hollow body closes, is arranged 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 volume of the hollow body and the further opening ( 4 a ) of the frustoconical hollow body ( 1 ) can be connected to a pipette. 2. Device according to claim 1, characterized in that the hollow body ( 1 ) is a pipette tip. 3. Device according to one of claims 1 or 2, characterized in that the devices ( 3 a , 3 b ), the Ma trix ( 2 ) fix 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. A method for separating and purifying molecules from a solution by adsorbing the molecules onto a matrix by means of a device in which the solution containing the molecules to be separated is pressed through a matrix arranged in a hollow body, characterized in that the matrix ( 2 ) is fixed by an upper and a lower device ( 3 a , 3 b ) and that the solution is sucked through the matrix ( 2 ) into a free space above the upper device ( 3 a ) and then through the Matrix ( 2 ) is pressed out of the hollow body ( 1 ). 11. The method according to claim 10, characterized in that Biopolymers are separated and cleaned. 12. The method according to claim 11, characterized in that Nucleic acids and / or proteins separated and purified will. 13. Use of a device according to one of claims 1 to 9 for the separation and purification of molecules, preferably wise biopolymers. 14. Use of a device according to claim 13 for doors Cleaning and purification of proteins and / or nucleic acid ren.