WO1988009201A1

WO1988009201A1 - Process and device for separating and cleaning molecules

Info

Publication number: WO1988009201A1
Application number: PCT/EP1988/000442
Authority: WO
Inventors: Metin Colpan; Ralf Piotrowiak
Original assignee: Diagen
Priority date: 1987-05-22
Filing date: 1988-05-19
Publication date: 1988-12-01
Also published as: DE3717211A1; DE3717211C2

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

Device and method for separating and cleaning

Molecules

The invention relates to a device and a method for separating and purifying molecules from a solution by adsorbing the molecules onto a matrix arranged in the device.

Chromatography materials of various types have long been used in a tried and tested way for sample preparation in chemically, biochemically and molecular biologically oriented laboratories. Frequently, the samples are obtained on a microscale, in particular in the bio-chemical and molecular biological, but also in the analytical-chemical laboratory. The chromatographic materials are also used to extract certain components from the samples. The procedure to be referred to as solid phase extraction can also be used to clean and separate substance mixtures in solution. Up to now, in addition to the chromatography columns, in particular in high-pressure liquid chromatography, cartridges, disposable syringes or small plastic chromatography columns, each filled with the desired chromatography material, have been used.

These tools, which are used in the low pressure range, are disadvantageously characterized in that they are not compatible with established laboratory equipment. Another disadvantage of these auxiliaries is that they only allow one direction of flow of the solution containing the molecules to be separated and purified. In the procedure according to the prior art, the sample must first be placed in a storage vessel, for example a disposable syringe, in order to then push it through the flux, in particular cartridges or extraction columns. Therefore, in practice, these aids are mainly used when you want to adsorb the unwanted products. Otherwise, the storage vessel, for example the disposable syringe, has to be removed, re-loaded with a solution eluting the adsorbed substance and then printed through the auxiliary in order to obtain the desired products. Removing and placing a storage container poses a risk of contamination for the employee, the environment and the sample itself. The danger exists particularly in the medical-technical field, for example when working with infectious material, or also in biochemical-molecular biological Laboratories, especially when handling radioactive substances.

In practice, there is a considerable need for a device which enables a method for purifying and separating molecules from a solution to be carried out in a simple manner.

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

- extract molecules from a solution,

to request no preferred extraction or elution direction, to avoid a separation and re-addition of a storage vessel,

- to carry out the separation and cleaning operations in a closed system, to avoid the risk of contamination for personnel, the environment and the sample, and at the same time to facilitate the overall working method.

The object is achieved according to the invention by a device which is characterized in that the Matrix 2 in a truncated cone-shaped hollow body 1 open at both ends 4a, 4b, to which a cylindrical hollow body adjoins optionally at the further opening 4a, permeable to the solution between two

Devices 3a, 3b is arranged, wherein the matrix 2 and the devices 3a, 3b together 5 to 50% of the

Fill the volume of the hollow body and the further opening 4a of the frustoconical hollow body 1 can be connected to a pipette.

Figures 1 and 2 show schematically the structure of a preferred embodiment of the device according to the invention. The matrix 2 is delimited by two devices 3a, 3b which simultaneously ensure that the matrix 2 is fixed in the selected position. The devices 3a, 3b 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 an adequate seal between the devices 3a, 3b and the wall of the hollow body 1. There is preferably a free space between the lower device 3b and the narrower opening 4b, this free space should be small compared to the total volume of the hollow body. The further opening 4a of the two openings 4a and 4b is designed such that they fit on a commercially available pipette. A preferred embodiment of the device according to the invention consists in 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, hydroxylapatite, dextran, agarose, acrylamide, polystyrene, polyvinyl alcohol ^. or other organic polymers, derivatives or from copolymers of the abovementioned 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, acrylic acid, polystyrene, polyvinyl alcohol or other organic polymers, derivatives or from copolymers of the abovementioned 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 of chromatography. For example, if a polar molecule is to be adsorbed on the matrix 2, it will only be adsorbed on the surface of the matrix 2 if there are corresponding interactions, for example likewise by polar groups on the surface of the matrix 2, but with opposite effects ¬ set 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 embodiments 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 μm, preferably 75 μm to 125 μm.

The devices 3a, 3b fixing the matrix 2 are preferably designed as porous frits with pore sizes of 10 μm to 1 mm, preferably 70 μm to 2000 μm. The porous frits can consist of plastics, in particular Teflon (PTFE), polyethylene, polypropylene, polystyrene, polyurethane etc. However, inorganic materials such as glass and / or sintered metal are also suitable materials for the production of the porous frits.

FIGS. 3a and 3b show a further embodiment of the device according to the invention, the simple manufacture of which is particularly advantageous. A removable cartridge 5 is inserted into the hollow body 1 and contains the matrix 2 between the devices 3a and 3b fixing the matrix. The device 3b is arranged above a net-like carrier 6, which preferably consists of the same material as the removable cartridge 5. In a particularly advantageous manner, the net-like carrier 6 and the cartridge 5 are produced in one operation, for example in the injection molding process, if cartridges are made of plastic, in particular polypropylene, the device 3a is arranged above the matrix 2 which in turn is arranged a porous, mesh-like cover 7. As a result, the arrangement in the removable cartridge 5, consisting of the first device 3b, matrix 2 and second device 3a, is additionally fixed. FIG. 3b shows the overall arrangement, consisting of hollow body 1 and removable cartridge 5. This overall arrangement, consisting of cartridge 5, devices 3a, b and cover 7 fixing the matrix 2 can be combined in a particularly advantageous manner with commercially available pipette tips become. In the simplest case, this is done by pushing the removable cartridge 5 into the corresponding hollow body 1, in particular a pipette tip. It goes without saying that the cartridge 5 is dimensioned so that it exerts a slight pressure on the inner walls of the pipette tip in order not to be pushed up when absorbing liquid or a sample or to provide the necessary seal to the inner wall of the hollow body 1, preferably a pipette tip, and the

To ensure the outer wall of the removable cartridge 5. The wall thickness of the removable cartridge 5 of the mesh-like carrier 6 and of the porous mesh-like cover 7 is matched to the pipette tip used.

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 designed, for example, as a pipette tip, the pipette tip is immersed in the sample, the sample in the pipette tip is sucked up through the matrix 2 and then pressed out. This results in a higher efficiency of the adsorption, since the sample passes through the chromatographic material twice. Since it is possible to work with a closed system, contamination of the sample or danger to the environment is avoided. The device according to the invention simplifies 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 reliably.

The method according to the invention is carried out in a special embodiment with a hollow body 1 designed as a pipette tip. The molecules containing, separating and cleaning the 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, then - 1 -

the pipette tip can also be connected to a disposable syringe using a silicone tube.

The method according to the invention is suitable, in particular, for separating and purifying biopolymers such as nucleic acids and proteins by using ion exchange chromatography materials as proposed in German patent application P 36 39 949.3 as the matrix 2. 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. If, however, one is interested in the analysis of the long-chain nucleic acids, after a few washing steps the long-chain nucleic acids can be eluted again by elution with buffers of high salt concentration (high ionic strength) and processed accordingly.

FIG. 4 shows an elution profile which can be obtained when anion exchange materials are used when using the device according to the invention. It has been shown 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 means of which nucleic acids with increasing chain length can be eluted with increasing ionic strength. Thus, 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 the phage M 13 is eluted at 1.1 M sodium chloride. The elution peaks are so sharp that even "baseline" separations are possible. At 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 ones

Nucleotides such as a nucleotide decamer used as a "left" elute.

A variant of the method consists in using 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 following advantages, in particular in molecular biological operations:

- performing DNA preparations in so-called "minipreps",

- purification of RNA, rRNA, viral RNA and RNA transcripts,

Cleaning of samples obtained after nick translation, end labeling or oligonucleotide-induced labeling (oligolabeling),

Removal of the DNA linker in cloning experiments,

- Purification of nucleic acids after gel extraction as well as rapid purification of nucleic acids after digestion with residual restriction enzymes, phosphatase treatments, polymerase reactions etc. instead of a time-consuming phenol treatment. The purity in routine preparations obtainable when using the device according to the invention is at least comparable to samples obtained by Caesiu chloride density gradient centrifugation. The isolated nucleic acids are free of proteins, polysaccharides and other cell metabolites and show no inhibition of enzymes in enzymatic reactions. The device according to the invention can be used for the isolation and purification of nucleic acids from oligonucleotides to plasmids, the time expenditure being restricted to fractions in comparison to other processes.

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, in particular of other cell components.

The use of the device according to the invention in various processes is explained in more detail in the following examples. The device according to the invention is used in a preferred embodiment, the pipette tip.

Example 1

A 70 μ polyethylene frit with a 4 mm diameter and a thickness of 1.6 mm is introduced into a 1 ml pipette tip, suitable for Gilson Pipetan, and clamped by pressing. Then about 70 mg of the chromatographic material, proposed in German patent application P 36 39 949, are filled in dry. The chromatographic material is closed with a 5.7 mm diameter frit as described above and the frit is fixed in place by pressing. The procedure is analogous for 200 μl pipette tips or 5000 μl pipette tips.

Example: 2

The general handling of the pipette tip produced in Example 1 is as follows:

First, the pipette tip is equilibrated once with 100 μl of a suitable adsorption buffer. With a sample volume of 100 to 200 μl to be processed, it is sufficient to push the sample through the chromatography material about five times. However, if a larger amount of liquid is to be processed, such as is obtained after gel elution, the sample can be drawn through the matrix using a silicone tube that connects the pipette tip and a disposable syringe. It is then sufficient for the sample to pass the chromatography material only twice. The flow rate of the solution should not be higher than 1 ml per minute.

Buffer A can be used as equilibration and adsorption buffer. Buffer A:

400 mM sodium chloride 15% ethanol 50 mM MOPS (3-N-morphine linopropanesulfonic acid) 1 mM EDTA (ethylenediaminetetraacetate) pH 7.0

The washing steps are carried out analogously to the adsorption process described above. In nucleic acid preparations, buffers B, C and D are typically used as washing buffers. Buffer B: Buffer C:

750 mM sodium chloride 1000 mM sodium chloride 15% ethanol 15% ethanol 50 mM MOPS 50 mM MOPS 1 M EDTA 1 mM EDTA pH 7.0 pH 7.0

Buffer D:

1000 mM sodium chloride 50 mM MOPS pH 7.0

An Eppendorf tube is filled with 1 ml of washing buffer and 150 μl are rinsed three times through the chromatography material in order to remove impurities. This step is repeated.

As in the two preceding steps, the adsorbed nucleic acids are eluted either with an Eppendorf or Gilson pipette or with a disposable syringe, care being taken that the pipette tip is connected to the disposable syringe by means of a silicone tube. It simply becomes the elution buffer E or F

Buffer E: Buffer F:

1100 mM sodium chloride 1500 mM sodium chloride 15% ethanol 15% ethanol 4 M urea 50 mM MOPS 50 M MOPS 1 mM EDTA 1 mM EDTA pH 7.50 pH 7.0

sucked or pressed through the pipette tip. This step is repeated once. The eluates are combined and the nucleic acid is precipitated therefrom. Example 3

A pipette tip, produced according to Example 1, is hydrated with 50 mM sodium phosphate buffer and equilibrated by passing 750 μl buffer several times through the

Pipette sucks. A sample containing 20 μg of Plasmid pBR322 DNA in 500 μl of 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 pressing 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 1 ml of the elution buffer three times and squeezing it out.

Example 4

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

The bacterial cells are incubated overnight and harvested the next day by centrifugation. The pellet is disrupted in 85 μl 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, the mixture is incubated 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 Isoa yl alcohol can be added. A pipette tip according to the invention from Gilson, Brand, Eppendorf, manufactured according to Example 1, is equilibrated with 100 μl buffer C. Then 100 μl 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 then the sample is centrifuged in an Eppendorf laboratory centrifuge for 30 minutes.

Example 5

The isolation of mRNA, rRNA and viral RNA from crude extracts takes place as follows:

The RNA extract is precipitated with ethanol and the pellet obtained is resuspended in TE buffer (10 mM Tris / 1 mM EDTA, pH 7.5). The adsorption conditions are adjusted by adding 0.1 part by volume (based on the re-suspended solution) of 5 M sodium chloride and 0.2 part by volume of 250 mM MOPS, pH 7.0. The pipette tip is equilibrated with 1 μl buffer A. The sample is then adsorbed onto the chromatography material by pipetting up and down five times. One rinses with buffer A in order to eliminate impurities. Elution is carried out with 300 μl of buffer C. The RNA is precipitated by adding 2.5 parts by volume of ethanol. After standing at -20 ° C. for 30 minutes, centrifugation in an Eppendorf centrifuge takes a total of 30 minutes at the highest speed. The pellet is carefully washed with ethanol before further processing. The sample should have a nucleic acid content of at most 15 μg. Example 6

Purification of mRNA for the synthesis of the corresponding cDNA is carried out as described in Example 5.

Example 7

Unused triphosphates in labeling reactions such as nick translations, end labels and oligonucleotide-dependent labels (oligolabeling) of DNA are removed as follows:

The labeling reaction of the DNA is terminated by adding 2 μl 0.5 M EDTA per 20 μl sample volume, and the adsorption conditions are adjusted by adding 1 volume part of the buffer C. The total final salt concentration should be approximately 500 mM. The pipette tip according to the invention is equilibrated with 100 μl buffer B. The sample is adsorbed onto the chromatography material by suction up five times and squeezed out and washed with a total of 10 ml of buffer B in order to separate the unreacted nucleotides. The flow rate of the washing buffer should preferably be approximately 5 ml per minute. The labeled DNA is then eluted with a total of 300 μl of the buffer F. The sample can be used directly if it is diluted at least 1:20 with the buffer intended for hybridization. Otherwise the sample is precipitated and dissolved in TE buffer.

Example 8

The removal of a DNA linker from a DNA with more than approx. 400 base pairs in a cloning experiment is carried out as follows: The sample to be cleaned is diluted with 1 part by volume of the buffer C so that the final concentration of salt is approximately 500 mM. The pipette tip according to the invention is equilibrated with 100 μl of buffer A. The sample is adsorbed as in the previous examples. The pipette tip according to the invention is rinsed with buffer B in order to remove the contaminants. The DNA is desorbed and eluted with a total of 300 μl of buffer F. Then the DNA is isolated by isopropanol precipitation (addition of 1 part by volume) and left on ice for 15 minutes, after which the sample is centrifuged in an Eppendorf centrifuge for the Duration of 30 minutes. Then wash carefully with 70% ethanol.

Example: 9

The rapid cleaning of a DNA sample after enzymatic modification (for example by phosphatase, restriction endonuclease, poly erases, etc., and cofactors) is achieved using the following procedure:

The underlying reaction volume is 50 μl and contains no more than 100 mM sodium chloride. 5 μl of 0.5 M EDTA, pH 8.0, are added to the reaction volume in order to terminate the modification reaction. A volume part of the buffer C is added in order to set the adsorption conditions with a salt concentration of about 500 mM. The pipette tip according to the invention is equilibrated, the sample is adsorbed and then eluted as described in Example 8. The further treatment of the sample is also carried out as described in Example 8. Example 1 0

The device according to the invention can also be used to efficiently separate agarose and acrylamide impurities. This is particularly necessary when gel elution of the sample has taken place. When using the procedure described below, the DNA is concentrated at the same time, even if the starting volume is a few ml. The DNA has a size of> 400 base pairs and can be present in an amount from 5 ng to 15 μg. The sample preparation and the equilibration of the pipette tip according to the invention is carried out as described in Example 8. The sample is then transferred to a disposable syringe and pressed twice through the pipette tip according to the invention. The flow rate should be around 250 μl / in. The pipette tip according to the invention is then washed with 5 ml of buffer B at a flow rate of 5 ml / min. The further treatment of the DNA sample is carried out as described in Example 8.

Claims

- 37-patent claims

1. 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 an open at both ends (4a, 4b), truncated cone-shaped hollow body (1) which is optionally connected to the further opening (4a) by a cylindrical hollow body, is arranged between two devices (3a, 3b) which are permeable to the solution, the matrix (2) and the devices (3a, 3b) altogether 5 to Fill 50% of the hollow body volume and the further opening (4a) 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 (3a, 3b) 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 (4b) and the lower device (3b) is a free space.

5. Device according to one of claims 1 to 4, characterized in that in the hollow body (1) a removable cartridge (5) is arranged, which is used for separation and cleaning matrix (2) between two devices (3a , b) fixed, the device (3b) being arranged above a net-like carrier (6), the removable cartridge being closed by a porous net-like cover (7), the lid (7) being arranged above the device (3a) and the removable cartridge (5) on the inner wall of the hollow body (1) closes tightly.

6. Device according to one of claims 1 to 5, characterized in that the removable cartridge (5), the mesh-like carrier (6) and the porous mesh-like cover (7) consist of the same material.

7. The device according to claim 6, characterized in that the material is made of plastics such as Teflon (PTFE), polyethylene, polypropylene, polystyrene and / or polyurethane.

8. Device according to one of claims 1 to 7, characterized in that the matrix of porous Chromatogra¬ phiematerial based on silica gel, aluminum oxide, titanium dioxide, hydroxylapatite, dextran, agarose, acrylamide, polystyrene, polyvinyl alcohol or other organic polymers, Derivatives or copolymers of the above-mentioned carrier materials.

9. Device according to one of claims 1 to 8, characterized in that the matrix is a superficially modified chromatography material made of 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.

10. The device according to claim 9, characterized in that the surface modified Chromatographiemate¬ rial is an ion exchanger, a reversed phase and / or an affinity chromatography material.

11. Device according to one of claims 1 to 10, characterized in that the pore size of the porous Chroma¬ tographiematerialien 20 to 1000 nm and the grain size of the materials is 10 to 2000 microns, preferably 75 to 125 microns. - ± 9 -

12. Device according to one of claims 1 to 11, characterized in that the matrix-fixing devices (3a, 3b) are porous frits with pore sizes of 10 μm to 1 mm, preferably 70 to 200 μm.

13. 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 (3a, 3b) and that the solution is drawn through the matrix (2) into a free space above the upper device (3a) and then through the Matrix (2) is pressed out of the hollow body (1).

14. The method according to claim 13, characterized in that biopolymers are separated and purified.

15. The method according to claim 14, characterized in that nucleic acids and / or proteins are separated and purified.

16. Use of a device according to one of claims 1 to 12 for the separation and purification of molecules, preferably biopolymers.

17. Use of a device according to claim 16 for the separation and purification of proteins and / or nucleic acids.