EP1180238A1 - Device and method for isolating electrically charged molecules - Google Patents

Abstract

The invention relates to a device which is open upward for electroelution of charged molecules in a reaction channel (2) comprising electrodes (3, 4) on the ends and at least one intermediate fitting (28). The device is particularly suitable for automating an xyz robot.

Description

Device and method for isolating electrically charged molecules

description

The present invention relates to methods and devices for the isolation of charged molecules with a wide range of applications, for example in the isolation of electrically charged molecules in receptor-ligand mixtures or in the sample preparation of nucleic acids.

Especially for the latter application, special electrophoretic processes, such as electroelution, can be used, as described in WO 97/34908 and WO 98/58251. In this process, charged molecules are first bound to an adsorber and then released again in an electroelution process, transferred to a removal volume and also concentrated there. Other devices for electroelution are e.g. in US 5340449 and US 4608147. However, the devices described there are not designed or provided for methods according to WO97 / 34908, but rather comprise closed and screwed chamber systems. These are then appropriately anchored in an electrophoresis buffer tank, the chamber system being hermetically sealed against the electrophoresis buffer tank. A major disadvantage of these devices is that they are not suitable for automation in a pipetting robot. This means that they cannot be used for standard procedures with a large sample throughput.

When developing electro-elution devices that are open at the top, one must take into account the fact that, depending on the design in detail, an electro-osmotic flow (EOF) must be expected. Details on this can be found, for example, in "Capillary Electrochromatography - Technology and Applications" (CEC guidebook Hewlett-Packard Publication No .: 5968-3231 E) Chapter 2 and "Analysis of Nucleic Acids by Capillary Electrophoresis" (Chr. Heller (Ed.) ISBN 3-528-06871 -X, 1 997) page 24 ff. This Flow, for example caused by capillary openings in the electro-elution area, can significantly influence the electro-elution process, even making it impossible.

The problem is particularly serious that, in conventional devices, due to the occurrence of an electroosmotic flow, one of the chambers runs empty before the desired insulation can be completed. In order to avoid such draining of one of the chambers, complicated measures, such as a liquid exchange between the anode and cathode chambers, have been necessary to compensate for the electroosmotic flow.

An object of the invention was therefore to provide a device for isolating charged molecules which can be automated and in which the problems of the prior art which arise due to the electroosmotic flow are at least partially eliminated.

This object is achieved according to the invention by a device and a method as described in the claims.

The device according to the invention for the isolation of electrically charged molecules consists of a downwardly closed base body with at least one upwardly open channel and at least two electrodes as the right and left outer boundaries of the channel, and is characterized in that one or more between the electrodes in the channel Intermediate installation (s) are arranged, which divide the channel into two or more chambers. The use of a channel which is open at the top in the sample entry and sampling areas enables the device according to the invention to be used in automated processes, for example with pipetting robots or x, y, z robots. Furthermore, the intermediate installations prevent or reduce the electroosmotic flow. The intermediate internals in particular allow only a small electroosmotic flow, which is desired for concentration, but which does not lead to the emptying of a chamber, so that electroelution of charged molecules is possible. The intermediate internals of the device according to the invention are thus permeable, in particular, to electrically charged molecules, but on the other hand represent a liquid barrier through which the electroosmotic flow can be limited. Depending on the application, it is possible to completely prevent the electroosmotic flow or to allow a low osmotic flow, which can advantageously be used for concentration. The intermediate installations of the devices according to the invention preferably limit the electroosmotic flow in such a way that the volume of the sample entry or removal chamber within a period of at most 60 minutes, preferably at most 30 minutes and particularly preferably at most 10 minutes and at least 30 seconds, preferably at least 1 minute and more preferably at least 2 minutes of electroelution does not decrease by more than 90% by volume, preferably not more than 50% by volume and particularly preferably not more than 30% by volume. However, it is also possible to limit the electroosmotic flow in such a way that the chamber volume does not decrease by more than 10% by volume, preferably not more than 5% by volume, in the specified time intervals.

While the device according to the invention can be operated at any voltages, voltages <250 V and in particular voltages <42 V are preferably used. The size of the device according to the invention can be chosen arbitrarily depending on the application, for example on a production scale from a few liters or larger to miniaturized devices with a few microliters of chamber volume, for example for use on conventional microtiter plates, as described below. The volume of the upwardly open channel is preferably 0.01 to 5 ml, more preferably 0.2 to 2 ml.

In principle, all electrically charged molecules, including their superordinate structures, such as complexes, clusters, specific binding pairs, etc., can be isolated with the device according to the invention.

Examples of preferred electrically charged molecules are biomolecules, such as nucleic acids, in particular DNA, RNA, proteins and others

Natural substances and dyes. So the device can e.g. for protein mixture analysis and for immunoassays. A particularly interesting field of application is the use of

Device for the elution of proteins from gels, for example from two-dimensional electrophoresis gels. This technique is used in particular in proteomics to determine the protein functions of the DNA sequences found in genome projects (e.g. Hugo).

According to the invention, however, the nucleic acid itself can also be isolated.

It has been found that the device according to the invention can be used to isolate molecules in a large molecular weight range from less than 100 Da to more than a few million Da. It has been observed that, in addition to small molecules, such as dye molecules or polypeptides, large molecules, such as genomic yeast DNA, can penetrate the internals and can thus be isolated or purified.

In a particularly preferred embodiment, at least one of the intermediate internals consists of a shape-stabilized gel. It was Surprisingly, it was found that gels, for example an agarose gel, outstanding in a low concentration, for example from 0.1% gel to 10% gel, preferably 0.2% gel to 1.2% gel, more preferably up to 0.8% gel Have separation or filtering properties, whereby charged particles are allowed to pass through, while the undesired electroosmotic flow is kept to a minimum. The problem with gels in such a low concentration, however, is that they are dimensionally stable or mechanically very unstable and no films or films can be produced therefrom that could be used as separating elements. It has now been found that mechanically stable elements with the above-mentioned advantageous properties can be obtained by stabilizing the shape of such gels. The invention therefore also relates to such dimensionally stabilized gels and their use as an intermediate installation in the device described above.

Preferred gels are easily deformable, liquid or gas-rich disperse systems composed of at least two components, consisting of a solid colloidally divided substance with long or / and highly branched particles and a liquid, in particular water. Examples of suitable gels are polysaccharide and protein gels such as gelatin, agar, carrageen, alginates, alginic acid, phyllophoran, furcellaran, agarose and others, as well as polymer gels such as polyacrylamide gels.

According to the invention, the stabilized gels are used as separating elements, for example as separating filters or separating membranes, and not as a carrier material. They are preferably used as thin disks with a density of at least 0.1 mm and preferably 0.5 mm, and at most 20 mm, preferably at most 5 mm and particularly preferably at most 2 mm. The molecules move across the gels across the thinnest dimension. The dimensional stability is preferably achieved by a support element on or into which the gel is introduced. The use of sintered plastic, for example sintered polypropylene or polyethylene with a pore size of 1 to 200 μm, preferably of 20 to 1 20 μm, has proven particularly advantageous. Excellent results can also be achieved by using fabrics, for example made of plastics such as polyester or polyamide, with a mesh size of 0.1 to 500 μm, preferably 0.5 to 10 μm, as the support element.

The permeability of the shape-stabilized gel can be adjusted by the degree of crosslinking of the gel. It is possible to choose the permeability in such a way that the desired charged molecule passes through the gel at the applied voltages and can then be obtained from the sampling chamber. For many applications, however, it can also be advantageous to choose the degree of crosslinking such that the charged molecule remains in the gel and is then recovered from the shape-stabilized gel in further steps. The desired analyte can get stuck in the gel by suitable derivatization with functional groups that bind specifically (e.g. streptavidin or biotin) or non-specifically (e.g. hydrophilic or hydrophobic groups) to the analyte.

It is also possible to use a liquid barrier as an intermediate installation, which can be opened and closed.

In a further embodiment, a fabric is used as an intermediate installation. It may be preferred here that a small electroosmotic flow from the anode to the cathode is generated through the tissue under the electroelution conditions. In order to achieve passage of electrically charged particles while avoiding excessive electroosmotic flow, the fabric preferably has a mesh size of 0.1 to 50 μm, preferably 0.5 to 5 μm. The absorption of electrically charged molecules on the tissue is preferably less than 40%, particularly preferably less than 10%, in order to allow the desired analyte to pass into the sampling chamber. Particularly advantageous results are obtained when the fabric has been treated by calendering.

In particular when separating analytes from biological samples from, for example, cell residues, the mesh size is adjusted so that biologically active particles of a defined size cannot penetrate the tissue.

The device according to the invention allows flexible handling and corresponding configurations, depending on the intended application. For example, multi-chamber systems can be used if the desired analyte, for example a nucleic acid or a protein, does not pass the intermediate installation as the first molecule of the sample mixture. This can be the case, for example, when using detergents to lyse cells. Here, the arrangement is advantageously chosen so that the detergents which first pass through the intermediate installation are electroeluted through the sampling space into a further chamber.

Furthermore, it is possible to provide desired molecules which have no charge even under the conditions applied, by means of carriers, for example by using specific charged antibodies or hybridization probes. Furthermore, it is possible to design the device according to the invention to be multidimensional, for example in the form of a cross, which enables a multidimensional separation.

A device with at least four chambers is particularly preferred, the anode and cathode chambers which are adjacent to the anode or Are cathode, compared to the intermediate sample entry or Sampling chambers are relatively large. The cathode and anode chamber preferably have a volume of 5 to 20: 1 to the volume of the sampling or sample insertion chamber. In this way, the sample volume can be kept small, while on the other hand the volume at the electrodes is large, so that any undesirable side reactions which may occur there are less important.

A reduction in the volume of the sampling or sample addition chamber in comparison to the anode or cathode chamber can be achieved, for example, in that the upwardly open channel has a taper in the middle.

In addition to the different volumes, a different buffer concentration is also preferably used in the individual chambers, the buffer concentration in the anode or cathode chamber preferably being 5 to 20: 1 to the volume of the sample entry or removal chamber. In addition to the buffer concentration, the pH in the individual chambers can also be set differently.

In a particularly preferred embodiment, as shown, for example, in FIG. 26, the device consists of four chambers. Starting from the cathode on the left, there is first a cathode chamber which is relatively large, for example has a volume of 1.6 ml and contains a high-molecular buffer, for example in a concentration of 100 mM. This chamber is separated from the sample entry chamber by a membrane, which has a smaller volume, for example 1 60 μl, and a lower buffer concentration, for example 10 mM. A shape-stabilized gel, for example an agarose gel separating filter, which consists of a sintered plastic filled with agarose gel, is located between the sample entry and the sampling chamber. The sampling chamber points again a small volume, for example 1 60 μl, and a low buffer concentration, for example 10 mM. In this preferred embodiment, a further stabilized agarose gel filter acts as a separation between the sampling chamber and the anode chamber. The anode chamber itself is again relatively large, for example 1.6 ml, and has a high buffer concentration, for example 100 mM. Passage of the desired analyte during electroelution through the second gel filter is prevented on the one hand by suitably adjusting the separation time and on the other hand by adjusting the buffer concentrations in the sampling chamber and the anode chamber. It was found that a high buffer concentration in the anode and cathode space compared to the sampling and sample introduction space has an additional favorable influence on the separation performance of the device and on a reduction in the osmotic flow. The sample taken from the sampling chamber can, for example, be analyzed by means of mass spectrometry or further processed by means of PCR.

Another important advantage of the device according to the invention is that the analyte can be processed during the transfer steps. An enzymatic reaction or a receptor / ligand binding preferably takes place in the chambers, in particular during the electrophoretic transfer of the charged molecules.

A further improvement in the separation properties can be achieved by reversibly applying a magnetic field in the vicinity of at least one electrode.

The electrodes can be made from conductive materials, for example metals, and preferably from conductive plastics. The entire device can also be assembled from individual modules in a modular system. The device according to the invention can be used, for example, for separating the free phase from the bound phase in receptor ligand assays, for isolating nucleic acids, for isolating DNA without contamination from RNA or for isolating RNA without contamination with DNA.

The invention further relates to a method for isolating electrically charged molecules, which is carried out in a device as described above.

The invention further relates to a method for isolating charged molecules in mixtures with electrophoretic agents in a reaction channel, which is characterized in that a mixture in liquid form is introduced into a reaction channel, is subjected to electrophoresis in the reaction channel, in this electrophoresis in the reaction channel is processed and after this processing the isolated charged molecules are removed from the reaction channel in a soluble form.

In the method according to the invention, the starting mixture is further processed during the electrophoresis, whereby a significant simplification of the overall process can be achieved. For example, ligand-receptor binding, an enzymatic reaction and / or concentration can take place in the reaction channel during electrophoresis.

By using a device as described above, in particular a device in which one or more shape-stabilized gels or other of the internals described above divide the reaction channel into different chambers, the electroosmotic flow can be reduced in electrophoresis in such a way that electroelution of charged molecules is possible without one of the chambers running empty. A lysed biological sample, for example lysed cells, is particularly preferably used as a mixture. It is also possible to separate the free and the bound phase from a ligand-receptor assay, in particular from an immunoassay, and thereby enable a quantitative determination.

Furthermore, a quantitative determination can also be carried out by separating components with a label, for example a radioactive label, an enzyme label, a fluorescent or luminescent label.

Electroelution is carried out in particular under conditions such that the concentration of detergent in the withdrawn solution with the isolated charged molecules is less than the concentration of detergent in the solution of a lysed sample originally introduced into the reaction channel. It may also be advantageous to add at least one further buffer component to the lysis mixture before the electroelution.

If, as described here, devices are used which are open at the top, sample mixtures, e.g. Nucleic acids or adsorbers (e.g. in particle form) to which nucleic acids are bound can be introduced. Such devices are generally suitable for automation using pipetting robots (e.g. from TECAN, Rosys, Canberra Packard and Beckman).

The devices according to the invention on the one hand enable automation with devices open at the top and on the other hand take into account the requirements of the electroosmotic flow. Surprisingly, they can be used for methods in which nucleic acids are bound to adsorbers, which are then introduced into a sample entry chamber and then into a corresponding one Electroelution with sample concentration are subjected. When preparing samples, especially of nucleic acids but also of other charged molecules, it has been shown that for the simplest possible handling - also with a view to automation - a further process step should be linked with the transfer, i.e. the spatial change of the charged molecules . This process step can change the charged molecules themselves or change the environment of the charged molecules. Such a process step linked to the transfer during electroelution is not provided in the prior art.

One of the most important applications of the devices and methods according to the invention is the isolation of nucleic acids from biological sample material. For this purpose, the sample material must first be lysed in order to release the nucleic acids from the biological compartments, such as the cell nucleus, mitochondria, virus envelopes, etc. For this lysis, in turn, the use of high concentrations of detergents of all kinds proved to be helpful. Ionically structured detergents are particularly suitable for the electrophoretic processes relevant here, in particular those with negatively charged detergent molecules, such as sodium dodecyl sulfate.

If one wants to process the released nucleic acid of the lysed sample further, e.g. in a PCR according to US 46831 95 or digestion with restriction enzymes, disrupt the high detergent concentrations and must be removed. This change in the environment of the charged molecules as a new process step can be carried out according to the invention together with the transfer step in corresponding devices and with the methods according to the invention.

Another application of a completely different kind is derived from molecular oncology. Here is the analysis of an oncogene's mRNA, for example important diagnostic parameter. It is crucial that the mRNA is isolated and separated from the analog genomic DNA, since residues of the genomic DNA interfere with the subsequent RT-PCR and falsify its informative value. There are currently two standard methods for obtaining the mRNA in a sample preparation process: a) hybridizing the mRNA with a solid-phase-bound oligo (dT), which binds to the naturally existing oligo (A) sequence of the mRNA and then releasing it after washing and b) methods which isolate the mRNA by selective precipitation.

However, both methods have serious disadvantages. Thus, the mRNA in a) tends to break, so that only parts of the mRNA are detected with this method, which makes it impossible for a quantitative analysis. The methods according to b) are complex to handle and difficult to automate, since centrifugation steps are necessary.

Surprisingly, it was found that by linking transfer and a further process step, namely the enzymatic digestion of DNA in this case, the isolation of the mRNA is possible without DNA contamination.

The present applications therefore also relate to methods and devices for the purpose of isolating charged molecules, which link further processing to the electrophoretic transfer, which processing may relate to the environment of the charged molecules or else to the molecules themselves.

Another preferred field of application is analytical test methods which are based on a ligand-receptor interaction, in particular those which use antibody-antigen reactions. Such methods are commonly referred to as immunoassays. There are basically two different applications: homogeneous and heterogeneous immunoassays. In the homogeneous immunoassays, methods are used in which, after incubation, a measurement signal is generated solely by dissolving the antibody and antigen in the form of specially labeled derivatives. In the case of heterogeneous immunoassays, a so-called bound / free separation, that is to say a separation of the antibody-antigen complexes formed during the incubation from the unbound free components, must take place after incubation. A summary of these techniques can be found, for example, in the "Handbook of Experimental Immunology", Weir, DM (Editor); Oxford; Blackwell Scientific and EP 0 1 63 1 22, as well as the prior art quotes contained therein.

The process step of bound / free separation is particularly difficult when the process is to be automated as simply as possible. In the past, various methods such as B. Double antibody techniques, techniques with solid phase bound antibodies such as with micro and macro particles, or coated reaction tubes (coated tubes).

Surprisingly, it has now been found that the devices described here enable simple automation of the bound / free separation of ligand / receptor assays. In contrast to all previous techniques, according to the invention there is no need for a washing step in the sense that the ligand-receptor complexes, which were initially separated in a first separation step, have to be mixed with a washing solution, suspended and re-separated in order to obtain the Also remove complex-attached residues from free components. In the prior art, this washing step generally has to be carried out twice and in the current immunoassay machines such as ES 600 from Boehringer-Mannheim now Röche Diagnostics or the device architect from Abbott, for example, represents the process step which is one Limiting the throughput. The devices of the present application enable automation on the one hand because they are open at the top and on the other hand take into account the requirements of the electroosmotic flow. Surprisingly, they can be used in such a way that the incubated mixture of a ligand-receptor mixture is introduced into a sample entry chamber, then subjected to a corresponding electroelution and a direct evaluation is possible without further washing steps.

Depending on the ligand or receptor markings used, detection can be carried out by measuring radioactivity (beta or gamma radiation), by photometric, fluorimetric or luminometric measurements, or else by mass spectrometric or nuclear magnetic resonance spectrometric methods.

The present applications therefore also relate to easily automated methods and devices for the purpose of isolating charged molecules in receptor-ligand mixtures.

Figure 1 a shows schematically the perspective view of an inventive device. It is a basic body (1) into which a

Reaction channel (2) was incorporated. The reaction channel (2) is characterized in that it is open at the top, a corresponding bottom and

Contains side walls so that the interior can be filled with electrolyte solution. The reaction channel (2) is preferably designed in a rectangular or cylindrical shape, in which the short sides each

Electrodes (3,4) are provided at the outer ends. Between these

Electrodes (3, 4) extend the reaction channel (2), which according to the invention can be provided with at least one intermediate installation (28), so that at least two liquid chambers (for example 10, 7 in FIG. 2a) can be generated. The channel (2) typically has a volume of 0.01-5 ml, preferably 0.2-2 ml. The base body (1) consists of electrically non-conductive material, generally of plastics such as polyacetal, Polycarbonate, polyamide, polystyrene, polyethylene, polypropylene, polyacrylates, polyvinyl chloride or the like and can be produced from semi-finished products or by injection molding or bubble drawing. In individual cases, glass fiber reinforced plastics or plastics with other additives can also be used.

Fig. 1 b shows an inventive variant of the basic shape of the channel (2) described above. Here it is constricted in the middle. Surprisingly, it was found that a particularly efficient concentration of charged molecules can be achieved with this shape. This form takes account of the following phenomena that occur during electroelution: a) The pH changes due to the electrolytic decomposition on the electrodes. The chambers around the electrodes must therefore have a sufficient volume to provide sufficient buffer capacity. B) In order to achieve a sufficient concentration, however, it is necessary to obtain the smallest possible sampling volume. Both can be achieved accordingly by the constriction (32) when the sampling space (7) is located on the constriction (32). The embodiment shown in Fig. 1 b is preferably intended as an injection molded part for single use and has slots (1 2) in the base body (1) for the intermediate fittings, such as the semipermeable membrane (1 4), which are described in more detail below are. According to the invention, a plurality of devices can also be present in a composite, for example in strip form or as plates, the 96-microtitration plate format as described in US Pat. No. 4,154,795 being a preferred solution. 1 h shows such an embodiment with microtitration plate strips (1 9) having 8 channels (2) and a suitable microtitration plate frame (1 8). In a miniaturized version, formats with 384 and 1 536 channels can also be used. The channel (2) can also be composed of various individual elements Fig. 1 cg and held together, for example, with a tensioning device (23) in Fig. 1g. This inventive design consists of individual basic elements (Fig.1c - 1f), which are strung together by means of sealing elements (25) on mating surfaces (20) and then pressed together with one or more clamping devices (23). The basic elements are usually open at the top and U-shaped. However, it is also possible to use individual elements which are closed at the top, such as a prefilled closed cathode and / or anode chamber.

1c shows the downwardly closed base body (1) with a corresponding recess (2) which then form the reaction channel (2) when a plurality of base elements connected to sealing elements (25) on the corresponding mating surface (20) by means of a clamping device (23) be pressed together.

1d shows an embodiment of an intermediate installation (28) for the reaction channel (2). It consists of a frame (29) with a rectangular or round window into which materials for the intermediate fittings (28) can be inserted. In a preferred embodiment, this element is manufactured in a layered structure, with laminating foils (29) on the outside and the corresponding intermediate installation materials (28) in the middle. The element is then welded with the aid of a laminator and thus has a smooth fitting surface (20) and thus also a good seal.

Fig. 1e represents a corresponding sealing element (25) with the two-sided fitting surfaces (20). According to the invention, this sealing element is preferably made of silicone or Teflon, depending on the use of the reactants.

Fig. 1f shows an embodiment for electrodes, which is constructed as a plate electrode or in a layered structure like Fig.1d. There is one electrical supply line (30) is provided for the electrodes (3, 4) located in the window.

1 g shows a top view of a reaction channel composed of basic elements with various intermediate internals. It has proven to be advantageous to insert the basic elements in a receptacle (26) to which a corresponding clamping device (23) is attached. Instead of the receptacle (26) with the clamping device (23), inventive elements such as FIGS. 1 i and 1 j can also be used, which can be plugged together via a corresponding clamping cone (9) with or without latches (1 6) and also without a clamping device (23) hold liquid-tight together.

1 i shows the basic element for delimiting the channel, which in this case can be produced in any length by plugging it together with the basic element for the extension from FIG. 1 j. Then on the opposite side there is a boundary with an identical basic element as FIG. 1 i. This either has corresponding slots for receiving electrodes (3) as shown in Fig. 1 i. According to the invention, however, two different variants have proven to be advantageous: In variant A, an electrode made of conductive plastic is injected directly into the production process in a two-component injection molding process. Surprisingly, variant B can also be used to produce the entire base element from conductive plastic. This is a particularly cost-effective variant. Materials with resistances of less than 1,000 ohms, such as, for example, Cabelec 3827 and Pre-Elec 1 362, have proven to be particularly advantageous as blend additives for polypropylene. The extension element according to Fig. 1j must accordingly be made of non-conductive plastics, preferably polypropylene. A further inventive variant is shown in FIG. 1 j, since here an opening (31) enables access to the reaction channel (2). This opening (31) can also be used for suctioning off solutions, for example during washing processes.

Several such reaction channels can be connected to one another via integrated connecting elements (21) as described above, preferably in the 96-microtitration plate format. Integration into a corresponding framework is also easy to achieve.

The reaction channel can also include branching so that, for example, multi-dimensional isolation can be carried out.

In the following, a wide variety of intermediate installations (28) in the channel (2) are described, which fulfill a wide variety of functions, an intermediate installation (28) dividing the reaction channel (2) into a sample entry space (10) and a sample removal space (7), as shown in Fig. 2 (Fig. 2 a: top view, Fig. 2b: sectional view)

In a device according to the invention, an intermediate installation (28) must meet the following criteria in order to be suitable, for example for electroelution. The intermediate installation must a) impart electrical conductivity, b) enable permeability to charged molecules c) represent a liquid barrier so that the two spaces (1 0, 7) have no liquid exchange when there is no voltage at the electrodes, d) if necessary Retain particles and e) if necessary generate an electroosmotic flow for concentration, but limit the electroosmotic flow in such a way that electroelution can be carried out. Surprisingly, it was found that an inventive device such as FIG. 3 fulfills these criteria, for example. A semipermeable membrane (14) is attached in the vicinity of the anode (4), so that an anode space (8) is created between the membrane (1 4) and the anode (4). In addition, a further element, namely a fabric (5) or, as described below, a shape-stabilized gel is introduced between the semipermeable membrane (1 4) and the cathode (3). This is preferably a precision sieve fabric with a defined mesh size and with a defined open sieve area, as described in “Synthetic monofilament precision sieve fabrics (company brochure from Sefar, Rüschlikon, Switzerland). This fabric (5) can be, for example, nylon and / or polyester fabric. These fabrics (5) can also be appropriately calendered or treated similarly. In the case of such fabrics, the fulfillment of the previously described requirements was only determined in a certain area for the mesh size and for the open screen area. The mesh width is 0.1 to 500 μm, but preferably 0.5 to 10 μm, and the open sieve area is 0.2% to 40%, but preferably 0.5 to 2%. The type of material also plays an important role. As shown in Example 1, an electroosmotic flow is generated on the tissue (5) which reduces the volume of the sampling space (7). This type of fabric (5) can also use other types of fabric, such as wool or cotton. Such a tissue (5) can be used in an intermediate installation (28) in a device according to FIG. 3 to carry out electroelution in such a way that, for example, particles are introduced into the sample entry space, which corresponds here to the cathode space (6), which act as adsorbers are loaded with electrically charged molecules, especially nucleic acids (Fig. 9c and Example 4). After the sampling space (7) and the anode space (8) have been filled with a suitable electrolyte, a separation of the charged molecules from the adsorber can be achieved by applying a voltage between the electrodes (3, 4). The fabric (5) hampers the electrical conductivity does not and allows charged molecules, especially nucleic acids, to pass. It also fulfills the task of retaining the particles in the sample entry space (10) so that a correspondingly clear nucleic acid solution can be removed from the sample removal space (7). In addition, the tissue ensures that when the nucleic acid is removed with a pipette, electrolyte solution flows in only to a limited extent from the sample entry chamber (10 here identical 6) and contaminates the isolated nucleic acid. 3 has a semipermeable membrane (1 4), which prevents charged molecules from a certain molecular weight from migrating into the anode compartment (8). This intermediate installation therefore has a protective function and prevents the charged molecules from penetrating into the anode compartment and from being oxidatively destroyed on the electrode surface.

Surprisingly, it was found that, as a result of the above-mentioned electrical osmotic flow, the buffer volume in the sampling chamber (7) is reduced if the tissues (5) are correctly selected and in this way a concentration of the tissue charged molecules takes place. This is a decisive advantage, especially in nucleic acid analysis. When using a device according to FIG. 3 with particles which are introduced into the cathode space (6), which corresponds to the sample entry space (10), it is a further inventive advantage that the particles are identical in the sample entry space (10) with a suitable tissue ) are retained so that a correspondingly clear nucleic acid solution can be removed from the sampling space (7). Care must be taken that the exclusion size of the mesh size must be adjusted accordingly to that of the particles used.

In a further variant, a liquid barrier (1 3) can also be used, which is guided through corresponding guides (1 2) in the Base body (1) can be introduced in a liquid-tight and movable manner, as shown in FIGS. 4 and 5a and 5b. Figure 5a shows the liquid barrier (1 3) in the open state, while Figure 5b shows the closed state, in which the reaction channel (2) is closed by pressing the liquid barrier (1 3) into the base body (1).

Figure 6 now illustrates the area of application of such a liquid barrier (1 3). In addition to the electrodes (3, 4) and the semipermeable membrane (1 4), the corresponding device provides a corresponding tissue (5) and / or a frit (27). The latter form a sample entry space (10) in the direction of the cathode (3), into which particles according to Example 4 can be introduced. After electroelution, the charged molecules accumulate in the sampling space (7), since the liquid barrier (1 3) is initially open. By pushing in the liquid barrier (1 3), a sampling space (7) is created that is liquid-tight against the space (6), from which, with the help of pipettes, e.g. the charged molecules, such as nucleic acid, can be removed.

Fig. 8 shows a special inventive device for the use of adsorbers with magnetic properties. These can be retained at certain points in the reaction channel (2) by means of permanent magnets (17). To do this, a permanent magnet (1 7) must be brought close to the reaction channel. For electroelution, it is therefore particularly appropriate to bring a suitable permanent magnet close to the sample entry space (10) so that the particles can be drawn onto the floor or the side wall after the charged molecules have been released. With this measure, the subsequent electroelution cannot be adversely affected by the fact that they clog tissues or other internals in the reagent channel by particles. In addition to electroelution (example 4), devices according to the invention can also be used to recover nucleic acid from gels (agarose or polyacrylamide) (example 2).

Disks or blocks made from conventional agarose or polyacrylamide gels represent particularly suitable intermediate internals, with devices for nucleic acid isolation e.g. It was found that gels with a low degree of crosslinking, that is to say small proportions of gel filler, are particularly advantageous. Again, it turned out that the thickness of the intermediate fittings should be correspondingly thin. They are in particular 0.1-20 mm, preferably 0.5-5 mm. The proportions of gel filler in shape-stabilized gels according to the invention, e.g. Agarose is 0.1-1.0%, preferably 0.1-12-4%. Attempts to produce such thin gel slices in a conventional manner fail because of the lack of dimensional stability. Surprisingly, it has now been found that these gels can be dimensionally stabilized in a wide variety of ways, so that they can then be introduced into an inventive device.

The shape stabilization takes place in particular by introducing support elements, two inventive variants being described below. In variant A, the space between two tissues (5) is poured out with the warm, liquid gel mass. After cooling, the gel mass becomes solid and is correspondingly stabilized in shape by the tissue, so that gel layers with low agarose concentrations can also be used. Examples 5 and 7 describe the production in detail. 7b shows this layer-like stabilization structure with the two tissues (5) and the gel layer (34) in between. This overall structure is referred to below as a shape-stabilized gel (33). Another inventive variant B (Example 7) is the use of a sintered plastic as a support element which is soaked with initially still liquid gel so that after the gel has solidified, its pores are filled with gel. Such sintered plastics are made of polyethylene, polypropylene or similar material and are foamed by a sintering process, so that a wide variety of capillary structures arise. The dimensions of the capillaries are in particular 1 to 200 μm, preferably 20 to 1 20 μm. Such form-stabilized gels serve the purpose that they are permeable to charged molecules, in particular biomolecules in certain molecular weight ranges, but that they also represent liquid barriers. Example 7 describes further details of the production of this variant B.

Such shape-stabilized gels are widely used in a wide variety of blotting techniques. Here, when using gels according to the prior art without shape stabilization, tearing can occur as a result of improper handling, since it is precisely for these techniques that gels have to be transferred manually several times. By using the shape stabilization according to the invention there is a great advantage in terms of simple and safe handling when transferring the gels, since protection is provided with regard to the destruction of the gel body.

When using the above-described open devices, account must be taken of the fact that, depending on the design in detail, an electroosmotic flow (EOF) must be expected. This flow, triggered, for example, by capillary openings in the electroelution area, can significantly influence the electroelution process, even making it impossible. In particular, the fact that individual chambers run empty in conventional systems, that is to say the transfer of almost the entire liquid volume from one chamber to another due to Electro-osmosis terminates the elution process. If the EOF is used in such a way that on the one hand it leads to a moderate reduction in the volume of the sampling space, but on the other hand it does not hinder the migration of the charged molecules too much, it can be concentrated, which is advantageous for the entire process. Surprisingly, it has been found that this can be achieved with shape-stabilized gels, for which further details are described in the examples.

Another problem is the use of a suitable voltage for the inventive transfer processes. For safety reasons, it makes sense here to remain below 42 V DC in the so-called low-voltage range, since then it is not necessary to cover the current-carrying reagent channel. Above all, this makes automation easier. Surprisingly, it was found that this can be achieved by using a buffer with higher ionic conductivity in the anode and cathode compartment. The buffer can be 2 - 30 times, but preferably 5 - 1 5 times more concentrated than in the inner chambers. A shape-stabilized gel between the sampling space (10) and an adjacent anode chamber (8), which is filled with a higher buffer concentration as described above, acts according to the invention like a semipermeable membrane and protects the charged molecules from reaching the anode (4) and, if necessary, to be decomposed. As an inventive advantage, the shape-stabilized gel is more permeable to detergents.

To solve special concentration and focusing questions, buffer gradients between the electrode chambers and the chambers in the interior can also be used according to the invention, the more concentrated buffers being used on the electrodes. Multi-buffer systems have also proven to be inventive in which adjacent chambers with different buffer substances and different pH values are used. This advantage can also be used by mixing different buffers during sample entry. The use of shape-stabilized gel layers in a reaction channel according to the invention, taking into account the boundary conditions described above, enables a variety of applications in which charged molecules can be transferred and at the same time the environment can be processed, as will be described in more detail below.

A device according to Fig. 7a with a tissue (5), a shape-stabilized gel layer (33) and a semipermeable membrane (1 4) as liquid barriers can be used as follows to isolate e.g. Nucleic acids are used. Nucleic acid is introduced into the sample entry space (10), the device is filled with electrolyte or electrophoresis buffer to such an extent that the intermediate fittings still protrude, and a DC voltage is applied to the electrodes. The nucleic acid travels through the shape-stabilized gel (33) and accumulates in front of the semipermeable membrane (1 4) in the sampling space (7). Due to the electroosmotic flow, the liquid level in chamber (7) is reduced and the nucleic acid is removed in a concentrated form. Depending on the proportion of gel filler in the stabilized gel, the nucleic acids can also be selected according to molecular weight.

7f is suitable for isolating nucleic acids from biological samples. Here, a semipermeable membrane (14) closes off the cathode space (6) and represents the connection to the sample entry space (1 0). Towards the anode there are two shape-stabilized gels (33) and (33a), which separate the sampling space (7) and the Limit anode space (8). A lysis mixture with released nucleic acids obtained from a biological sample is introduced into the sample entry chamber (10). The lysis mixture contains to release the nucleic acids and destroy the biological structures detergents such as lithium, sodium dodecyl sulfate, cetylammonium bromide, ionic surfactants or ionic glycosides or the like. These are used in concentrations of 0.1-1.0%, but preferably 0.1-2%. However, chaotropic salts such as guanidinium thiocynate, guanidinuium hydrochloride, iodine salts, perchlorates or the like can also be used. The device is filled with electrolyte solution and a voltage is applied. The shape-stabilized gel (33) has a small proportion of gel filler, which is 0.1 2% -0.2% and not only retains coarser cell debris but also larger protein aggregates. The released nucleic acids and the detergent, eg sodium dodecyl sulfate, passes through this intermediate installation, the detergent passing through the chamber (7) into the anode chamber (8) faster than the nucleic acid due to its smaller molecular weight. The shape-stabilized gel (33a) thus separates the nucleic acids from the detergent. The nucleic acids are retained by this form-stabilized gel (33a) and removed from the chamber (7). In this case, a semipermeable membrane has to be omitted: on the one hand, the protection provided by the shape-stabilized gel (33a) is sufficient; on the other hand, it has surprisingly been found that detergents such as SDS form-stabilized gels generally pass easily, while presumably due to mycelial formation, semipermeable membranes as detachments retain the detergent . The electrophoretic process takes 1 to 30 minutes, but preferably 2 to 1 2 minutes, and can be automated very easily by placing the device according to the invention on a pipetting robot together with a suitable power supply unit. The liquids can then be pipetted automatically, and for the isolation process an electrical signal is provided by the pipetting robot to turn the power supply on and off. The isolated nucleic acid can then be removed with the robot and used for further processing.

If the direct depletion of the detergent from the lysis mixture is not sufficient, then in a device according to FIG. 7d with two form-stabilized ones Gel layers (33) and (33a) are used as liquid barriers, a modified process for isolating nucleic acids as follows. In this case, particles, preferably those which have magnetic properties, are introduced into the cathode compartment (6) which have been previously loaded with nucleic acid and, if appropriate, also treated with washing solutions. The first fabric (5) is designed so that it retains the particles. After filling with electrolyte and applying a voltage to the electrodes (3,4), charged biomolecules migrate through the tissue into the chamber (35) and through the shape-stabilized gel (33) as before, but are then retained by the shape-stabilized gel (33a) , while detergent residues that are adsorbed on the particles, for example, pass through them. The detergent-free nucleic acid is removed from the sampling space (7). To remove larger amounts of detergent, for example greater than 2% in the case of sodium dodecyl sulfate, special chambers can also be used according to the invention which contain detergent-binding agents or agents which precipitate detergent. It is also possible to add detergent-binding agents after lysis and then to separate them by means of electroelution. Hydrophobic macroporous particles with a large inner surface, such as, for example, detergent adsorber gel [Fa. Röche Diagnostics, Mannheim Cat. No.: 1 500 678] can be used. In addition, the aforementioned materials can also be used in sintered form as an intermediate installation (28) in order to bind detergents.

Depending on the concentration of the agarose in agarose gels or the polyacrylamide in polyacrylamide gels, the capillary structures of the gels can be changed and these materials are used as molecular sieves in the electrophoresis process ("Electrophoresis Theory, Technics and Biochemical and Clinical Applications" AT Andrews (Ed.) ISBN 0- 0- 1 9-854633-5, Clarendon Press, Oxford 1 988. However, this requires correspondingly large hikes (2-80 cm). Surprisingly, however, it was found that with the device according to FIG. 7, even in thin form-stabilized gel layers of 0, 1 - 20 mm, but preferably 0.5 - 5 mm, separation of charged molecules can take place, so that in addition to the liquid barrier described above, the function as molecular sieves can also be used for the isolation of charged molecules.

In addition, the shape-stabilized gel layer can also be used to protect the anode and replace the semipermeable membrane (14). If agarose is used, the agarose concentration for this should be 2-5%, preferably 2.5-3.5%.

Another application is the isolation of ribonucleic acid (RNA), preferably messenger RNA (mRNA) from biological samples, ie in the presence of genomic homologous DNA. In this case, the processing of the lysis mixture consists according to the invention in a DNase digestion during the nucleic acid transfer step. Since the electrolyte solution generally has a pH of 7.25 ± 0.5, this process can be carried out in a device according to FIG. 7d when using a DNase with a p1> 7, preferably p1 8-9. The detergent-containing lysis mixture resulting from the biological sample is filled into the cathode chamber (6), the reaction channel is filled with electrolyte and the transfer is started by applying a voltage to the electrodes. Cell residues and coarse impurities remain on the tissue (5). Instead of the tissue, a third shape-stabilized agarose gel can also be used. Nucleic acids, in this case DNA and RNA, pass this intermediate installation together with the detergent if it is also negatively charged. However, the detergent migrates faster and sometimes also passes through the subsequent shape-stabilized gel (33). In this way, the chamber (35) becomes detergent-free and DNA degradation can be carried out using DNase, which is inhibited by detergents. For this purpose, the voltage is interrupted and a DNase solution is added to the reaction chamber (35), if necessary briefly incubated, and the transfer process is then continued. After the DNA has been broken down, the Suitable inventive selection of the p1 value of the DNase can be achieved so that it is separated from the remaining RNA and an RNA preparation can be removed DNase-free from the chamber (7). The corresponding detergent has meanwhile penetrated through the shape-stabilized gel (33a) into the anode chamber (8). Due to its pl value, the DNase is, for example, positively charged and migrates in the opposite direction to the RNA. In a somewhat modified application, the DNA is digested by means of DNase bound to the solid phase, which is pipetted into chamber (35). This variant has the advantage that it is independent of the pl of the DNase used. This also applies when using thermolabile DNases, which can be destroyed in the subsequent RT-PCR, for example in a first heating step.

Another application that avoids the previously necessary interruption of the power supply for a pipetting step, which in turn is a hindrance to rapid automatic processing, uses a DNase with a pI less than the pH of the electrolyte. In this case the nuclease migrates together with the nucleic acid. By using a device according to FIG. 7e with an additional form-stabilized gel (33b) compared to FIG. 7d at the cathode (3), in which the nuclease is filled with the electrolyte into the reaction chamber (35b) and the lysed sample into the sample entry chamber (10) the nuclease migrates slower than the detergent but faster than the DNA from the sample and destroys it.

In addition to the above-mentioned possibilities of deactivating the nuclease, according to the invention there is the possibility of introducing solid-phase-bound antibodies against the nuclease or solid-phase-bound proteinase K into the shape-stabilized gel (33), which bind or digest and thus deactivate the enzyme. Such processes would then proceed, for example, as follows in devices according to FIG. 7d: The chamber (6) is filled with the lysed sample and the remaining chambers with electrolyte. The electro elution is started and interrupted after 1 -5 min, but preferably 1 -2 min. Solid phase-bound DNase is then introduced into the reaction chamber (35) and incubated. The electroelution is then continued and the DNA-free sample is removed from the sampling chamber (7). It has been shown that in practice a small proportion of DNase can detach itself in this process and, depending on the pI value, migrate with the sample into the sampling chamber (1 0), which is unfavorable for further processing. In this case, solid phase-bound proteinase K according to the invention or antibodies against the DNase in soluble or solid phase-bound form can be added to the reaction chamber (35) before the electroelution is started again, so that the DNase is deactivated. Care must be taken that proteinase K in particular does not get into the sampling chamber (10), since this can, for example, disrupt the subsequent PCR in that the Taq polymerase used there is deactivated. The pI of the complete proteinase K is at pH 6.60, whereas the pI of a particularly active partial fragment (amino acids 106-384) is 8.25. This fragment is particularly suitable for inventive use, since it is positively charged at neutral pH and migrates to the cathode and does not migrate into the sampling space.

A further possibility is also that solid phase-bound proteinase K or else solid phase-bound antibodies against the DNase are added to the still liquid gel and are thus kept in a shape-stabilized gel layer, which in turn saves a pipetting step. A comparable application is the removal of RNA from a DNA / RNA mixture by treatment with RNase or the isolation of protein mixtures using analogous methods.

Another inventive solution is the use of buffers with different pH values in the different chambers of the devices described here. Depending on the pl values of the added proteins or enzymes, an adjacent chamber can be filled with a pH such that the desired charged molecules such as nucleic acids pass through this chamber, but the additives do not. A conceivable application is shown in perspective in FIG. 7g and in section in FIG. 7h. It is an agarose flat bed gel (36) with three incorporated pockets, which are used as a sample entry chamber (1 0), reaction chamber (35) and sample removal chamber (7). Care must be taken to ensure that the electrolyte does not rise above the upper edge of the flatbed gel in order to avoid mixing of the various reactants, especially in chamber (35). The processes described here cannot be carried out in devices of this type, since the chambers (35) and (7) run empty as a result of EOF and thus no stable electroelution is possible.

The invention is further illustrated by the attached figures and examples. The figures show:

Fig. 1 a Perspective view: Rectangular reaction channel with intermediate installation Fig. 1 b Top view: Rectangular reaction channel with constriction

Fig. 1 c basic element

Fig. 1 d intermediate installation

Fig. 1 e sealing element

Fig. 1 f electrode holder Fig. 1 g holder with clamping device

Fig. 1 h microtitration frame with 48 reaction channels

Fig. 1 i basic element with electrode holders and clamping

Fig. 1 j basic element with opening and clamping

Fig. 2a Top view: reaction channel with intermediate installation Fig. 2b section: reaction channel with intermediate installation

Fig. 3 reaction channel with two internals

Fig. 4 reaction channel with liquid barrier Fig. 5a liquid barrier open

Fig. 5b liquid barrier closed

Fig. 6 reaction channel with liquid barrier and membrane

7a device for electroelution with dimensionally stabilized gel according to variant A.

Fig. 7b device for electroelution with shape-stabilized gel after

Variant B Fig. 7c device with three chambers Fig. 7d device with two shape-stabilized gel layers

Fig. 7e device with three form-stabilized gel layers. Fig. 7f perspective view of a flat bed gel

Reaction chambers Fig. 7g section through a flat bed gel with reaction chambers

Fig. 8 reaction channel with permanent magnet

Fig. 9a device for electroelution Fig. 9b device for electroelution with frit

Fig. 9c device for electroelution with particles

Fig. 10a pieces of agarose gel before and after electroelution Fig. 10b Agarose gel for electroelution with device Fig. 9a Fig. 1 1 Agarose gel for electroelution with device Fig. 9b Fig. 1 2 Agarose gel for nucleic acid isolation from yeast by means of

Electroelution in Fig. 9c Fig. 1 3 standard agarose gel for analysis of electroelution after

Example 5 Fig. 14 Standard agarose gel for analysis of electroelution according to example 7

FigJ 5 standard agarose gel on a support made of sintered

Plastic (Example 1 0) Fig.1 6 standard agarose gel for analysis of genomic isolation

DNA and RNA from yeast Example 1 1. Fig. 1 7 standard agarose gel after PCR with different

Electroeluates Fig. 1 8 standard agarose gel for analysis of the isolation genomic

DNA from yeast before amplification. Fig. 1 9 standard agarose gel for analysis of the isolation genomic

DNA from yeast after amplification. Fig. 20 standard agarose gel for analysis of the isolation genomic

DNA from yeast before amplification using a detergent adsorber. Fig. 21 Standard agarose gel for analysis of genomic isolation

DNA and RNA from yeast after amplification using a detergent adsorber

Fig. 22 Standard agarose gel for analysis of genomic isolation

DNA from yeast before amplification using a

Two-component buffer system.

23 Standard agarose gel for analyzing the isolation of genomic DNA and RNA from yeast after amplification using a

Two-component buffer system. Fig. 24 Standard agarose gel for analysis of genomic isolation

DNA from yeast after amplification in a kinetic. 25 electroelution of a fluorescent dye in a device with four chambers, the charged dye migrating through a shape-stabilized gel. The structure of the device used is as follows (from left to right :)

Cathode, cathode compartment, membrane, sample entry chamber, shape-stabilized gel, sampling chamber, shape-stabilized gel,

Anode chamber, anode.

It should be noted that after a short time of 1 20 s, the charged dye has already been completely transferred to the sampling chamber without a fluorescent dye being visible in the anode chamber.

26 shows a schematic representation of the device from FIG. 25. Fig. 27 Results of a radioimmunoassay using electroelution.

Reference symbol list:

1) Base body closed at the bottom

2) Channel open at the top

3) Electrode A, e.g. cathode

4) electrode B, e.g. anode

5) tissue

6) Cathode room or chamber

7) Sampling room or chamber

8) Anode compartment or chamber

9) Clamping cone or chamber

1 0) Sample entry space or chamber

1 1) Electrophoresis separation section

1 2) Guide in base body for built-in elements

1 3) Liquid barrier

1 4) Semipermeable membrane

1 5) reaction chamber or chamber (open)

1 6) Latch

1 7) Permanent magnet

1 8) Microtitration plate frame

1 9) Microtitration plate strips

20) mating surface

21) Integrated connecting element

22) Solid phase

23) tensioning device

24) basic element

25) sealing element

26) Recording

27) frit

28) Intermediate internals

29) Laminating film (30) Electrical supply line

(31) Opening to the reaction channel

(32) Constriction

(33) shape stabilized gel (34) gel layer

(35) reaction chamber

(36) agarose flat bed gel

Examples 1.) Measurement of electroosmotic flow

For this application, a device according to Fig. 9a with two fabrics (5) [No. 07/5/1 from SEFAR, Rüschlikon, Switzerland] and a semipermeable membrane (14) [Size 3; Medicell Int. Ltd., London, UK], which then delimit a cathode space (6), a sample entry space (10) and an anode space (8). The device was made up of individual basic elements according to FIG. 1 c [No. : 01 1 1 1 20 005 05; J. Pützfeld B.V., Amsterdam, Netherlands]. 3 elements were glued for the anode and cathode compartments, the tissue (5) and the semipermeable membrane (14) were each placed in a perforated laminating film [250 μm thick, No. 54x86; Böttcher, Jena] with a laminator [Lamirel MAXIPLAST 335 E] welded and with silicone seals [1 mm thick No. 6084.0810, H. Wegener, Hamburg] between the above. Basic elements clamped liquid-tight with a screw clamping device. Aluminum plates served as electrodes (3,4) and an electrophoresis transformer [Fa. Hölzel] used.

The device was filled with electrophoresis buffer (10 mM Tris / acetate [from Roth, Karlsruhe], 1 mM EDTA [from Sigma], pH 8.0) and a voltage of 40-80 was applied to the electrodes (3,4) V applied at a constant 10 mA for 10 min. The following table shows the volumes in the individual chambers before and after electroelution.

This experiment shows the reduction in the buffer volume in the sampling space (7). For all of the examples below, materials such as Example 1) were used, unless stated otherwise.

2.) Electroelution of DNA from agarose gels

2.5 μg DNA (λ Hind IM) [No. # SM01 01; MBI Fermentas, St. Leon-Roth] was in a standard flat bed agarose gel (0.8% agarose [Sigma, Munich]) in an electrophoresis buffer (see above) with a running time of 10 min. (approx. 3 V / cm) transferred into the gel and the band with SYBR ^ gold [S-1 1 494; Mo Bi Tee, Göttingen] stained. The corresponding gel fragment i containing the DNA was cut out with a scalpel and is shown in Fig. 10a before and after electroelution.

The excised agarose gel was brought into the sample entry space and the entire device according to Fig. 9 a was filled with 1.9 ml electrophoresis buffer (see above). A voltage between 40-60 V at a constant 10 A for 7 or 10 minutes was applied to the electrodes with an electrophoresis transformer. created.

Fig. 10 b shows the result of the electroelution in a standard agarose gel (see above):

3) Electroelution with a device according to Fig. 9b

For this application, a device according to Fig. 9b with a fabric (5), a sintered plastic frit [No. XS-561 6, from Porex. Technologies GmbH, Singwitz] and a semipermeable membrane (1 4), which then delimit a cathode space (6), a sample entry space (1 0) and an anode space (8). The device was assembled as described in Example 1.

2.5 μg DNA (λ Hind IM) was brought directly into the sample entry space and the entire device according to FIG. 9 b was filled with 1.75 ml electrophoresis buffer (see Example 1). A voltage of 70 V at a constant 10 mA for 10 minutes was applied to the electrodes with an electrophoresis transformer. created.

Fig. 1 1 shows the result of the electroelution in a standard agarose gel (see above) with withdrawals from the sample entry space and immediately next to it, sample removal space in a kinetics of 0-1 0 min. M denotes the track of the marker as in Example 1. 4.) Isolation of nucleic acid from yeast using magnetic particles and electroelution

For this application, a device with a fabric (5) [Fa. SEFAR see Example 1] and a dialysis membrane [Fa. Medicell see Example 1] as used in Fig. 9c.

1 x ¹⁰ 0 yeast cells per ml (Saccharomyces cereviciae) were in Lyticase buffer (1 M sorbitol [from AppliChem, Darmstadt], 100 mM sodium citrate [from ICN, Aurora, USA], 60 mM EDTA [from Sigma], 50 mM dithioerythritol [from Roth], pH 7.5) and 10 μl (1 0 ⁸ cells) thereof with 1 μl lyticase [No. 1 372 467, Röche Diagnostics, Mannheim] and incubated for 1 5 min at 37 ° C.

The lysis and the binding and washing of the magnetic particles were carried out in accordance with US Pat. No. 5,705,628. After the last washing step, the magnetic particles were separated, the washing solution was carefully removed and the particles were taken up in 150 μl elution buffer (see Example 1 for electrophoresis buffer). The suspension was then filled into the sample entry chamber (in this case identical to the cathode compartment (6)) of a device according to FIG. 9 c that had already been filled with elution buffer. The electroelution was carried out by applying a voltage of 40-70 V at 1 mA current flow for 5 min. The nucleic acid was pipetted from the removal chamber and subjected to agarose gel electrophoresis as in Example 1. Fig. 1 2 shows the associated gel picture.

5.) Electroelution with a device according to Fig. 7a

For this application, a device according to FIG. 7a with a fabric (5) [No. 07/5/1 from SEFAR, Rüschlikon, Switzerland] and a semipermeable membrane (14) [Size 3; Medicell Int. Ltd., London, UK], which initially delimit a cathode compartment (6) and an anode compartment (8). In addition, a shape-stabilized gel (33) is introduced between these two internals, which delimits a sample entry space (10) and a sample removal space (7), each with a volume of approximately 250 μl. The device was made up of individual basic elements [No. : 01 1 1 1 20 005 05 J. Pützfeld B: V., Amsterdam, Netherlands]. For each of the anode (8) and cathode compartments (6), 3 elements with a volume of approx. 750 μl were glued, the tissue (5) and the semipermeable membrane (14) were each placed in a perforated laminating film [250 μm thick, No. 54x86 ; Böttcher, Jena] welded in with a laminator [Lamirel MAXIPLAST 335 E] and with silicone seals [1 mm thick No. 6084.081 0, H. Wegener, Hamburg] between the above. g. Liquid-tight elements are clamped in with a screw clamping device.

According to variant A, the gel was dimensionally stabilized as follows: A sintered plastic [pore size 45-90 μm, No. X-4899, from Porex., Singwitz] was mixed in a heated mixture of agarose (0.4% - 3%) [ Fa. Sigma, Munich] and electrophoresis buffer 1 0 mM Tris / acetate [Fa. Roth, Karlsruhe] with 1 mM EDTA [Fa. Sigma] pH 8.0), the agarose was allowed to solidify and the shape-stabilized gel (33) was introduced into the device according to FIG. 7a.

2.5 μg DNA (λ Hind IM) [No. # SM01 01; MBI Fermentas, St. Leon-Rot] was brought directly into the sample entry space and the entire device was filled with 1.75 ml electrophoresis buffer (see before). To the

Electrodes were with an electrophoresis transformer [Fa. Hölzel, Dorfen] a voltage of 50 - 500 V at a constant 10 mA for 10 min. created.

Fig. 13 shows the result of the electroelution with different agarose concentrations in the shape-stabilized gel, wherein both withdrawals from the sample entry space (10) and from the sample removal space (7) were analyzed using a 0.8% standard agarose gel (see above) with a kinetics of 0-10 min. Under the heading "gel traces" is the time after which DNA was detectable on a gel. Agarose conc. Gel traces in stable shape. Gel (33)

Marker (Λ-Hindlll): 23.1 j 9.416.614.412.312.01 0.56 KB 0.4% sample entry space (10) before electroelution 0 min. 0.4% sampling chamber (7) after electroelution 0 min

0.4% sample entry space (10) before electroelution 1 min.

0.4% sampling chamber (7) after electroelution 1 min

0.4% sample entry space (10) before electroelution 2 min.

0.4% sampling chamber (7) after electroelution 2 min 0.4% sample entry space (10) before electroelution 3 min.

0.4% sampling chamber (7) after electroelution 3 min

0.4% sample entry space (10) before electroelution 4 min.

0.4% sampling chamber (7) after electroelution 4 min

0.4% sample entry space (10) before electroelution 5 min. 0.4% sampling chamber (7) after electroelution 5 min

0.4% sample entry space (10) before electroelution 7 min.

0.4% sampling chamber (7) after electroelution 7 min

0.4% sample entry space (10) before electroelution 10 min.

0.4% sampling chamber (7) after electroelution 10 min marker (Λ-Hindlll): 23.1 | 9.4 | 6.6 | 4.4 | 2.3 | 2.0 | 0.56KB , 8% sample entry space (1 0) before electroelution 0 min. , 8% sampling chamber (7) after electroelution 0 min, 8% sample entry space (10) before electroelution 1 min. , 8% sampling chamber (7) after electroelution 1 min, 8% sample entry space (1 0) before electroelution 2 min. , 8% sampling chamber (7) after electroelution 2 min, 8% sample entry space (1 0) before electroelution 3 min. , 8% sampling chamber (7) after electroelution 3 min, 8% sample entry space (10) before electroelution 4 min. , 8% sampling chamber (7) after electroelution 4 min, 8% sample entry space (1 0) before electroelution 5 min. , 8% sampling chamber (7) after electroelution 5 min, 8% sample entry space (1 0) before electroelution 7 min. , 8% sampling chamber (7) after electroelution 7 min, 8% sample entry space (10) before electroelution 10 min. , 8% sampling chamber (7) after electroelution 10 min marker (Λ-Hindlll): 23, 1 | 9.4 | 6.6 | 4.4 | 2,3 | 2.0 | 0, 56KB, 2% sample entry space (1 0) before electroelution 0 min. , 2% sampling chamber (7) after electroelution 0 min, 2% sample entry space (10) before electroelution 1 min. , 2% sampling chamber (7) after electroelution 1 min, 2% sample entry space (1 0) before electroelution 2 min. , 2% sampling chamber (7) after electroelution 2 min, 2% sample entry space (1 0) before electroelution 3 min. , 2% sampling chamber (7) after electroelution 3 min, 2% sample entry space (1 0) before electroelution 4 min. , 2% sampling chamber (7) after electroelution 4 min, 2% sample entry space (10) before electroelution 5 min. , 2% sampling chamber (7) after electroelution 5 min, 2% sample entry space (1 0) before electroelution 7 min. . 2% sampling chamber (7) after electroelution 7 min 1, 2% sample entry space (1 0) before electroelution 10 min.

1, 2% sampling chamber (7) after electroelution 10 min

Markers (Λ-Hindlll): 23, 1 | 9.4 | 6.6 | 4.4 | 2,3 | 2.0 | 0.56KB 3.0% sample entry space (1 0) before electroelution 0 min. 3.0% sampling chamber (7) after electroelution 0 min

3.0% sample entry space (1 0) before electroelution 1 min.

3.0% sampling chamber (7) after electroelution 1 min

3.0% sample entry space (1 0) before electroelution 2 min.

3.0% sampling chamber (7) after electroelution 2 min 3.0% sample entry space (10) before electroelution 3 min.

3.0% sampling chamber (7) after electroelution 3 min

3.0% sample entry space (10) before electroelution 4 min.

3.0% sampling chamber (7) after electroelution 4 min

3.0% sample entry space (10) before electroelution 5 min. 3.0% sampling chamber (7) after electroelution 5 min

3.0% sample entry space (1 0) before electroelution 7 min.

3.0% sampling chamber (7) after electroelution 7 min

3.0% sample entry space (1 0) before electroelution 1 0 min.

3.0% sampling chamber (7) after electroelution 10 min

You can see the decrease in the amount of DNA (Λ Hind III) in the sample entry area (1 0) and the increase in the sample removal area (7). In addition, the bands with a smaller molecular weight are retained at higher agarose concentrations, so that selection of certain nucleic acids can be achieved in this way with a shape-stabilized gel. 6.) Measurement of the electroosmotic flow with a device according to Fig. 7a

For this application, a device according to FIG. 7a and with a shape-stabilized gel of variant A with 0.8% agarose was operated. All other experimental conditions are as in Example 1. The final volumes of the sample entry space (1 0) and the sampling space (7) are in a kinetics of 2 - 1 0 min. compiled in the following table:

By using a shape-stabilized gel, the volume in the sampling space (7) is reduced, so that the eluted nucleic acid is concentrated accordingly.

7.) Electroelution with a device according to Fig. 7b

For this application, a device according to FIG. 7b was used, which contains a shape-stabilized gel according to variant B. The preparation was carried out as follows: Two layers of tissue (5) were placed in the device at a distance of approximately 2 mm and the interspace was filled with a heated mixture of agarose (0.4%) and electrophoresis buffer. The agarose was cooled and thus allowed to solidify.

2.5 μg DNA (Λ Hind III) was brought directly into the sample entry space (10) and the entire device was operated as in Example 5 and the result was taken from the sampling chamber (7) and analyzed. Fig. 1 4 shows the result of electroelution, both Withdrawals from the sample entry space (10) and from the sampling chamber (7) were analyzed using an 0.8% standard agarose gel (see above) with an elution rate of 0-1 0 min. Agarose conc. Gel traces in stable shape.

Gel (33)

Markers (Λ-Hindlll): 23, 1 | 9.4 | 6.61 4.4} 2.3 j 2.01 0.56KB 0.4% sample entry space (10) before electroelution 0 min.

0.4% sampling chamber (7) after electroelution 0 min 0.4% sample entry space (10) before electroelution 1 min.

0.4% sampling chamber (7) after electroelution 1 min

0.4% sample entry space (1 0) before electroelution 2 min.

0.4% sampling chamber (7) after electroelution 2 min

0.4% sample entry space (1 0) before electroelution 3 min. 0.4% sampling chamber (7) after electroelution 3 min

0.4% sample entry space (1 0) before electroelution 4 min.

0.4% sampling chamber (7) after electroelution 4 min

0.4% sample entry space (10) before electroelution 5 min.

0.4% sampling chamber (7) after electroelution 5 min 0.4% sample entry space (1 0) before electroelution 7 min.

0.4% sampling chamber (7) after electroelution 7 min

0.4% sample entry space (1 0) before electroelution 10 min

0.4% sampling chamber (7) after electroelution 10 min

The result corresponds to the data in Example 5.

8.) Measurement of the electroosmotic flow with a device according to Fig. 7b

For this application, a device according to FIGS. 7b and 7 was used with a shape-stabilized gel of variant B with 0.4% agarose used. The entire device according to FIG. 7b was operated as in Example 1.

The final volumes of the sample entry space (1 0) and the sampling chamber (7) are after 10 min. compiled in the following table:

By using a shape-stabilized gel, the volume in the sampling chamber (7) is reduced, so that the eluted nucleic acid is concentrated accordingly.

9.) Electroelution with a device according to Fig. 7c with lower voltages

A device according to FIG. 7c and Example 5 with a shape-stabilized gel (33) of variant A with 0.8% agarose was used for this application. In contrast to Example 5, the entire device was operated with a 10-fold concentrated electrophoresis buffer in the anode compartment (6). 1 0 mA were achieved with this arrangement with a voltage of 20 - 25 V. The volumes were determined before us after electroelution as follows:

This example shows a minimum of electroosmotic flow at low voltage range for electroelution, which can also be advantageous for some applications.

This result shows that electroelution can be carried out with a considerably lower voltage, which is a considerable simplification with appropriate automation, since different safety guidelines apply to these voltages. If the anode compartment and cathode compartment are charged with a buffer of higher concentration, as in Example 11, the voltage drops to below 10 V.

10.) Use of an agarose gel on a carrier made of sintered plastic

A standard agarose gel (0.8%) was poured onto a sintered plastic [X 4588, from Porex, Singwitz] and allowed to cool. DNA (DNA Hind IM) was applied with this gel, electrophoresis was carried out and stained (see above). Fig. 1 5 shows the result.

1 1.) Electroelution with a device according to FIG. 7f in the presence of sodium dodecyl sulfate (SDS)

For this application, a device according to FIG. 7f with a dialysis membrane (14) [Fa. Medicell see Example 1] and two dimensionally stabilized gels (33 and 33a) were used. The agarose concentration was 0.2% in (33) and 2% in (33a) with shape stabilization according to variant A.

30 μl (1 0 ⁸ cells) of a yeast cell suspension (Saccharomyces cereviciae) were mixed with 1 1 5 μl BILATEST lysis buffer I consisting of 5% sodium dodecyl sulfate [L4390 from Sigma, Munich], 100 mM Tris / HCl [T2584 L4390 from Sigma , Munich], 10 mM EDTA [E51 34 from Sigma, Munich] and 5 μl of a Proteinase K solution [1 373 1 96, from Röche, Mannheim] incubated for 1 hour at 55 ° C. and into the sample entry chamber (1 0) transferred. Anode (8) and cathode compartments (6) were each filled with 600 μl of a 10-fold concentrated electrophoresis buffer as in Example 1 and the remaining chambers (7) with 1 50 μl of a 1-fold concentrate. The electroelution was carried out by applying a voltage of 7.5 V at 10 mA current flow for 5 min or 10 min. The nucleic acid was pipetted from the removal chamber (7) and subjected to agarose gel electrophoresis as in Example 1. Fig. 1 6 shows the associated gel picture.

Lane content M marker (Λ-Hindlll): 23, 1 1 9.4 j 6.614.4 ' _t 2.31 2.0 | 0.56 KB

0- sample entry space (10) before electroelution

5- sample entry space (1 0) after electroelution for 5 min.

5 + sampling chamber (7) after electroelution for 5 min.

1 0 sample entry space (1 0) after electroelution for 1 0 min. 1 0 + sampling chamber (7) after electroelution for 1 0 min.

M markers (Λ-Hindlll): 23, 1 | 9.4 | 6.6 | 4.4 | 2,3 | 2.0 | 0.56 KB

The gel picture shows the genomic DNA and RNA.

12) Depletion of sodium dodecyl sulfate using electroelution

In this experiment, a device according to FIG. 7f with an analog structure as example 11 was used. Shape-stabilized gel (33) was prepared with 0.4% agarose and (33a) with 4% agarose. A sodium dodecyl sulfate solution in electrophoresis buffer (see above) was used in the sample entry chamber (10) in a concentration of 0.1% and 1%, respectively, and subjected to electroelution as in Example 11. Thereafter, 1 μl of the solutions from the sample entry chamber (10), the sampling chamber (7) and the anode chamber (8) were added to a standard PCR of the BCY gene of the yeast, as described in more detail in Example 1 3. The PCR products were analyzed using an agarose gel and FIG. 17 shows the result.

Control Add 1 μl of 0.1% SDS solution for PCR Add 10 μl of 0.1% SDS solution for PCR Add 1 μl of 1% SDS solution for PCR Add 10 μl of 1% SDS PCR solution

The result shows that 1 .mu.l of a 0.1% SDS solution does not interfere with the PCR (control), while all higher SDS amounts no longer allow a PCR product. On the other hand, in this example after electroelution from the sampling chamber, 1 μl of a 1% SDS solution can be used for the PCR without interference as a result of the SDS depletion.

13.) Simple isolation of DNA from yeast with subsequent amplification. For this application, a device according to FIG. 7f with a dialysis membrane (14) [Fa. Medicell see Example 1] and two dimensionally stabilized gels (33 and 33a) were used.

The agarose concentration was 0.15% in (33) and in (33a) with shape stabilization according to variant A.

30 μl (1 0 ⁸ cells) of a yeast cell suspension (Saccharomyces cereviciae) were mixed with 1 1 5 μl BILATEST lysis buffer I consisting of 2% sodium dodecyl sulfate [L4390 from Sigma, Munich], 1 00 mM Tris / HCl [T2584 L4390 from Sigma , Munich], 10 mM EDTA [E51 34 from Sigma, Munich] and 5 μl of a Proteinase K solution [1 373 1 96, from Röche, Mannheim] incubated for 1 hour at 55 ° C. and into the sample entry chamber (10) transferred.

Anode (8) and cathode compartments (6) were each filled with 600 μl of a 10-fold electrophoresis buffer as in Example 1 and the remaining chambers (7) with 1 50 μl of a 1-fold concentrate.

The electroelution was carried out by applying a voltage of 7.5 V at 1 mA current flow for 2 min. The nucleic acid was pipetted from the removal chamber (7) and subjected to agarose gel electrophoresis as in Example 1. Fig. 1 8 shows the associated gel picture. Trace content

M markers (Λ-Hindlll): 23, 1 | 9.4 | 6.6 | 4.4 | 2,3 | 2.0 | 0.56 KB

1 sample entry area (10) before electroelution

2 sampling chamber (7) after electroelution for 2 min. 3 anode compartment (8) after electroelution for 2 min.

M markers (Λ-Hindlll): 23, 1 1 9.4j 6.6 j 4.4 | 2.31 2, O | 0.56 KB

With this result, it should be pointed out that no DNA can be observed in the anode chamber (8). Surprisingly, a shape-stabilized gel (33a) together with an anode chamber (8), filled with a buffer concentration 10 times higher than in the sampling chamber (1 0), represents a barrier to the nucleic acid, comparable to a semipermeable membrane.

With this isolated yeast DNA a PCR (US 4,683,195) with the primers KV80: 5 '-GCG GAT CCT TAA GTC CAA TCG TCA AAA TT- 3' KV102: 5 "-GCG AAT TCG TAT CTT CTT TGC CCA AGG AA-3 '[MWG Biotech AG, Ebersberg near Munich] was carried out using these primers to amplify a 496bp fragment from the yeast BCY gene. The PCR batches contain the following components:

0.5 μL primer solution 1 (50 pmol μL ^"1 in H ₂ O bidest)

0.5 μL primer solution 2 (50 pmol μL ^"1 in H ₂ O bidest)

2 μL dNTP solution, concentration of the nucleotides each

5mM (Eurogentec, Seraing, Belgium)

4 μL MgCI ₂ solution 25mM (Eurogentec)

5 μL 1 0x PCR buffer (Eurogentec) 0.5 u Taq polymerase (Eurogentec) up to 30 μL DNA or sample solution

in a total volume of 50 μL. During the preparation, the PCR plate is cooled to 4 ° C. After adding the last solution, the Mix samples once with a pipette. The PCR is carried out in thermosprint plates [Fa. Innova GmbH, Mannheim) in the Primus 96 Plus [MWG Biotech AG, Munich]. The program includes the following steps:

Cover heating to 1 1 0 ° C

3 min 94 ° C

27 cycles with

30 s 94 ° C 30 s 50 ° C 2 min 72 ° C

5 min 72 ° C

Cooling to 4 ° C. After the PCR, 1 5-20% gel loading buffer with EDTA [Sigma, Munich] is added. The amplificates were analyzed on a standard agarose gel (1.6%). Fig. 1 9 shows the result.

In this experiment, genomic yeast DNA was obtained from a SDS-containing lysis mixture with electroelution, which was subsequently used directly in a PCR.

14.) Simple isolation of DNA from yeast with subsequent amplification and use of a detergent adsorber

For this application, a device according to FIG. 7f according to Example 1 3 was carried out. In contrast to Example 1 3, 50 μl detergent adsorber [Röche Diagnostics Mannheim, Cat. No. 1 500 678] was added after lysis and the resulting mixture was added to the sample entry chamber. The electroelution was carried out at a voltage of 1 0, 1 V and 1 0 mA current for 1 0 min.

A standard agarose gel was carried out to analyze the amplification (FIG. 20):

A PCR was then carried out using the eluates according to Example 13 and analyzed as above (FIG. 21).

1 5.) Simple isolation of DNA from yeast with subsequent amplification and use of a two-component buffer system (Tris / HCl and phosphate buffer)

For this application, a device according to FIG. 7f according to Example 1 3 was carried out. In contrast to Example 1 3, 50 μl of a phosphate buffer (250 mM pH 7.0) consisting of Na ₂ HPO ₄ and NaH ₂ PO ₄ was obtained after lysis [Merck, Darmstadt, Cat. No. 1 .06580J 000 or 1 .06346J 000] was added and the resulting mixture was added to the sample entry chamber. The electroelution was carried out at a voltage of 8.6 V and 10 mA current for 10 minutes.

A standard agarose gel was carried out for analysis before amplification (FIG. 22):

A PCR was then carried out with the eluates according to Example 13 and analyzed as above (FIG. 23).

16.) Simple isolation of DNA from yeast with subsequent amplification and use of a two-component buffer system (Tris / HCl and citrate buffers). A device according to FIG. 7f according to Example 13 was carried out for this application. In contrast to Example 1 3, 5 μl of a citrate buffer (1 M pH 4.5) [Appischem, Darmstadt, Cat. No. A 2337] was added after lysis 1 and the resulting mixture into the sample entry chamber given. The electroelution was carried out at a voltage of 8.5 V and 10 mA current with sampling from 0-6 min.

A PCR according to Example 13 was then carried out with the withdrawals and analyzed as above (FIG. 24).

Trace content

M markers (Λ-Hindlll): 23.1 | 9.4 | 6.614.4 j 2.312.01 0.56 KB

1 sampling chamber (7) after electroelution for 0 min. after PCR

2 sampling chamber (7) after electroelution for 0 min. after PCR 3 anode compartment (8) after electroelution for 0 min. after PCR

M marker (Λ-Hindlll): 23.119.416.614.4 j 2.3 j 2.01 0.56 KB

1 sampling chamber (7) after electroelution for 2 min. after PCR

2 sampling chamber (7) after electroelution for 2 min. after PCR

3 anode compartment (8) after electroelution for 2 min. after PCR M marker (Λ-Hindlll): 23.1 | 9.4 | 6.6 | 4.4 | 2.3 | 2.0 | 0.56 KB

1 sampling chamber (7) after electroelution for 4 min. after PCR

2 sampling chamber (7) after electroelution for 4 min. after PCR

3 anode compartment (8) after electroelution for 4 min. after PCR M marker (Λ-Hindlll): 23.1 j 9.416.6 j 4.412.3 | 2.0 | 0.56 KB 1 sampling chamber (7) after electroelution for 6 min. to

PCR

2 sampling chamber (7) after electroelution for 6 min. after PCR

3 anode compartment (8) after electroelution for 6 min. after PCR M marker (Λ-Hindlll): 23.1 | 9.4 | 6.6 | 4.4 | 2.3 | 2.0 | 0.56 KB This experiment shows that amplified material was only detected in the sampling chamber (7).

17.) Production of shape-stabilized gels According to variant A, the gel was shape-stabilized as follows: A sintered plastic [pore size 45-90 μm, No. X-4899, from Porex., Singwitz] was mixed in a heated mixture of agarose (0, 4% - 3%) [Fa. Sigma, Munich] and electrophoresis buffer 10 mM Tris / acetate [Fa. Roth, Karlsruhe] with 1 mM EDTA [Fa. Sigma] pH 8.0), the agarose was allowed to solidify and the shape-stabilized gel (33) was introduced into the device according to FIG. 7a.

According to variant B, the production of a shape-stabilized Gesl was carried out as follows: Two layers of tissue (5) were placed in the device at a distance of approx. 2 mm and the space in between was filled with a heated mixture of agarose (0.4%) and electrophoresis buffer . The agarose was cooled and thus allowed to solidify. Variant C with acrylamide:

18.) Use of shape-stabilized gels for the separation of an immunologically active fluorescent dye

25, 26 consisting of two electrode chambers, each with a volume of 600 μl, an entry chamber (1 0) [1 60μl] and a removal chamber [1 60μl], with the intermediate fittings dialysis membrane (1 4), and two shape-stabilized gels (33, 33a) according to variant A with 0.2% agarose, the electrode chambers were each with twice 600μl 1 0 times concentrated electrophoresis buffer from Example 17, the removal chamber (7) with 1 50μl electrophoresis buffer (see before) and the entry chamber (10) with a solution of Cascade ^® -Blue-biocytin (0.067 mg / ml) [Molecular Probes, Eugene, USA; Order No .: C-6949] filled in electrophoresis buffer as in Example 1. The device according to the invention was on a UV light table [Fa. Vilbert Lourmat, Marne la Vallee, France; Order No .: TFX-20M] and provided with a money detection system [EDAS 1 20, Kodak, Rochester USA]. 25a shows the arrangement at the time 0 seconds as the starting point. A fluorescent band of the dye can be seen in the entry chamber (10). A voltage of approximately 7.5 V was then applied at a constant 15 mA current flow for 120 seconds. Figure 25b shows a band of the dyes in the sampling chamber.

In addition, 1 50 μl of the starting solution and the solution of the sampling chamber after electroelution were placed in a microtitration plate [Fa. NUNC, Wiesbaden; Maxisorb] transferred and recorded accordingly.

19.) Use of shape-stabilized gels for separating an immunological mixture of fluorescent dye and antibody. An anti-biotin antibody [Fa. Sigma, St. Louis, USA; Order No .: B 7653] was diluted 1: 100 in electrophoresis buffer from Example 2; 1 0Oμl thereof were -Blue-biocytin solution incubated with 00μl of 1 Cascade ^® from Example 1 8 at room temperature for 30 min and then subjected to 1 8 electroeluted in an arrangement as Example. After electroelution of 1 20 sec., Hardly any fluorescence can be detected in the removal chamber, since the dye binds to the antibody and, due to the increased molecular weight, cannot migrate through the shape-stabilized gel under the specified conditions. Since the fluorescent property of the dye is quenched as a result of the antibody binding and only the unbound dye is detectable, a protein determination according to Bradford was carried out using a test kit [Fa. BioRad, Munich order no.: 300 0006] in a photometer [BioPhotometer; Eppendorf, Hamburg; Order No .: 61 31 000 42]. After that, the protein concentrations were as follows:

The experiment was repeated with a shape-stabilized gel with 2% agarose:

These experiments show that the free fluorescent dye passes through the shape-stabilized gel and the complex of antibody and fluorescent dye remains in the shape-stabilized gel after electroelution

20.) Use of shape-stabilized gels to carry out a radioimmunoassay using electroelution

A test set for the detection of triiodothyronine (T3) [Fa. Medipan, Selchow; T3-RIA magnum] was used as follows: 50 .mu.l sample or standard were with 50 .mu.l anti-T3 antiserum and 50 .mu.l T3 tracer each from the above test kit for 1.5 hours at room temperature incubated. Subsequently, 80 μl of the incubation mixture and 80 μl electrophoresis buffer (see above) were mixed and filled into the sample entry space (1 0) of a 4-chamber device as shown in FIG. 26 with anode (8) and cathode space (6) each 1, 2 ml volume with 10-fold concentrated electrophoresis buffer and a sampling chamber (7) filled with 1 60 μl electrophoresis buffer. Two agarose filters with 0.2% agarose and a dialysis membrane were used. The electroelution conditions were 30 mA at 20 V and an air time of 1 80 sec. Then 10 μl were removed from the sample entry space (1 0) and in a gamma counter (Berthold Wildbad; LB 21 1 1) for 2 min. measured. The results are shown in Fig. 27.

Since the T3 tracer of this test kit is chemically modified on the carboxyl group and the molecule is therefore uncharged, the bound phase with the negatively charged antibody migrates into the form-stabilized gel. With standard "0" tracer is bound and removed from the sample entry space (10), while with standard 4 e.g. Tracer from the antibody is displaced by cold T3 and a larger amount remains in the sample entry space (1 0).

Claims

claims

1 . Device for isolating electrically charged molecules consisting of a base body (1) closed at the bottom with a channel (2) open at the top and two electrodes (3, 4) as right and left outer boundaries of the channel (2), characterized in that one or more intermediate installation (s) (28) are arranged between the electrodes (3, 4) in the channel (2) and divide the channel into two or more chambers.

2. Device according to claim 1, characterized in that at least one of the and in particular all intermediate fittings (28) on the one hand represents a liquid barrier, on the other hand is permeable to electrically charged molecules.

3. Apparatus according to claim 1 and 2, characterized in that at least one of and in particular all intermediate internals (28) limited by their nature the electroosmotic flow so that electroelution of charged molecules is possible.

4. Apparatus according to claim 1 to 3, characterized in that it is a shape-stabilized gel in the intermediate installation

(33) acts.

5. Apparatus according to claim 1 to 3, characterized in that it is a fabric in the intermediate installation.

6. The device according to claim 5, characterized in that an electroosmotic flow from the anode to the cathode is generated by the tissue (5).

7. The device according to claim 5 or 6, characterized in that the fabric (5) has a mesh size of 0.1 to 50 microns, preferably from 0.5 to 5 microns.

8. The device according to claim 5 to 7, characterized in that the fabric (5) is matched in its mesh size so that biologically active particles of a defined size cannot penetrate through the fabric.

9. The device according to claim 5 to 8, characterized in that the absorption of the electrically charged molecules on the tissue (5) is less than 40%, but preferably less than 1 0%.

1 0. Device according to claim 5 to 9, characterized in that the fabric (5) has been treated by calendering.

1 1. Device according to claim 1 to 3, characterized. that the intermediate installation is a liquid barrier (1 3) that can be opened and closed.

2. Device according to claim 1 to 1 1, characterized in that the volume of a sampling space (10) is reduced by electroosmotic flow.

3. Apparatus according to claim 1 to 1 2, characterized in that a buffer with a higher concentration is used in the anode compartment (8) and in the cathode compartment (6) than in the other chambers.

4. Apparatus according to claim 1 to 1 3, characterized in that buffers with different pH are kept in the chambers.

5. Apparatus according to claim 1 to 1 4, characterized in that an enzymatic reaction or a receptor / ligand binding takes place in the chambers.

6. The device according to claim 1 5, characterized in that the enzymatic reaction or a receptor / ligand binding takes place during the electrophoretic transfer of charged molecules.

7. The device according to claim 1 to 1 6, characterized. that an electroosmotic flow is generated from the anode to the cathode, which leads to a concentration in the sampling space (7).

1 8. Apparatus according to claim 1 to 17, characterized in that the environment can be reversibly exposed to at least one electrode a magnetic field.

1 9. Apparatus according to claim 1 to 1 8, characterized in that the channel open at the top has a taper in the middle.

20. The device according to claim 1 to 1 9, characterized in that the volume of the upwardly open channel (2) is 0.01 - 5 ml, preferably from 0.2 - 2 ml.

21. Apparatus according to claims 1 to 20, characterized in that the channel which is open at the top is rectangular or cylindrical.

22. The apparatus according to claim 1 to 21, characterized in that it is composed of several basic elements liquid-tight.

23. Shape-stabilized gel for electrophoretic purposes, characterized in that the shape stabilization is achieved by a support element.

24. Dimensionally stabilized gel according to claim 23, characterized in that the support element consists of a sintered plastic.

25. Shape-stabilized gel according to claim 23 or 24, characterized in that the sintered plastic consists of polypropylene or ethylene.

26. Shape-stabilized gel according to claim 23 to 25, characterized in that the sintered plastic has a pore size of 1 to 200 microns, preferably 20 to 120 microns.

27. Shape-stabilized gel according to claim 23, characterized in that the support element consists of one or more fabrics (n).

28. Shape-stabilized gel according to claim 27, characterized in that the fabric consists of a plastic, in particular polyester or amide.

29. Shape-stabilized gel according to claim 28, characterized in that the fabric has a mesh size of 0.1 to 500 μm, preferably 0.5 to 10 μm.

30. Use of a shape-stabilized gel according to claim 23 to 29 for separating the free from the bound phase

Immunoassays.

31 Use of the shape-stabilized gel according to claims 23 to 29 for gel blotting.

32. Use of a shape-stabilized gel according to claims 23 to 29 in devices according to claims 1 to 4.

33. Use of a device according to claim 1 to 22 for separating the free from the bound phase in receptor-ligand assays.

34. Use of a device according to claim 1 to 22 for the isolation of nucleic acids.

35. Use of a device according to claim 1 to 22 for the isolation of deoxyribonucleic acids (DNA) without contamination by

Ribonucleic acids (RNA).

36. Use of a device according to claim 1 to 22 for the isolation of ribonucleic acids (RNA) without contamination by deoxyribonucleic acids (DNA).

37. Process for the isolation of charged molecules in mixtures with electrophoretic agents in a reaction channel, in particular in a device according to claim 1 to 22, characterized in that the mixture is introduced in liquid form in a reaction channel, is subjected to electrophoresis in the reaction channel this electrophoresis is further processed in the reaction channel and after this processing the isolated charged molecules are removed from the reaction channel in soluble form.

38. The method according to claim 37, characterized in that a lysed biological sample is used as a mixture.

39. The method according to claim 37 or 38, characterized in that one or more shape-stabilized gels divide the reaction channel into different chambers.

40. The method according to claim 37 to 39, characterized in that a ligand-receptor binding takes place in the electrophoresis in the reaction channel.

41. Process according to Claims 37 to 40, characterized in that an enzymatic reaction takes place in the reaction channel during electrophoresis.

42. The method according to claim 37 to 41, characterized in that a concentration takes place in the reaction channel during electrophoresis.

43. The method according to claim 37 to 42, characterized in that the electroosmotic flow is reduced in the electrophoresis so that an electroelution of charged molecules is possible.

44. The method according to claim 37 to 43, characterized. that the free and the bound phase are separated in a ligand-receptor assay, in particular in an immunoassay, and thereby a quantitative determination is made possible.

45. The method according to claim 37 to 44, characterized in that a quantitative determination is carried out by separating components with a label from a mixture.

46. The method according to claim 45, characterized in that the label consists of a radioactive isotope, an enzyme, a fluorescent or luminescent substance.

47. The method according to claim 37 to 46, characterized in that the concentration of detergent in the removed solution with the isolated charged molecules is smaller than the concentration of the solution of the lysed sample originally introduced into the reaction channel.

48. The method according to claim 37 to 47, characterized in that a second buffer component is added to the lysis mixture and then an electroelution takes place.

49. Use of a method according to claim 37 to 48 for the isolation of deoxyribonucleic acids (DNA) without contamination by ribonucleic acids (RNA).

0. Use of a method according to claim 37 to 48 for the isolation of ribonucleic acids (RNA) without contamination by deoxyribonucleic acids (DNA).

1 . Use of a method according to claims 37 to 48 for carrying out immunoassays.