DE19948049A1 - 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

The subject of the present patent specification are methods and apparatus for isolating charged molecules with a wide range of applications, for example in Sample preparation of nucleic acids. Special electrophoretic processes can be used for this such as electroelution as described in WO 97/34908 and WO 98/58251. In this, charged molecules are first attached to one Bound adsorber and then released again in an electroelution process Extraction volume can be transferred and concentrated there. Other devices for Electroelution are described in US 5340449 and US 4608147. Both are devices however not designed for methods according to WO 97/34908, but show devices with closed and screwed chamber systems. These are then in one Electrophoresis buffer tank anchored accordingly, the chamber system being hermetic is sealed off against the electrophoresis buffer tank: especially with regard to one Automation in a pipetting robot is not suitable for these devices.

If, on the other hand, devices are used as described here that are open at the top With the help of a pipetting device, nucleic acids or adsorbers (e.g. in Particle shape), to which nucleic acids are bound, can be introduced. Such Devices are generally used for automation with the use of Pipette robots (e.g. from TECAN, Rosys, Canberra Packard and Beckman) suitable.

When developing such electroelution devices that are open at the top, the fact must Are taken into account that, depending on how the design looks in detail, with an electroosmotic flow (EOF) must be expected. Details are described in "Capillary Electrochromatography - Technology and Applications" (CEC- Guidebook Hewlett-Packard Publication No .: 5968-3231E) Chapter 2 and "Analysis of Nucleic Acids by Capillary Electrophoresis "(Chr. Heller (Ed.) ISBN 3-528-06871-X, 1997), Page 24 ff. This river, triggered z. B. through capillary openings in the Electroelution area, can significantly affect the electroelution process, even to make impossible.

The devices of the present application take into account on the one hand the Requirements for automation with open devices and wear on the other hand, the requirements of the electroosmotic flow. You let yourself Surprisingly use for processes in which nucleic acids are bound to adsorbers  are then placed in a sample entry chamber and then one be subjected to appropriate electroelution with sample concentration.

When preparing samples specifically from nucleic acids but also from other charged ones Molecules have been shown to be as easy to use as possible - also in Consideration for automation - with the transfer of the spatial change of the charged molecules still another process step should be linked. This Process step can be a change in the charged molecules themselves or the Change the environment of the charged molecules.

Such a process step linked to the transfer in the electroelution is in the given the current state of the art.

One of the most important applications is the isolation of nucleic acids from biological Sample material. For this purpose, a lysis of the sample material is first necessary for the Nucleic acids from the biological compartments such as cell nucleus, mitochondria, To release virus envelopes etc. For this lysis, in turn, the use of high was found Concentrations of detergents of any kind are helpful. For the relevant here electrophoretic processes are particularly suitable for ionic detergents, especially those with negatively charged detergent molecules such as Sodium dodecyl sulfate. If the nucleic acid released in the lysed sample is to be passed on Process z. B. in a PCR according to US 4683195 or digestion with restriction enzymes, disrupt the high detergent concentrations and must be removed. This change The environment of the charged molecules as a new process step can be inventively together with the transfer step in appropriate device and with Perform the inventive method. Another application entirely different is derived from molecular oncology. Here is the analysis of an oncogene's mRNA e.g. B. an important diagnostic parameter. It is crucial that the mRNA of the analog genomic DNA is isolated and separated, because residues of the genomic DNA the subsequent RT-PCR interferes and falsifies its informative value. For the extraction of the There are currently two standard methods for mRNA in a sample preparation process: a) the Hybridization of the mRNA with a solid phase-bound oligo (dT), which is similar to that of nature binds from existing oligo (A) sequence of the mRNA and subsequent release after Washing, and methods that isolate the mRNA by selective precipitation. Both procedures have disadvantages, it can be said that a) the mRNA tends to break and one detects only parts of the mRNA with this method, what a quantitative analysis  is not suitable. The methods according to b) are complicated to handle and can be difficult to automate as centrifugation steps are necessary.

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

The present application therefore also relates to methods and devices for the purpose of isolating charged molecules using the electrophoretic Link another processing, whereby this processing is based on the Environment of the charged molecules or can refer to the molecules themselves.

Fig. 1a shows schematically a perspective view of an inventive device. It is a base body ( 1 ) into which a reaction channel ( 2 ) has been incorporated. The reaction channel ( 2 ) is characterized in that it is open at the top, contains a corresponding bottom and side walls, so that the interior can be filled with electrolyte solution. The reaction channel ( 2 ) is designed in a rectangular or cylindrical shape, in which the short sides are each provided with electrodes ( 3 , 4 ) at the outer ends. Between these electrodes ( 3 , 4 ) extends 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 (e.g. 10 , 7 in Fig. 2a) can be generated . The channel ( 2 ) typically has a volume of 0.01-5 ml, but 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. 1b is an inventive variant of the above-described basic form shows the channel (2). 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, which surprisingly occur in 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. 1b is preferably intended as an injection molded part for single use and has slots ( 12 ) in the base body ( 1 ) for the intermediate fittings, such as the semipermeable membrane ( 14 ), which are described in more detail below: 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.

Fig. 1h illustrates such an embodiment with Mikrotitrationsplattenstreifen (19) A 8 channels (2) and a matching Mikrotitrationsplattenrahmen (18). In a miniaturized version, formats with 384 and 1536 channels can also be used.

The channel ( 2 ) can also be composed of various individual elements Fig. 1c-g and z. B. are held together 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 generally open at the top and U-shaped, but can also be closed at the top in individual cases.

, Fig. 1c to in the bottomed base (1) with a corresponding recess (2) therein (2) form the reaction channel when several basic elements associated with sealing elements (25) on the corresponding mating surface (20) by means of clamping device (23) be pressed together.

Fig. 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 mating 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 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 as Fig. 1d. An electrical feed line ( 30 ) is provided for the electrodes ( 3 , 4 ) located in the window.

Fig. 1g 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 . 1i and 1j can also be used, which can be plugged together with or without locking lugs ( 16 ) via a corresponding clamping cone ( 9 ) and also without a clamping device ( 23 ). hold liquid-tight together. In Fig. 1i, the base element is shown for limiting the channel, which in this case, by mating with the base member on the extension of Fig. 1j may be produced in any length. Then on the opposite side there is a boundary with an identical basic element as FIG. 1i. This either has corresponding slots for receiving electrodes ( 3 ) as shown in Fig. 1i. According to the invention, however, two different variants have proven to be advantageous: In variant A, an electrode made of conductive plastic is directly injected during 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 represents a particularly cost-effective variant. Materials with resistances of less than 1000 ohms, such as Cabelec 3827 and Pre-Elec 1362, 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. 1j, since here an opening ( 31 ) enables access to the reaction channel ( 2 ). This opening ( 31 ) can also be used to extract solutions, e.g. B. used in washing operations.

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.

The following are the various intermediate components (28) are described in the channel (2) closer to fulfill the various functions, wherein an intermediate mounting (28), the reaction channel (2) into a sample introduction chamber (10) and into a sampling chamber (7), as shown in Fig. 2 ( Fig. 2a: top view, Fig. 2b: sectional view).

In a device according to the invention, an intermediate installation ( 28 ) must meet the following criteria in order, for. B. to be suitable 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 ( 10 , 7 ) have no fluid exchange when there is no voltage at the electrodes,
• (d) if necessary, retain particles and
• e) if necessary, generate an electroosmotic flow for concentration.

Surprisingly, it was found that an inventive device such as Fig. 3 z. B. meets these criteria. A semipermeable membrane ( 14 ) is attached in the vicinity of the anode ( 4 ), so that an anode space ( 8 ) is created between the membrane ( 14 ) and the anode ( 4 ). In addition, a further element, namely a tissue ( 5 ) is introduced between the semipermeable membrane ( 14 ) and the cathode ( 3 ). This is preferably a precision screen fabric with a defined mesh size and with a defined open screen area, as described in "Synthetic Monofilament Precision Screen Fabric" (company brochure from Sefar, Rüschlikon, Switzerland). This fabric ( 5 ) can be 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 size is 0.1-500 µm, but preferably 0.5-10 µm, the open screen area 0.2% -40%, but preferably 0.5-2%. The type of material also plays an important role. As Example 1 (p. 10) shows, an electroosmotic flow is generated on the tissue ( 5 ), which reduces the volume of the sampling space ( 7 ). With this fabric ( 5 ) other types of fabrics, such as. B. wool, cotton are used. Such a tissue ( 5 ) can be used in an intermediate installation ( 28 ) in a device according to FIG. 3 for performing an electroelution in such a way that in the sample entry space, which corresponds to the cathode space ( 6 ), for. B. particles are introduced which are loaded as an adsorber with electrically charged molecules, in particular nucleic acids ( Fig. 9c and Example 4, p. 11 ff). After the sampling space ( 7 ) and the anode space ( 8 ) have been filled with an appropriate electrolyte liquid, the charged molecules can be separated from the adsorber by applying a voltage between the electrodes ( 3 , 4 ). The tissue ( 5 ) does not hinder the electrical conductivity and allows charged molecules, in particular nucleic acids, to pass through. 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. Furthermore, the execution of FIG. 3 is a semipermeable membrane (14), the charged molecules at a certain molecular weight prevents it to migrate 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 electroosmotic flow mentioned above, the buffer volume in the sampling chamber ( 7 ) is reduced if the tissues ( 5 ) are correctly selected and the charged molecules are concentrated in this way. 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 ), a further inventive advantage is that the particles with a suitable tissue in the sample entry space ( 10 identical 6 ) 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 adapted to that of the particles used.

In a further variant, a liquid barrier ( 13 ) can also be used, which can be introduced into the base body ( 1 ) in a liquid-tight and movable manner by corresponding guides ( 12 ), as shown in FIGS. 4 and 5a and 5b. FIG. 5a shows the liquid barrier ( 13 ) in the open state, while FIG. 5b shows the closed state in which the reaction channel ( 2 ) is closed by pressing the liquid barrier ( 13 ) into the base body ( 1 ).

Fig. 6 now illustrates the area of application of such a liquid barrier ( 13 ). In addition to the electrodes ( 3 , 4 ) and the semipermeable membrane ( 14 ), 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 ( 13 ) is initially open. By pressing in the liquid barrier ( 13 ), a sample-taking space ( 7 ) which is closed in a liquid-tight manner against the space ( 6 ) is formed, from which, with the aid of pipettes, z. B. the charged molecules can usually be removed nucleic acid. Disks or blocks made of conventional agarose or polyacrylamide gels are particularly suitable intermediate internals, with devices for nucleic acid isolation e.g. B. was found that especially gels with a low degree of crosslinking, ie small amounts of gel filler are particularly advantageous. Again, it turned out that the thickness of the intermediate fittings should be correspondingly thin. They are 0.1-20 mm, but preferably 0.5-5 mm. The proportions of gel filler in shape-stabilized gels according to the invention such as. B. Agarose is 0.1-10%, but preferably 0.12-4%. Attempts in the conventional manner to produce such thin gel slices fail due to the lack of dimensional stability. Surprisingly, it was 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 is stabilized by introducing support elements, there being two inventive variants. In variant A, the space between two tissues ( 5 ) is poured out with the warm, liquid gel mass. After cooling, the gel mass solidifies 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. Fig. 7b shows this sheet-like structure stabilizing the two fabrics (5) and the intermediate layer of gel (34). 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 capillaries are 1-200 µm in size, but preferably 20-120 µm. Such shape-stabilized gels serve the purpose that they are permeable to charged biomolecules in certain molecular weight ranges, and that they also represent liquid barriers. Example 7 describes further details of the production of this variant B. A wide use of such dimensionally stabilized gels is given in the most varied 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 with regard to simple and safe handling when transferring the gels, since there is protection against destruction of the gel body.

When using the above-described open devices, the Fact that, depending on what the design looks like in detail, with an electroosmotic flow (EOF) must be expected.

This river, triggered z. B. by capillary openings in the electroelution area, can significantly affect the electroelution process, even make it impossible. 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 was 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 compartments. The buffer can be 2-30 times, but preferably 5-15 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 can also buffer gradients according to the invention between the electrode chambers to the chambers in Interior are used, the more concentrated buffer to each Electrodes are used. Multi-buffer systems have also proven to be inventive, in which neighboring chambers with different buffer substances and different pH values are used. This advantage can also be achieved through Mixture of different buffers can be used for sample entry.  

The use of shape-stabilized gel layers in an inventive Reaction channel taking into account the boundary conditions described above enable a variety of applications in which charged molecules are transferred and at the same time the environment can be processed, as described in more detail below becomes.

A device according to FIG. 7a with a tissue ( 5 ), a shape-stabilized gel layer ( 33 ) and a semipermeable membrane ( 14 ) as liquid barriers can be used for the isolation of nucleic acids as follows. 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 internals still protrude, and a DC voltage is applied to the electrodes. The nucleic acid migrates through the shape-stabilized gel ( 33 ) and accumulates in front of the semipeimeable membrane ( 14 ) 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 shape-stabilized gel, the nucleic acids can also be selected by molecular weight.

For isolating nucleic acids from biological samples is suitable, inter alia, Fig. 7f. Here, a semiperineable membrane ( 14 ) closes off the cathode compartment ( 6 ) and represents the connection to the sample insertion compartment ( 10 ). Towards the anode there are two dimensionally stabilized gels ( 33 ) and ( 33 a), which separate the sampling compartment ( 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 ). To liberate the nucleic acids and destroy the biological structures, the lysis mixture contains detergents such as lithium, sodium dodecyl sulfate, cetylammonium bromide, ionic surfactants or ionic glycosides or the like. These are used in concentrations of 0.1-10%, but preferably 0.1-2% % used. 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 low proportion of gel filler, which is 0.12% -0.2% and not only retains coarser cell debris but also larger protein aggregates. The released nucleic acids and the detergent, e.g. B. Sodium dodecyl sulfate passes 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 ( 33 a) thus separates the nucleic acids from the detergent. The nucleic acids are retained by this shape-stabilized gel ( 33 a) and out of the chamber. ( 7 ) taken. In this case, a semipermeable membrane has to be omitted: on the one hand, the protection provided by the shape-stabilized gel ( 33 a) is sufficient; on the other hand, it has surprisingly been found that detergents such as SDS form-stabilized gels generally pass easily, while as a result of mycelial formation, semipermeable membranes are presumably used as intermediate fittings Withhold detergent. The electrophoretic process takes 1-30 minutes. however, preferably 2-12 min. 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 an electrical signal from the pipetting robot that switches the power supply unit on and off is necessary for the isolation process. The isolated nucleic acid can then be removed with the robot and used for further processing.

Ranges direct depletion of the detergent from the lysis mixture is not sufficient, 7d with two form-stabilized gel layers (33) and (33 a) can be used in a device according to Fig. As a liquid barrier, a modified process for the isolation of nucleic acids be used 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 move through the tissue into the chamber ( 35 ) and through the shape-stabilized gel ( 33 ) as before, but are then replaced by the shape-stabilized gel ( 33 a) retained while detergent residues the z. B. adsorbed on the particles, they happen. The detergent-free nucleic acid is removed from the sampling space ( 7 ). To remove larger amounts of detergent, for sodium dodecyl sulfate z. B. greater than 2% can be used according to the invention also special chambers that contain detergent-binding agents or agents that fail detergent. It is also possible to add detergent-binding agents after the 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. Roche Diagnostics, Mannheim Cat. No .: 1 500 678]. 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 Chlinical Applications" AT Andrews (Ed :) ISBN 0- 19-854633-5, Clarendon Press; Oxford 1988). For this, however, correspondingly large hiking trails (2-80 cm) are required. Surprisingly, however, it was found that with the device according to FIG. 7, even in thin, dimensionally 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 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 must 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 in accordance with 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, when using a DNase with a pI <7, preferably PI 8-9, this process can be carried out in a device according to FIG. 7d. 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 form-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 then continues. After the DNA has been broken down, it can be achieved through a suitable inventive choice of the pI value of the DNase that this is separated from the remaining RNA and an RNA preparation can be removed from the chamber ( 7 ) without DNase. The corresponding detergent has meanwhile penetrated through the shape-stabilized gel ( 33 a) into the anode chamber ( 8 ): Due to its pI value, the DNase is e.g. B. 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 pI of the DNase used. This also applies when using thermolabile DNases, which are used in the subsequent RT-PCR. B. can be destroyed 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 fast 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 respect to Fig. 7d additional form-stabilized gel (33 b) in the cathode (3), in which filled the nuclease with the electrolyte in the reaction chamber (35 b) and the lysed sample in sample introduction 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, there is the inventive 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 the enzyme and thus deactivate it. Such processes would then proceed as follows in the devices according to FIG. 7d: The chamber ( 6 ) is filled with the lysed sample and the remaining chambers with electrolyte. The electroelution is started and interrupted after 1-5 min, but preferably 1-2 min. DNase bound to solid phase 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 become detached in this process and, depending on the pI value, migrate with the sample into the sampling chamber ( 10 ), which is unfavorable for further processing. In this case, according to the invention, solid phase-bound proteinase K 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 to ensure that proteinase K in particular does not get into the sampling chamber ( 10 ), since this z. B. can interfere with 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.

Another possibility is that solid phase-proteinid K or else Solid phase-bound antibodies against the DNase were added to the still liquid gel and thereby being held in a form-stabilized gel layer, which in turn is a Would save 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 with analog methodologies.

Another inventive solution is the use of buffers with different pH Values in the various chambers of the devices described here. Depending on the PI An adjacent chamber with a pH Value are filled in such a way that the desired charged molecules such. B. Nucleic acids pass through this chamber, but the aggregates 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 built-in receiving pockets, which are used as a sample entry chamber ( 10 ), 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.

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 ( 17 ) must be brought close to the reaction channel. For electroelution, it is therefore particularly appropriate to bring a suitable permanent magnet in the vicinity of the sample entry space ( 10 ), so that the particles after the charged molecules have been released, for. B. can be pulled to the floor or to the side wall. 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 for Recovery of nucleic acid from gels (agarose or polyacrylamide) used (Example 2).

Illustrations

FIG. 1a perspective view: Rectangular reaction channel with intermediate installation,

FIG. 1b supervisory: Rectangular reaction channel constriction,

Fig. 1c base member,

Fig. 1d intermediate installation,

Fig. 1e sealing element,

Fig. 1f electrode holder,

Fig. 1g receiving with clamping device,

Fig. 1h Mikrotitrationsrahmen with 48 reaction channels,

Fig. 1i basic element with electrode holders and clamping,

Fig. 1j base member having opening and clamping,

FIG. 2a supervisory: reaction channel with intermediate installation,

FIG. 2b-section: the reaction channel with intermediate installation,

Fig. 3 reaction channel with two intermediate baffles,

Fig. 4 reaction channel with liquid barrier,

FIG. 5a liquid barrier open,

Fig. 5b liquid barrier closed,

Fig. 6 with reaction channel and liquid barrier membrane,

Fig. 7a device for electroelution with form-stabilized gel according to variant A,

Fig. 7b device for electroelution with form-stabilized gel according to variant B,

Fig. 7c apparatus having three chambers,

Fig. 7d device with two form-stabilized gel layers,

Fig. 7e device having three dimensionally stabilized gel layers,

Fig. 7f perspective view of an Flachbettgels with reaction chambers,

Fig. 7g-section through a Flächbettgel 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 Agarosegelstückchen before and after electroelution

Fig. 10b agarose gel for electroelution with device Fig. 9a

Fig. 11 for agarose gel electroelution apparatus with Fig. 9b

Fig. 12 agarose gel for isolation of nucleic acids from yeast by electroelution in Fig. 9c,

13 standard agarose gel , for analysis of the electroelution according to example 5,

14 standard agarose gel for analysis of the electroelution according to example 7,

Fig. 15 standard agarose gel on a carrier made of sintered plastic. (Example 10),

Fig. 16 Standardagarosegel to analyze the isolation of genomic DNA and RNA from yeast Example 11

17 standard agarose gel after PCR with various electroeluates,

Fig. 18 Standardagarosegel to analyze the isolation of genomic DNA from yeast before amplification,

Fig. 19 Standardagarosegel to analyze the isolation of genomic DNA from yeast after amplification,

Fig. 20 Standardagarosegel to analyze the isolation of genomic DNA from yeast before amplification with use of a Detergensadsorbers,

Fig. 21 Standardagarosegel to analyze the isolation of genomic DNA and RNA from yeast after amplification with use of a Detergensadsorber,

Fig. 22 Standardagarosegel to analyze the isolation of genomic DNA from yeast before amplification with use of a two-component buffer system,

Fig. 23 Standardagarosegel to analyze the isolation of genomic DNA and RNA from yeast after amplification with use of a two-component buffer system,

Fig. 24 Standardagarosegel to analyze the isolation of genomic DNA from yeast after amplification in kinetics.

Reference list

1

Base body closed at the bottom

2nd

Channel open to the top

3rd

Electrode A, e.g. B. cathode

4th

Electrode B, e.g. B. Anode

5

tissue

6

Cathode room or chamber

7

Sampling room or chamber

8th

Anode room or chamber

9

Clamping cone or chamber

10th

Sample entry space or chamber

11

Electrophoresis separation section

12th

Guide in basic body for installation elements

13

Liquid barrier

14

Semipermeable membrane

15

Reaction space or chamber (open)

16

Latch

17th

Permanent magnet

18th

Microtitration plate frame

19th

Microtitration plate strips

20

Mating surface

21

Integrated connector

22

Solid phase

23

Jig

24th

Basic element

25th

Sealing element

26

admission

27

Frit

28

Intermediate internals

29

Laminating film

30th

Electrical supply

31

Opening to the reaction channel

32

Constriction

33

Dimensionally stabilized gel

34

Gel layer

35

Reaction chamber

36

Flat agarose gel

Examples 1. Measurement of the 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. 1c [no .: 011 11120 005 05; J. Pützfeld BV, Amsterdam, Netherlands]. 3 elements were glued in each case 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. 54 × 86; Böttcher, Jena] with a laminator [Fa. Lamirel MAXIPLAST 335 E] welded in and clamped with silicone seals [1 mm thick No. 6084.0810, H. Wegener, Hamburg] between the above basic elements in a liquid-tight manner 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 V was applied to the electrodes ( 3 , 4 ) 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 ). Materials such as Example 1 were used for all of the following examples, unless stated otherwise.

2. Electroelution of DNA from agarose gels

2.5 µg DNA (λ Hind III) [No. # SM0101; MBI Fermentas, St. Leon-Roth] was placed in a standard flat bed agarose gel (0.8% agarose [from Sigma, Munich]) in an electrophoresis buffer (see above) with a running time of 10 min. (approx. 3 V / cm) was transferred into the gel, and the band was prepared using SYBR®-Gold [S-11494; Mo Bi Tec, Göttingen] stained. The corresponding piece of gel 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. 9a was filled with 1.9 ml of electrophoresis buffer (see above). A voltage between 40-60 V at a constant 10 mA for 7 or 10 min was applied to the electrodes with an electrophoresis transformer. created.

Fig. 10b shows the result of electroelution in a Standardagarosegel (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-5616, Fa: Porex. Technologies GmbH; Singwitz] and a semipermeable membrane ( 14 ) which then delimit a cathode space ( 6 ), a sample entry space ( 10 ) and an anode space ( 8 ). The device was assembled as described in Example 1.

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

FIG. 11 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 with a kinetics of 0-10 min. M denotes the track of the marker as in Example 1.

4. Isolation of nucleic acid from yeast using magnetic articles 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 × 10 ¹⁰ yeast cells per ml (Saccharomyces cereviciae) were in Lyticase buffer (1M 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 (10 ⁸ cells) thereof with 1 μl of lyticase [No. 1372 467, Roche Diagnostics, Mannheim] and incubated for 15 min at 37 ° C.

The lysis and the binding and washing of the magnetic particles were carried out in accordance with US Pat. No. 5,7605,628. After the last washing step, the magnetic particles were separated off, 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. 9c that was already filled with elution buffer. The electroelution was carried out by applying a voltage of 40-70 V at 10 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. 12 shows the associated gel image.

5. Electroelution with a device according to FIG. 7a

For this application, a device according to FIG. 7a with a tissue ( 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. 011 1120 005 05 J. Pützfeld. B: V., Amsterdam, Netherlands]. For the anode ( 8 ) and cathode compartment ( 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. 54 × 86; Böttcher, Jena] welded in with a laminator [Fa. Lamirel MAXIPLAST 335 E] and with silicone seals [1 mm thick No. 6084.0810, H. Wegener, Hamburg] between the. The above-mentioned elements are clamped liquid-tight 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%) [from . Sigma, Munich] and electrophoresis buffer 10 mM Tris / acetate [Fa. Roth, Karlsruhe] with 1 mM EDTA [Fa. Sigma] pH 8; 0) placed; 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 III) [No. # SM0101; MBI Fermentas, St. Leon-Rot] was directly in the Sample entry space was brought and the entire device was filled with 1.75 ml Electrophoresis buffer (see before) filled. The electrodes were attached with a Electrophoresis transformer [Fa. Hölzel, Dorfen] a voltage of 50-500 V at constant 10 mA for 10 min. created.

Wherein analyzed in kinetics of 0-10 min both withdrawals from sample introduction chamber (10) and from the sampling chamber (7) by means of a 0.8% Standardagarosegel (see above) Fig. 13 shows the result of electroelution with various Agarosekonzeritrationen in the form stabilized gel were:

One can see the decrease in the amount of DNA (λ Hind III) in the sample entry space ( 10 ) and the increase in the sample removal space ( 7 ). In addition, the bands with a smaller molecular weight are retained at higher agarose concentrations, so that in this way a selection of certain nucleic acids can be achieved 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 ( 10 ) and the sample removal space ( 7 ) are in a kinetics of 2-10 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 production was carried out as follows:

Two layers of tissue ( 5 ) were placed in the device at a distance of approx. 2 mm and the intermediate space 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. 14 shows the result of the electroelution, wherein 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) in an elution rate of 0-10 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 with a shape-stabilized gel of variant B with 0.4% agarose was used. The entire device according to FIG. 7b was operated as in Example 1.

The final volumes of the sample entry space ( 10 ) 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. 7e 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 ). 10 mA was 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 for electroelution when the voltage range is low electroosmotic flow, which can also be advantageous for some applications.  

This result shows that with much lower voltage an electroelution can be carried out, which is significant with appropriate automation Simplification, since different safety guidelines apply to these voltages. Become anode compartment and cathode compartment as in Example 11 with a higher concentration buffer fed, 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 (λ Hind III) was applied with this gel, electrophoresis was carried out and stained (see above). Fig. 15 shows the result.

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

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

30 μl (10 ⁸ cells) of a yeast cell suspension (Saccharomyces cereviciae) were mixed with 115 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 [E5134 from Sigma, Munich] and 5 μl of a proteinase K solution [1 373 196; Roche, Mannheim] incubated for 1 hour at 55 ° C and transferred to the sample entry chamber ( 10 ).

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 150 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. 16 shows the corresponding gel.

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 ( 33 a) with 4% agarose. In the sample entry chamber ( 10 ), a sodium dodecyl sulfate solution in electrophoresis buffer (see above) was used in a concentration of 0.1% or 1% and subjected to electroelution as in Example 11. Then 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 13.

The PCR products were analyzed using an agarose gel and FIG. 17 shows the result.

The result shows that 1 µl of a 0.1% SDS solution does not interfere with the PCR (control), while all higher SDS quantities no longer allow a PCR product. In contrast, in this example after electroelution from the sampling chamber due to the SDS Depletion 1 µl of a 1% SDS solution can be used on the PCR without interference.

13. Simple isolation of DNA from yeast with subsequent amplification

A device according to FIG. 7f with a dialysis membrane ( 14 ) [Fa. Medicell see Example 1] and two shape-stabilized gels ( 33 and 33 a) were used. The agarose concentration was 0.15% in ( 33 ) and in ( 33 a) with shape stabilization according to variant A.

30 μl (10 ⁸ cells) of a yeast cell suspension (Saccharomyces cereviciae) were mixed with 115 μl BILATEST lysis buffer I consisting of 2% sodium dodecyl sulfate [L4390 from Sigma, Munich], 100 mM Tris / HCl [T2584 L4390 from Sigma, Munich], 10 mM EDTA [Es 5134 from Sigma; Munich] and 5 μl of a proteinase K solution [1 373 196, Roche, Mannheim] incubated for 1 hour at 55 ° C. and transferred to the sample entry chamber ( 10 ).

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 150 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 2 min. The nucleic acid was pipetted from the removal chamber ( 7 ) and subjected to agarose gel electrophoresis as in Example 1. Fig. 18 shows the corresponding gel.

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

This isolated yeast DNA is used for a PCR (US 4,683,195) with the primers KV 80:
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 '
[Fa. MWG Biotech AG, Ebersberg near Munich]. A 496bp fragment from the yeast BCY1 gene is amplified with these primers.

The PCR approaches 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 nucleotides 5 mM each (Eurogentec, Seraing, Belgium)
4 µl MgCl ₂ solution 25 mM (Eurogentec)
5 µl 10x 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 batch, the PCR plate is cooled to 4 ° C. After adding the last solution, the samples are mixed once with a pipette. The PCR is carried out in thermosprint plates [Fa. Innova GmbH, Mainheim] in the Primus 96 Plus [MWG Biotech AG, Munich]. The program includes the following steps:
Lid heating to 110 ° 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, the samples are mixed with 15-20% gel loading buffer with EDTA [Sigma, Munich]. The amplificates were analyzed on a standard agarose gel (1.6%). Fig. 19 shows the result.

In this experiment, genomic yeast DNA from an SDS containing lysis mixture with electroelution, which is then directly in a PCR was used.  

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

A device according to FIG. 7f according to Example 13 was carried out for this application. In contrast to Example 13, 50 .mu.l of detergent adsorber [Roche Diagnostics, Mannheim, Cat. No. 1500 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 10.1 V and 10 mA current for 10 min.

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

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

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

A device according to FIG. 7f according to Example 13 was carried out for this application. In contrast to Example 13, 50 μl of a phosphate buffer (250 mM pH 7.0) consisting of Na ₂ HPO ₄ and NaH ₂ PO ₄ [Merck, Darmstadt, Cat. No. 1.06580.1000 or 1.06346.1000] was added after lysis and the resulting mixture is added to the sample entry chamber. The electroelution was carried out at a voltage of 8.6 V and 10 mA current for 10 min.

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

A PCR was then carried out according to Example 13 with the eluates 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 buffer)

A device according to FIG. 7f according to Example 13 was carried out for this application. In contrast to Example 13, 15 l of a citrate buffer (1M pH 4.5) Appischem, Darmstadt, Cat. No. A 2337] was added after lysis and the resulting mixture was added to the sample entry chamber. The electroelution was carried out at a voltage of 8.5 V and a current of 10 mA 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).

This experiment shows that amplified material was only detected in the sampling chamber ( 7 ).

Claims (42)

1. Device for the isolation of electrically charged molecules consisting of

• - A base body ( 1 ) closed at the bottom with a channel ( 2 ) open towards the top
• - Two electrodes ( 3 , 4 ) as right and left outer boundaries of the channel ( 2 ), characterized in that one or more intermediate installation (th) ( 28 ) are arranged between the electrodes ( 3 , 4 ) in the channel ( 2 ) divide the channel into two or more chambers.

2. Apparatus according to claim 1, characterized in that all the internals ( 28 ) on the one hand represent a liquid barrier, on the other hand are permeable to electrically charged molecules. 3. Apparatus according to claim 1 and 2, characterized in that all intermediate internals ( 28 ) limit the electroosmotic flow by their nature so that electroelution of charged molecules is possible. 4. Apparatus according to claim 1 to 3, characterized in that it is in the Intermediate installation is a fabric. 5. The device according to claim 1 to 3, characterized in that it is in the intermediate installation to a liquid barrier ( 13 ) which can be opened and closed. 6. The device according to claim 1 to 3, characterized in that it is a dimensionally stabilized gel ( 33 ) in the intermediate installation. 7. The device according to claim 4, characterized in that the fabric ( 5 ) has a mesh size of 0.1 to 50 microns, but preferably 0.5 to 5 microns. 8. The device according to claim 4, characterized in that the mesh ( 5 ) is matched in its mesh size so that biologically active particles of a defined size cannot penetrate through the mesh. 9. The device according to claim 4, characterized in that the absorption of the electrically charged molecules on the tissue ( 5 ) is less than 40%, but preferably less than 10%. 10. The device according to claim 4, characterized in that the fabric ( 5 ) has been treated by calendering. 11. Shape-stabilized gel for electrophoretic purposes, characterized in that the Shape stabilization is achieved by a support element. 12. Dimensionally stabilized gel according to claim 11, characterized in that the support element consists of a sintered plastic. 13. Shape-stabilized gel for according to claim 11 and 12, characterized in that the sintered plastic is made of polypropylene or ethylene. 14. Shape-stabilized gel according to claim 11 to 13, characterized in that the sintered plastic has a pore size of 1-200 µm, but preferably 20-120 µm. 15. Dimensionally stabilized gel according to claim 11, characterized in that the support element consists of one or more fabrics. 16. Dimensionally stabilized gel according to claim 15, characterized in that the tissue Polyester or amide. 17. Shape-stabilized gel according to claim 16, characterized in that the tissue is a Mesh size of 0.1-500 microns, but preferably 0.5-10 microns. 18. Use of the shape-stabilized gel according to claim 11 to 17 for gel blotting. 19. Use of a shape-stabilized gel according to claim 11 to 17 in devices according to claims 1 to 3. 20. The apparatus of claim 1 to 10 and claim 19, characterized in that the volume of a sampling space ( 10 ) is reduced by electroosmotic flow. 21. The apparatus of claim 1 to 10 and claims 19 and 20, 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. 22. The apparatus according to claim 1 to 10 and claim 19 to 21, characterized in that buffers with different pH values are kept in the chambers. 23. The device according to claim 1 to 10 and claim 19 to 22, characterized in that in the chambers an enzymatic reaction or a receptor / ligand binding takes place. 24. The device according to claims 23, characterized in that the enzymatic reaction or a receptor / ligand binding during the electrophoretic transfer of charged molecules takes place. 25. The device according to claim 1 to 10 and claim 19 to 24, characterized in that an electroosmotic flow is generated from the anode to the cathode, which leads to a concentration in the sampling space ( 7 ). 26. The device according to claim 1 to 10 and claim 19 to 25, characterized in that the surroundings of at least one electrode are reversibly exposed to a magnetic field can be. 27. The device according to claim 1 to 10 and claim 19 to 26, characterized in that that the upwardly open channel has a taper in the middle. 28. The device according to claim 1 to 10 and claim 18 to 27, characterized in that the volume of the upwardly open channel ( 2 ) is 0.01-5 ml, but preferably 0.2-2 ml. 29. Use of a device according to claim 1 to 10 and claim 19 to 28 for Isolation of nucleic acids. 30. Use of a device according to claim 1 to 10 and claim 19 to 29 for Isolation of deoxyribonucleic acids (DNA) without contamination by Ribonucleic acids (RNA).   31. Use of a device according to claim 1 to 10 and claims 19 to 29 for Isolation of ribonucleic acids (RNA) without contamination by Deoxyribonucleic acids (DNA). 32. Method for isolating charged molecules from lysed biological samples using electrophoretic agents in a reaction channel, characterized in that the lysed Sample in liquid form is introduced into a reaction channel, an electrophoresis in the Reaction channel is subjected to this electrophoresis in the reaction channel is processed and after this processing the isolated charged molecules in soluble Form can be removed from the reaction channel. 33. The method according to claim 32, characterized in that one or more shape-stabilized gels divide the reaction channel into different chambers. 34. The method according to claim 32 and 33, characterized in that the concentration Detergent in the extracted solution with the isolated charged molecules is smaller than the concentration of the lysed solution originally introduced into the reaction channel Sample. 35. The method according to claim 32 to 34, characterized in that in the electrophoresis an enzymatic reaction takes place in the reaction channel. 36. The method according to claim 32 to 35, characterized in that in the electrophoresis ligand-receptor binding takes place in the reaction channel. 37. The method according to claim 32 to 37, characterized in that in the electrophoresis a concentration takes place in the reaction channel. 38. The method according to claim 32 to 37, characterized in that in the electrophoresis the electroosmotic flow is reduced so that electroelution of charged Molecules is possible. 39. The method according to claims 32 to 38, characterized in that a for the lysis mixture second buffer component is added and then an electroelution takes place.   40. Use of a method according to claim 32 to 39 for the isolation of Nucleic acids. 41. Use of a method according to claim 32 to 39 for the isolation of Deoxyribonucleic acids (DNA) without contamination by ribonucleic acids (RNA). 42. Use of a method according to claims 32 to 39 for the isolation of Ribonucleic acids (RNA) without contamination by deoxyribonucleic acids (DNA).