EP1960519A1

EP1960519A1 - Method for the sorption of at least one nucleic acid molecule using acid-activated phyllosilicates

Info

Publication number: EP1960519A1
Application number: EP05819297A
Authority: EP
Inventors: Ulrich Sohling; Thomas Scheper; Cornelia Kasper; Kirstin Suck; Daniel Riechers
Original assignee: Sued Chemie AG
Current assignee: Sued Chemie AG
Priority date: 2005-12-09
Filing date: 2005-12-09
Publication date: 2008-08-27
Also published as: US20090221809A1; WO2007065461A1

Abstract

The invention relates to a method for sorbing at least one nucleic acid molecule from a liquid medium. Said method comprises the following steps: a) a liquid medium is provided that contains at least one nucleic acid molecule; b) a layer is provided which contains at least one acid-activated phyllosilicate, is permeable to the liquid medium, and has a minimum thickness of 1 mm; c) the liquid medium containing the at least one nucleic acid molecule according to step (a) is conducted through the layer according to step (b) in order to sorb the at least one nucleic acid molecule in the layer.

Description

METHOD FOR SORPING AT LEAST ONE

NUCLEIC ACID MOLECULE USED

ACID ACTIVATED S CH I CHTS IL I CATE

description

The invention relates to a method for the sorption of at least one nucleic acid molecule from a liquid medium using a layer which comprises at least one acid-activated layered silicate. The method can be used in particular for the enrichment, depletion, removal, extraction or separation of nucleic acid molecules from or in liquid media.

The separation or purification of biomolecules has an ever increasing technical and scientific importance. Separation processes for cleaning up or wiping off _ ^ _ DNA is important on the one hand for basic research, whereby genetic material has to be isolated and purified, for example, in order to produce genetically modified organisms. However, this is currently increasingly being done in industrial applications. Some active ingredients used in medicine are already genetically engineered.

Another field of application for such separation processes and the adsorbents used for this is the depletion of DNA in waste water, especially in production processes with genetically modified organisms, such as Bacteria or fungi.

A large number of adsorbents are already known in the prior art, in particular those based on silanized silicate particles (silica gel) or functionalized celluloses.

US Pat. No. 4,029,583 describes a chromatographic carrier material made of silica gel which is suitable for the separation of proteins, peptides and nucleic acids and which has a cavity diameter of up to 50 nm and on which a stationary phase with an anion or cation exchanger is formed by means of a silanizing reagent Groups are bound that interact with the substances to be separated. The silanized silica gel is brought into contact with water, with the risk that the stationary phase polymerizes and closes the pores of the carrier material.

According to EP-B 0 104 210, nucleic acid mixtures can be separated into their constituents with high resolution and at high throughput speed if a chromatographic support material is used in which the diameter of the cavities is one to twenty times the largest dimension of the nucleic acid to be isolated or the largest dimension of the largest of all nucleic acids contained in the mixture. To produce the chromatographic support material, this is first reacted with a silanizing reagent which has a flexible chain group, which in turn is converted to the finished support material by reaction with a reagent which forms an anion or cation exchanger.

EP 0 496 822 (WO 91/05606, DE 393 50 98) describes a chromatographic support material whose cavities are one to twenty times the size of the largest dimension of the nucleic acids to be separated, which can be obtained by using a starting support material with a cavity size of 10 up to 1000 nm, a specific surface area of 5 to 800 m ² / g and a grain size of 3 to 500 μm with a silanizing reagent, which is characterized in that the silanizing reagent has at least one reactive group already reacted with a primary or secondary hydroxyalkylamine or contains a reactive group which can be reacted with a hydroxyalkylamine, such as an epoxy group or halogen atoms, which is reacted with a hydroxyalkylamine in a further reaction step.

The article by TG Lawson et al., "Separation of synthetic oligonucleotides on columns of microparticulate", Analytical Biochemistry (1983), 133 (1), 85-93, describes the separation of synthetic oligonucleotides on columns based on microparticulate silicon dioxide or silica gel, which has been coated with cross-linked polyethyleneimine. The coating was achieved by pumping the polyethyleneimine solution through the silica gel column. The procedure described in this article can only be used for small quantities deploy. Furthermore, this article only refers to silica gel particles modified with polyethyleneimine.

Further adsorbent systems are described in US 2003003272, EP 1 162 459 and EP 281 390. The article "Nucleic acid purification by cation complexation" by Prof. Dr. Michael Lorenz, Molzym GmbH & Co. KG, Bremen in the laboratory world. No. 4/2003, page 40, describes a new process for the purification of nucleic acid with special mini spin columns. Lt. Information in the article is based on a matrix in which a clay mineral was mixed with sand. Nothing is said about the type of clay minerals.

A disadvantage of the prior art sorption systems is that they are either relatively expensive or do not meet the requirements in terms of the binding capacity, the binding kinetics and / or the recovery rate of the absorbed nucleic acid (s). Due to the increasing importance of separating or purifying nucleic acids from different media, there is a constant need for improved sorbents for nucleic acids.

It was therefore an object of the present invention to provide an improved method for the sorption of nucleic acids which can be used efficiently and simply for enrichment or depletion, for removal or extraction, or for the separation of nucleic acids and the disadvantages of the prior art avoids.

Surprisingly, it has now been found that sorbents which comprise at least one acid-activated phyllosilicate can be used particularly advantageously to achieve this object. Such acid-activated phyllosilicates show a surprisingly high binding capacity for nucleic acids "" * O """exceeds that of commercial adsorption systems of the prior art. Furthermore, they exhibit particularly rapid binding kinetics. It is also advantageous that the bound nucleic acid can be removed again virtually quantitatively from the sorbent.

It has furthermore been found that the acid-activated layered silicate used according to the invention as a sorption agent can be used particularly advantageously for the sorption of at least one nucleic acid molecule from a liquid medium if it is present in a layer with a layer thickness of at least one millimeter. For sorption, the liquid medium with the at least one nucleic acid molecule can then be passed through the layer containing the at least one acid-activated layered silicate.

One aspect of the present invention thus relates to a method for the sorption of at least one nucleic acid molecule from a liquid medium, comprising the following steps: a. Providing a liquid medium containing at least one nucleic acid molecule;

b. Providing a layer containing at least one acid-activated layered silicate, the layer being permeable to the liquid medium and the layer thickness being at least 1 mm;

c. Passing the liquid medium with the at least one nucleic acid molecule according to step a. through the layer according to step b. in order to sorb the at least one nucleic acid molecule in the layer.

In the method according to the invention, the liquid medium with the at least one nucleic acid molecule is thus passed through the Layer containing the at least one acid activated

Layered silicate, passed through. This passage can be done in any way. In many cases, the capillary forces or gravity will be sufficient to allow the liquid medium with the at least one nucleic acid molecule to flow through the layer with the acid-activated one

Layer silicate to accomplish. If necessary, a pressure can also be applied to enable or accelerate the passage of the liquid medium through the layer, depending on the viscosity of the liquid medium containing the at least one nucleic acid molecule. In the same way, a vacuum or vacuum can be applied below the layer, so that the liquid medium with the at least one nucleic acid molecule through the layer with the acid-activated

Layered silicate is sucked. As a result, the desired flow rate can easily be routinely set by the person skilled in the art, depending on the liquid medium used. During the passage of the liquid medium with the at least one nucleic acid molecule, this can in the

Layer are sorbed with the acid-activated layered silicate.

The sorbents disclosed here are thus suitable both for separating nucleic acids and for enriching or enriching them from corresponding solutions / media in order to obtain or remove them. The almost quantitative recovery rate when eluting with conventional, usually high-salt buffers shows that it is also possible to recover the bound nucleic acid. Preferred elution buffers have a pH of 8 or more. The areas of application for such sorbents are diverse. Without restricting this invention to the following examples, a few possible applications should be mentioned: For example, separation of nucleic acids from a multicomponent gene is conceivable. mix or the depletion of DNA from wastewater from biotechnological production residues with genetically modified organisms. In general, the sorbent and method according to the invention can also be used for all molecular biological, microbiological or biotechnological methods in connection with nucleic acids, in particular their enrichment or depletion, separation, transient or permanent immobilization or other utilization. Exemplary methods and procedures can be found in relevant textbooks such as Sambrook et al. , "Molecular Cloning: A Laboratory Manual", CoId Spring Harbor Press 2001 and are familiar to the person skilled in the art. The present method can also be used in the context of the chromatographic separation of nucleic acids. Nucleic acids are to be understood primarily as DNA and RNA species, including genomic DNA and cDNA and their fragments, mRNA, tRNA, rRNA and other nucleic acid derivatives of natural or synthetic origin of any length.

In principle, however, the method according to the invention is also suitable for the separation or purification of proteins and other biomolecules. In the context of the present invention, biomolecule is understood to be a molecule which has, as building blocks, nucleotides or nucleosides (nucleobases), amino acids, monosaccharides and / or fatty acids. According to one aspect of the present invention, the reference in the description to "nucleic acid (molecule)" also includes other biomolecules. However, use for the sorption of nucleic acids is particularly preferred.

As starting materials for the used according to the invention

Layered silicates can be used all natural or synthetic layered silicates or mixtures thereof, which can be activated by an acid, ie in which Katio - - can be exchanged for protons in the intermediate layers. Two-layer and in particular three-layer silicates are preferred. Acid-activatable sheet silicates are familiar to the person skilled in the art and include, in particular, the smectic or montmorillonite-containing sheet silicates, such as bentonite. Generally, both so-called nature-active and non-nature-active layered silicates can be used, in particular di- and trioctahedral layered silicates of the serpentine, kaolin and talc pyrophylite group, smectites, vermiculites, II-lite and chlorites as well as those of the sepiolite-palygorskite group, such as montmorillonite , Natronite, saponite and vermiculite or hectorite, beidellite, palygorskite as well as alternating storage minerals (mixed layer mineral). Mixtures of two or more of the above materials can of course also be used. Furthermore, the layered silicate used according to the invention can also contain further constituents (including, for example, non-acid-activated layered silicates) which do not impair the intended use of the acid-activated clay, in particular its sorption capacity, or even have useful properties.

Layered silicates from the montmorillonite / beidellite series, such as e.g. Montmorillonite, bentonite, natronite, saponite and hectorite. Bentonites are most preferred, since surprisingly particularly advantageous binding capacities and kinetics for nucleic acids are achieved here.

In the context of the present invention, it was also found that acid-activated phyllosilicates in particular can advantageously be used in the processes according to the invention which have an iron content, calculated as Fe ₂ O ₃ , based on the total amount of acid-activated phyllosilicate used, of less than 6% by weight. %, preferably less than 4% by weight, more preferably less than 3% by weight, in particular _ y - have less than 2.5 wt .-%. It was found that when using such an acid-activated layer silicate with a low iron content a particularly gentle and

, 1 efficient sorption or purification of the nucleic acid molecule _, Ie is possible. Without the invention being restricted to the assumption of this theoretical mechanism, it is believed that the low iron contamination of the acid-activated layered silicate avoids undesired redox reactions which could be catalyzed by iron. Harmful influences of such redox reactions on the nucleic acid molecules or other components in the liquid medium can thereby be minimized or avoided. A method for determining the iron content in the context of the silicate analysis is given below in the method section.

According to a preferred embodiment of the invention, the layer with the at least one acid-activated layered silicate has a layer thickness of more than 0.3 cm, preferably more than 0.5 cm. The one chosen in the individual case

Layer thickness, as can be seen by the person skilled in the art, will depend on the volume of the liquid medium with the at least one nucleic acid molecule and the concentration of the nucleic acids contained in the liquid medium. In many cases, however, the layer thickness will be between about 0.1 and 100 cm.

According to a further aspect of the present invention, it was found that acid-activated phyllosilicates in particle form with a dry sieve residue of less than 10%, in particular less than 5%, more preferably less than 1% at 5 μm, in particular at 10 μm, more preferably at 35 μm (using appropriate fine sieves), particularly advantageous for the production of a layer or column packing. The exact preferred particle size is Oil packs are often influenced by the porosity of the frits used and in some cases can also be between 5 and 10 μm, determined on the basis of the dry sieve residue (as stated above). It has been shown that with such a particle size the permeability of the layer with the acid-activated layered silicate is particularly favorable for the preferred aqueous or alcoholic media and at the same time good sorption of the nucleic acid molecules onto the. particulate acid-activated layered silicate is made possible.

According to a preferred embodiment of the invention, the acid-activated layered silicate has a swellability of less than 15 ml / 2 g, in particular less than 10 ml / 2 g. A swellability of about 1 to 15 ml / 2 g, in particular of 2 to 10 ml / 2 g, is further preferred. Such swellability of the acid-activated layered silicate makes it particularly easy to use layers suitable for sorption of nucleic acids, e.g. produce in the form of columns. A method for determining the swellability (sediment volume) is given below in the method section.

According to one embodiment of the invention, weathering products of clays with a specific surface area of more than 200 m ² / g, a pore volume of more than 0.5 ml / g and a cation exchange capacity of more than 35 meq / 100 g in acid-activated form have also proven to be useful .

According to this special embodiment, particular preference is given to raw clays for acid activation whose cation exchange capacity is above 40 meq / 100 g, preferably in the range from 45 to 85 meq / 100 g. The specific BET surface area is particularly preferably in the range from 170 to 280 m ² / g, in particular between 180 and 260 m ² / g. The pore volume is preferably in the range from 0.7 to 1.0 ml / 100 g, in particular in the range of 0.80 to 1.0 ml / 100g. Acid activation of such raw clays can be carried out as specified herein. Such clays are described, for example, in DE 103 56 894.8 by the same applicant, which in this regard is expressly incorporated by reference into the present description.

It has also been found in the context of the present invention that in particular the two-layer and the three-layer layer silicates can advantageously be used in the layers for sorption of nucleic acids and other biomolecules even without acid activation. The smectitic layered silicates (see above) such as bentonite are particularly preferred. According to a further aspect of the present invention, a non-activated layered silicate can be used instead of the acid-activated layered silicate, or a mixture of both as the sorbent according to the invention. Otherwise, the information given in relation to the method and the use of the sorbent apply accordingly in the present description.

According to a preferred embodiment of the invention, however, the sorbent used in the layer according to the invention or the layer itself is based on at least one acid-activated layered silicate, ie at least 50% by weight, preferably at least 75% by weight, more preferably at least 90% by weight, in particular at least 95% by weight or even at least 98% by weight of the sorbent or layer according to the invention consist of one (or more) acid-activated layered silicate (s), as defined herein. According to a preferred embodiment, no silica or silica gel is used. According to a further preferred embodiment, the sorbent according to the invention or the layer consists essentially or completely of at least one _ I ₂ - acid activated layered silicate. However, the sorbent used according to the invention can also be used together with other sorbents or other components which appear suitable to the person skilled in the art, for example in the context of the method according to the invention.

!

According to a preferred embodiment of the invention, the acid-activated layered silicate has an average pore diameter, determined by the BJH method (DIN 66131), between approximately 2 nm and 25 nm, in particular between approximately 4 and approximately 10 nm.

According to a preferred embodiment of the invention, the pore volume, determined by the CCl ₄ method according to the method part, of pores up to 80 nm in diameter is between approximately 0.15 and 0.80 ml / g, in particular between approximately 0.2 and 0.7 ml / G. The corresponding values for pores up to 25 nm in diameter are in the range between approximately 0.15 and 0.45 ml / g, in particular 0.18 to 0.40 ml / g. The corresponding values for pores up to 14 nm are in the range between approximately 0.10 and 0.40 ml / g, in particular approximately 0.12 to 0.37 ml / g. The pore volumes for pores between 14 and 25 nm in diameter can be, for example, between 0.02 and 0.3 ml / g. The pore volume of pores with 25 to 80 nm can be in the same range, for example.

The porosimetry of the acid-activated layered silicates can also be influenced in a targeted manner via the conditions in the acid activation of the layered silicates, ie in particular the amount or concentration of the acid used, the temperature and the duration of the acid treatment. For example, a stronger acid activation with an increased amount of acid or at an elevated temperature over a longer period of time can result in a larger one - -

Porosity of the layered silicates are brought about, especially in the region of the smaller pores with a diameter of less than 50 nm, in particular less than 10 nm, determined by the CCl ₄ method according to the method part. By increasing the amount of acid used to activate the acid, the micropore volume of the layered silicate can be increased. At the same time, the cation exchange capacity is decreasing. Thus, for the nucleic acid species of interest in each case, the sorption capacity of the acid-activated layered silicate or its absorption and desorption rate via acid activation in individual cases can be optimized on the basis of routine examination of a number of differently acid-activated layered silicates. For example, via the acid activation, the pores / cavities in the sorbents according to the invention can be modified in the manner provided in accordance with EP 0 104 210 or US 4,029,583 (see above).

The acid-activated phyllosilicates used according to the invention are generally produced by:

Layered silicates treated with at least one acid. For this purpose, the layered silicates are brought into contact with the acid (s). In principle, any method for the acid activation of phyllosilicates which is known to the person skilled in the art can be used here, including the methods described in WO 99/02256, US Pat. No. 5,008,226 and US Pat. No. 5,869,415, which are expressly incorporated by reference into the description. In general, any organic or inorganic acids or mixtures thereof can be used. For example, acid can be sprayed on using a so-called SMBE process (Surface Modified Bleaching Earth). Here, the activation takes place on the surface of the layered silicates without working in a solution or dispersion. In a first embodiment, the activation of the layered silicate is thus carried out in the aqueous phase. For this purpose, the acid is brought into contact with the layered silicate as an aqueous solution. The procedure can be such that the layered silicate, which is preferably provided in the form of a powder, is first slurried in water. Then the acid (eg in concentrated form) is added. However, the layered silicate can also be suspended directly in an aqueous solution of the acid, or the aqueous solution of the acid can be applied to the layered silicate. According to an advantageous embodiment, the aqueous acid solution can, for example, be sprayed onto a preferably broken or powdered layered silicate, the amount of water preferably being chosen to be as small as possible and, for example, a concentrated acid or acid solution being used. The amount of acid can preferably be selected between 1 and 10% by weight, particularly preferably between 2 and 6% by weight, of an acid, in particular a strong acid, for example a mineral acid such as sulfuric acid, based on the anhydrous layered silicate (atro). If necessary, excess water can be evaporated and the activated layered silicate can then be ground to the desired fineness. As already explained above, no washing step is required in this embodiment of the method according to the invention, but is possible. After the aqueous solution of the acid has been added, drying is only carried out, if necessary, until the desired moisture content has been reached. The water content of the acid-activated layered silicate obtained is usually adjusted to a proportion of less than 20% by weight, preferably less than 15% by weight.

For the activation described above with an aqueous solution of an acid or a concentrated acid, the acid itself can be chosen as desired. Both mineral acids and organic acids or mixtures of the above acids can be used - - be used. Conventional mineral acids can be used, such as hydrochloric acid, phosphoric acid or sulfuric acid, with sulfuric acid being preferred. Concentrated or diluted acids or acid solutions can be used. Solutions of, for example, citric acid or oxalic acid can be used as organic acids. |

Another preferred activation option is the boiling of the layered silicates in an acid, in particular hydrochloric or sulfuric acid. Different degrees of activation can be set here by the appropriate concentrations of acid and cooking times, and the pore volume distribution can be set in a targeted manner. Such activated

Layered silicates are often referred to as bleaching earths. After the materials have dried, they are ground using standard methods.

In the "classic" activation, which is preferable in the invention in many cases, is activated at temperatures of about 100 ⁰ C to the boiling point (boiling point). In contrast, the SMBE process is usually carried out at room temperature, with elevated temperatures allowing better acid activations in individual cases. However, the influence of temperature is far less with the SMBE process than with the "classic" activation (so-called HBPE process). The dwell time (duration of acid activation) in the HBPE process is, for example, between about 8 hours, for example when using hydrochloric acid, and 12 to 15 hours, for example when using sulfuric acid. In contrast to the SMBE process, the HBPE process attacks the layer structure, which results in areas with silica, in addition to structurally largely unchanged areas. In the SMBE process, for example, 3% by weight of H ₂ SO _{4 are added} (100 + 3). The analysis of the processed material then usually reveals acidity - - keep in the range of 0.4 to 1.0%, ie a large part of the acid is consumed (exchange of H ⁺ ions for other cations etc.). A small part may be consumed by the lime it contains. In the SMBE process, the contact times with the acid are often around 15 minutes in the laboratory.

It has been found that, depending on the layered silicate used, activation with small amounts of acid may already be sufficient to obtain surprisingly good sorbents.

According to a particularly preferred embodiment according to the invention, the layered silicate is activated in such a way that the cation exchange capacity (CEC) of the acid-activated layered silicate used is less than 50 meq / 100 g, in particular less than 40 meq / 100 g. Activation is particularly preferably carried out by means of an at least 1 molar, in particular at least 2 molar acid solution at elevated temperature, in particular at more than 30 ^° C., more preferably more than 60 ^° C. According to a further preferred embodiment, the layer silicates are advantageously activated an acid with a pKa value of less than 4, in particular less than 3, more preferably less than 2.5 is used. For example, strong mineral acids, in particular hydrochloric acid, sulfuric acid or nitric acid or mixtures thereof, in particular in concentrated form, are preferably used. The preferred amount of acid is more than 1% by weight, in particular more than 2% by weight, particularly preferably at least 3% by weight of acid, more preferably at least 4% by weight of acid, based on the amount of layered silicate to be activated (determined from Drying at 130 ⁰ C). According to a particularly preferred embodiment according to the invention, the exchangeable (metal) cations (interlayer cations) are essentially completely replaced by protons by the acid activation of the layered silicate, ie more than 90%, in particular more than 95%, particularly more preferably more than 98%. This can be determined on the basis of the CEC and its ion proportions before and after acid activation.

According to one embodiment, the acid activation does not require that the excess acid and the salts formed during the activation are washed out. Rather, after the acid has been fed in, as is customary for acid activation, no washing step is carried out, but the treated layered silicate is dried and then, if necessary, ground to the desired particle size.

The sorbent used according to the invention (acid-activated layered silicate) can be used in the form of a powder, granulate or any shaped body. In general, the sorbents can be used in any form, including supported or immobilized forms. For example, use in the separation of different nucleic acid components on the basis of their molecular weight is also conceivable. The application form of the adsorbents according to the invention is not limited to the examples given.

In many cases, the layer used according to the invention with the at least one acid-activated layered silicate will be a layer in a column or cartridge, as is usually used for the passage of a liquid medium. For example, these can be chromatography columns, including gravity or centrifugation columns, solid phase chromatography, filter cartridges or membranes.

In general, the grain or shaped body size of the acid-activated layered - - Licenses depend on the respective application. All grain sizes or agglomerate sizes are possible here. For example, the acid-activated layered silicate in powder form, in particular with a D ₅ ~ o value of 1 to 1,000 are used in particular from 5 to 500 microns in order. Typical usable granules are in the range (D ₅₀ , by volume) between 100 μm to 5,000 μm, in particular 200 to 2,000 μm particle size. However, the above-mentioned dry sieve residues are particularly preferred for the layers or column packs used according to the invention. In many applications, it is advantageous to use moldings made from or with the acid-activated layered silicates, for example in the case of chromatography columns, including gravity or centrifugation columns,

Solid phase chromatography, filter cartridges, membranes etc.

According to a particularly preferred embodiment of the invention, as mentioned above, the sorbent used according to the invention can be in immobilized form. For example, the sorbent can be placed in a filter cartridge, a

HPLC cartridge or a comparable dosage form can be embedded. Embedding in gels, such as agarose gels or other gel-like or matrix-like structures, is also preferably possible. Such applications are often sold as part of so-called kits for the purification of nucleic acid molecules, such as, for example, the products from Quiagen, such as Quiagen genomic tip or the like. The medium containing the nucleic acid molecules of interest is generally sent through a column or filter cartridge or the like containing the sorbent. It can then be washed with suitable buffers to remove adhering contaminants. Finally there follows an elution step to obtain the nucleic acid molecules of interest. _

According to a further preferred embodiment of the invention, the acid-activated layered silicate has a BET surface area (determined according to DIN 66131) of at least 50 to 800 m ² / g, in particular at least 100 to 600 m ² / g, particularly preferably of at least 130 to 500 m ² / g g, on. Interaction with the nucleic acid is apparently facilitated by the high surface area, with the possibility of desorption surprisingly being retained.

According to a preferred embodiment of the invention, the nucleic acids are DNA or RNA molecules in double-stranded or single-stranded form with one or more nucleotide units.

With regard to nucleic acids, the method according to the invention is particularly advantageous in the case of media which contain oligonucleotides or nucleic acids with at least 10 bases (pairs), preferably with at least 100 bases (pairs), in particular at least 1000 bases (pairs). Of course, the method according to the invention can also be used with nucleic acids between 1 and 10 bases (pairs) or with quite large nucleic acid molecules, such as plasmids or vectors with, for example, 1 to 50 kB or longer genomic or c-DNA fragments. Restriction-digested DNA and RNA fragments, synthetic or natural oligo- and polymers from nucleic acids, cosmids etc. are also included.

For example, the chromatographic separation of biological macromolecules such as long-chain oligonucleotides, high-molecular nucleic acids, tRNA, 5S-rRNA, other rRNA species, single-stranded DNA, double-stranded DNA (eg plasmids or fragments of genomic DNA) etc. is of interest Surprisingly, an improved resolution can be achieved at a high throughput speed. The The carrier materials used can be used in a wide temperature range and are highly loadable. The carrier material also shows high pressure resistance and a long service life.

The need for high-purity nucleic acids, such as high-purity plasmid DNA for modern biotechnological, but also medical development, e.g. in the field of gene therapy. The protocols known in the prior art for the highly pure purification of nucleic acids are often costly and / or time-consuming, are not suitable for use on a large scale or are not safe enough for therapeutic purposes, since toxic solvents or enzymes of animal origin, such as e.g. RNAse can be used.

According to the invention, a liquid medium is understood to mean any non-solid medium, including low or high viscosity and fluid media. It will preferably be polar media in which the biomolecules or nucleic acid molecules of interest are generally contained. For example, it can also be a colloidal solution, a suspension, a dispersion, a solution or an emulsion.

According to the invention, the particularly preferred aqueous or alcoholic media are understood to mean all media containing water or alcohol, including aqueous-alcoholic media. In general, all media are also included in which water is completely miscible or completely mixed with other solvents. In particular, alcohols such as methanol, ethanol and C ₃ - to Cio-alcohols with one or more OH groups or acids are to be mentioned. Solvents that are completely miscible with water and their mixtures with water and alcohol are also conceivable. In particular acts - - In practice, these are aqueous, aqueous-alcoholic or alcoholic media.

Typical examples are aqueous or alcoholic buffer systems as used in science and industry, industrial or non-industrial waste water, process waste water, fermentation residues or media, media from medical or biological research, liquid or fluid contaminated sites and the like.

The sorbent according to the invention can contain further components in the layer as long as these do not unacceptably impair the adsorption of the nucleic acids and, if provided, their desorption. Such additional components can include, but are not limited to, organic or inorganic binders (see below), other sorbents for biomolecules or other inorganic or organic substances of interest from the medium which are known to the person skilled in the art, or also carriers such as glass, plastic or ceramic materials or the like include.

Thus, according to an advantageous embodiment according to the invention, the sorbent particles can be combined in the layer used to form larger agglomerates, granules or shaped bodies or applied to a carrier by means of a suitable binder. The shape and size of such superordinate structures, which contain the primary sorbent or layered silicate particles, depends on the particular application desired. It is therefore possible to use all shapes and sizes familiar to the person skilled in the art and suitable in individual cases. For example, in many cases agglomerates with a diameter of more than 10 μm, in particular more than 50 μm, may be preferred. In the case of a bed for chromatography columns and the like, one can - -

Spherical shape of the agglomerates may be advantageous. Possible carriers are, for example, calcium carbonate, plastics or ceramic materials.

Any binder familiar to the person skilled in the art can also be used as long as the attachment or incorporation of the biomolecules in or onto the sorbent in the layer is not impaired too much or ensures the stability of the particle agglomerates or moldings required for the particular application is. For example, without being limited to this, the following can be used as binders: agar-agar, alginates, chitosans, pectins, gelatins, lupine protein isolates or gluten.

As already stated above, it was surprisingly found according to one aspect of the invention that the acid-activated phyllosilicates themselves already provide particularly favorable surfaces for the sorption of nucleic acids. According to the invention, there is therefore preferably no (additional) use or treatment of the layered silicate with cationic polymers and / or polycations (multivalent cations). Furthermore, according to the invention, preferably no other polymers (eg polysaccharides), polyelectrolytes, polyanions and / or complexing agents (for modifying the layered silicate) are used. According to a particularly preferred embodiment of the invention, in particular no cationic polymer such as an aminated polysaccharide polymer or polycation is used. In particular, according to a further preferred embodiment of the invention, the acid-activated layered silicate used in accordance with the invention is not modified or treated with a (cationic) polymer or a polycation. - -

The method according to the invention can be used both for enrichment (i.e. increasing the concentration of the desired nucleic acid molecule (s)) as well as for enrichment (i.e. reducing the concentration of the desired nucleic acid molecule (s)) or for separating several different nucleic acid molecules.

If the method according to the invention aims to remove or dispose of nucleic acid molecules, the layer containing the nucleic acid molecules can be disposed of in a further step. Disposal can be carried out, for example, by thermal treatment to remove the layered silicate containing the biomolecules, the layered silicate being able to be disposed of after the thermal disintegration of the nucleic acid molecules.

According to a first aspect of the invention, it is possible to remove nucleic acids from media in a targeted manner. This plays a major role in wastewater treatment, for example, since in most countries there are strict legal regulations for removing nucleic acids and other biomolecules from wastewater.

According to a further preferred embodiment of the invention, the depletion or removal of nucleic acid molecules from culture media can also be carried out. For example, an undesirable increase in viscosity can occur in bioreactors due to the high concentration of nucleic acid molecules contained in the medium, in particular high-molecular nucleic acids. Efficient and biologically compatible removal of the disruptive nucleic acid molecules from the culture medium can take place here using the method according to the invention. - 4 -

Likewise, in many cases it is desirable to increase the concentration of nucleic acid molecules in a medium or to obtain these nucleic acid molecules as pure as possible.

For example, the extraction or purification of desired nucleic acids from solutions is one of the standard procedures in biological and medical research. In a further step, according to the invention, the nucleic acid molecule can be desorbed or recovered from the sorbent in the layer, as a result of which the layer can also be used again, if appropriate after renewed acid activation of the layered silicate contained therein.

According to a preferred embodiment of the invention, after the sorption of the at least one nucleic acid molecule in the layer with the acid-activated layered silicate, at least one washing step can take place. A conventional aqueous or alcohol-containing buffer can be used to remove impurities that are deposited in the layer next to the nucleic acid molecules. A non-limiting example of a suitable buffer is 50 µm citrate buffer (pH 4.0).

In the context of the present invention, it was also unexpectedly found that the acid-activated phyllosilicates have a high binding capacity for nucleic acid molecules over a very wide pH range. Advantageously, liquid media with both acidic and basic pH for sorption of the nucleic acid molecules contained therein can thus be passed through the layer with the acid-activated layered silicate. According to a preferred embodiment of the invention, the liquid medium is passed through or the nucleic acid molecule is sorbed in the layer at a pH between about pH 3 and pH 8, in particular between about pH 6 and pH 8. These conditions can be simple - 2 - can be provided by adjusting the pH of the liquid medium. According to a further aspect of the invention, the advantageous binding of the nucleic acid molecules to the layer with the acid-activated layered silicate over a wide pH range can also be used to separate a nucleic acid molecule from protein constituents which are also contained in the liquid medium, for example. After the liquid medium (containing the nucleic acid molecule) has been passed through, the layer can be washed with a series of buffers adjusted to different pH values or a pH gradient buffer in order to detach and wash out proteins with different isoelectric points from the layer. As far as the expected protein impurities are known, the pH value at which these protein impurities (as a rule depending on their isoelectric point) have the lowest sorption on the acid-activated layered silicate can be determined on the basis of preliminary tests.

Another aspect of the present invention relates to a layered composition comprising at least one acid-activated layered silicate and at least one nucleic acid molecule, the layer thickness being at least one millimeter. As stated herein, such a layered composition can advantageously be used, for example, to separate, recover or purify a nucleic acid molecule from a liquid medium.

As stated above, a low iron contamination of the acid-activated layered silicate used is advantageous. Another aspect of the present invention thus also relates to the use of an acid-activated

Layered silicate with a low iron contamination, as defined above, for sorption, in particular for separation - 6 - Preparation, extraction or purification of a nucleic acid molecule from a liquid medium. Another aspect relates to the use of such an acid-activated layered silicate as an inorganic vector for introducing biomolecules into cells or as a pharmaceutical composition, in particular as a reservoir for the storage and controlled release of biomolecules, preferably nucleic acids.

It has surprisingly been found that the sorbents according to the invention are also suitable for efficiently introducing these biomolecules into prokaryotic or eukaryotic cells. Apparently, according to the method according to the invention, biomolecules, in particular nucleic acids, can be “packaged” in a particularly advantageous manner for introduction into cells. The basic mechanism of such an introduction using the example of DNA-LDH nanohybrids is described, for example, in the reference Choy et al., Angew.

Chem. 2000, 112 (22), pages 4207-4211, and also described in EP 0 987 328 A2, to which reference is made in this regard and which are hereby incorporated into the description by reference to the method. The use as a pharmaceutical composition, in particular as a reservoir for the storage and controlled release of biomolecules, preferably of nucleic acids, is as such in WO

01/49869, to which reference is made and which is hereby incorporated by reference into the description.

Method part

1. BET surface

The BET surfaces specified here were in accordance with DIN

66131 determined. - 7 -

2. Porosimetry

The specified (average) pore diameters, volumes and areas were determined using a fully automatic nitrogen adsorption measuring device (ASAP 2000, company Micretrics) according to the manufacturer's standard program (BET, BJH, t-plot and DFT). The percentages of the proportion of certain pore sizes relate to the total pore volume of pores between 1.7 and 300 nm in diameter (BJH Adsorption Pore Distribution Report).

As far as indicated, the porosimetry was carried out according to the CCl ₄ method as follows:

Reagents:

Carbon tetrachloride (CCl ₄ )

Paraffin (liquid), Merck, (Order No. 7160.2500)

Execution :

1 to 2 grams of the dried material to be tested in a small weighing bottle at 130 ⁰ C in a drying cabinet. It is then cooled in the desiccator, weighed exactly and the glass is placed in a vacuum desiccator, which contains the following paraffin / carbon tetrachloride mixture ratios, depending on the micropore volume to be measured: -

Paraffin (ml) CCI ₄ (ml) micropores (A)

26 184 800

47.9 162.1 390

66.5 143.5 250

82.5 127.5 180

96.4 113.6 140

108.7 101.3 115

The desiccator connected to the graduated cold trap, manometer and vacuum pump is now evacuated to the boil of the contents. 10 ml of carbon tetrachloride are evaporated and collected in the cold trap.

The contents of the desiccator are then left to balance for 16 to 20 hours at room temperature, and then air is slowly let into the desiccator. After removing the desiccator lid, the weighing jar is closed immediately and weighed back on the analytical balance.

Evaluation:

The values are calculated in milligrams of carbon tetrachloride adsorption per gram of substance by weight gain. By dividing by the density of the carbon tetrachloride, you get the

Pore volume in ml / g substance. Weight-out - weight-in = weight gain

g substance x weight x density CCl ₄ = ml / g substance.

(Carbon tetrachloride at 2O ⁰ C, d = 1.595 g / cm ³ ) - -

3rd Measurement of the zeta potential

An aqueous suspension with dist. Water. The suspension to be measured was adjusted to pH 7 in each case. The zeta potential of the particles was determined using the Zetaphoremeter II from Particle Metrix according to the principle of microelectrophoresis. The rate of migration of the particles was measured in a known electric field. The particle movements that take place in a measuring cell are observed with the aid of a microscope. The direction of migration provides information about the type of charge (positive or negative) and the particle velocity is directly proportional to the electrical interfacial charge of the particles or to the zeta potential. The particle movements in the measuring cell are recorded by means of image analysis and after the measurement the particle paths covered are calculated and the resulting particle speed is determined.

Taking into account the suspension temperature and the electrical conductivity, the zeta potential (given in mV) was calculated from this.

Surprisingly, it was found in the context of the present invention that good results can also be achieved with layered silicates with a negative zeta potential.

4. Determination of particle sizes and particle size distribution

Unless otherwise stated, the particle sizes (distribution) are determined using the Malvern method. For this purpose, a Malvern Mastersizer was used according to the manufacturer's instructions. Approx. 2- Put 3 g (1 coffee spoon) of the sample to be examined in the "dry powder feeder" and set the correct measuring range depending on the sample (the coarser the sample, the higher the weight).

For determination in water, a sample (approx. 1 knife tip) is placed in the water bath until the measuring range is reached (the coarser the higher the weight), and stirred for 5 minutes in the ultrasonic bath. The measurement is then carried out.

5. Cation Exchange Capacity (CEC)

Principle: The clay is treated with a large excess of aqueous NH ₄ C1 solution, washed out and the amount of NH ₄ ⁺ remaining on the clay is determined by elemental analysis.

Me ⁺ (tone) ^~ + NH ₄ ⁺ NH ₄ ⁺ (tone) ^" + Me ⁺

(Me ⁺ = H ⁺ , K ⁺ , Na ⁺ , 1/2 Ca ²⁺ , 1/2 Mg ²⁺ ....)

Devices: sieve, 63 μm; Erlenmeyer ground flasks, 300 ml; Analytical balance; Membrane filter, 400 ml; Cellulose nitrate filter, 0.15 µm (from Sartorius); Drying cabinet; Reflux condenser; Hot plate; Distillation unit, VAPODEST-5 (Gerhardt, No.

6550); Volumetric flask, 250 ml; Flame carrion

Chemicals: 2N NH ₄ C1 solution; Nessler's reagent (Merck,

Art. No. 9028); Boric acid solution, 2%; Sodium hydroxide solution, 32%; 0.1 N hydrochloric acid; NaCl solution, 0.1%; KCl solution, 0.1%.

Procedure: 5 g of clay are sieved through a 63 μm sieve and dried at 110 ^° C. Then exactly 2 g are weighed on the analytical balance in differential weighing into the Erlenmeyer ground flask and mixed with 100 ml of 2N NH4Cl solution. The suspension is boiled under reflux for one hour. With strong CaCO ₃ - - - Containing bentonites may develop an ammonia. In these cases, NH ₄ C1 solution must be added until there is no longer a smell of ammonia. An additional check can be carried out with a damp indicator paper. After a standing time of approx. 16 h, the NH ₄ ⁺ bentonite is filtered off through a membrane filter and washed with deionized water (approx. 800 ml) until it is largely free of ions. Evidence of the freedom from ions in the wash water is carried out on NH ₄ ⁺ ions with the sensitive Neßlers reagent. The number of washes can vary between 30 minutes and 3 days depending on the key. The washed NH ₄ ⁺ clay is removed from the filter, h dried, milled at 110 ⁰ C 2, screened (63 micron sieve), and again at 110 ⁰ C for 2 hours dried. The NH ₄ ⁺ content of the clay is then determined by elemental analysis.

Calculation of the CEC: The CEC of the clay was determined in a conventional manner via the NH ₄ ⁺ content of the NH ₄ ⁺ clay, which was determined by elemental analysis of the N content. For this purpose, the Vario EL 3 device from Elementar-Heraeus, Hanau, DE, was used according to the manufacturer's instructions. The information is given in mval / 100 g clay (meq / 100g).

Example: nitrogen content = 0.93%;

Molecular weight: N = 14.0067 g / mol

0.93 x 1000

CEC = = 66.4 mVal / 100g

14.0067

CEC = 66.4 meq / 100 g NH ₄ ⁺ bentonite - -

6. Swelling capacity (sediment volume)

The swellability was determined as follows: A calibrated 100 ml measuring cylinder is washed with 100 ml dist. Filled with water. 2.0 g of the substance to be measured are slowly added to the water surface in portions of 0.1 to 0.2 g. After the material has dropped, the next quantum is given up. After the addition has ended, wait 1 hour and then read off the volume of the swollen substance in ml / 2 g.

7. Silicate analysis: (a) sample digestion

This analysis is based on the total digestion of the layered silicate. After the solids have dissolved, the individual components are analyzed using conventional specific analysis methods, e.g. ICB, analyzed and quantified.

For sample digestion, about 10 g of the sample to be examined are finely ground and dried in a drying cabinet at 120 ^° C. for 2 hours to constant weight. Approx. 1.4 g of the dried sample are placed in a platinum crucible and the sample weight is determined to an accuracy of 0.001 g. The sample is then mixed in the platinum crucible with 4 to 6 times the amount by weight of a mixture of sodium carbonate and potassium carbonate (1: 1). The mixture is provided with the platinum crucible into a Simon-Müller furnace and 2 - melted 850 C ⁰ - 3 hours at 800th The platinum crucible with the melt is taken out of the oven with a platinum tongs and left to cool. The cooled melt is rinsed into a saucepan with a little distilled water and carefully mixed with concentrated hydrochloric acid. After the evolution of gas has ended, the solution is evaporated to dryness. The backlog will - - again in 20 ml conc. Hydrochloric acid was taken up and again evaporated to dryness. Evaporation with hydrochloric acid is repeated once more. The residue is mixed with approx. 5 - 10 ml hydrochloric acid

(12%) moistened with approx. 100 ml dist. Water added and heated. Insoluble SiO ₂ is filtered off, the residue washed three times with hot hydrochloric acid (12%) and then (dest.), Washed with hot water until the filtrate water is Dfree chlori _j.

(b) Silicate determination

The SiO ₂ is incinerated and weighed with the filter.

(c) Determination of iron (and aluminum, calcium and magnesium)

The filtrate collected in the silicate determination is transferred to a 500 ml volumetric flask and supplemented with distilled water up to the calibration mark. The iron determination (or also for aluminum, calcium and magnesium) is then carried out from this solution using FAAS.

(d) Determination of potassium, sodium and lithium

500 mg of the dried sample are weighed to the nearest 0.1 mg in a platinum dish. Then the sample with approx. 1 - 2 ml dist. Moisturized water and 4 drops of concentrated sulfuric acid added. Then it is concentrated three times with approx. 10 - 20 ml. HF evaporated to dryness in a sand bath. Finally, moisten with H ₂ SO ₄ and smoke to dryness on the oven plate. - After briefly glowing the platinum shell, approx. 40 ml dist. Add water and 5 ml of hydrochloric acid (18%) and boil the mixture. The solution obtained is transferred to a 250 ml volumetric flask and up to the calibration mark with dist. Water added. The sodium, potassium and lithium content is determined from this solution using EAS. - -

8. DNA quantification by fluorescent labeling

To determine the DNA content of solutions that were used for the adsorption studies in the dynamic system, the method of fluorescence-photometric quantification of DNA using the fluorescence marker Hoechst 33342 (2 '- (4-ethoxyphenyl) -5- (4-methyl- l-piperazinyl) -2,5'- bi-1H-benzimidazole ^• 3HCl) was used (from Sigma, Steinheim). This is based on the non-intercalative binding of the dye to the base pair adenine-thymine in the DNA. The binding of the fluorochrome to the DNA multiplies its fluorescence intensity by a factor of 60. The location of the maximum excitation and emission wavelength of unbound Hoechst dye shifts from 340 nm and 510 nmi to 355 nm and 465 nm for dye bound to DNA . The fluorescence intensity of a sample incubated with the fluorochrome can be determined by means of a fluorescence photometer. Since the wavelength of the excitation and emission maximum of the fluorochrome shifts when it is attached to the DNA, the bound dye can be selectively excited, thereby avoiding a strong background signal.

The following stock solutions were used for this process:

Hoechst 33342 stock solution. : 10 mg-ml-1 Hoechst 33342 in ddH2O Ix TNE buffer: 10 mM Tris, 1 mM EDTA, 0.2 M NaCl in 1000 ml ddH2O, pH 7.4)

A calibration line for fluorescence photometric determination of the DNA concentration was now created as follows:

• 10 μl sample and a standard series are placed on a black 96-hole plate (Nunc, Roskilde, Denmark) - - applied with DNA concentrations of 100 μg-ml ^"1 to 4750 μg-ml ^x three times.

• The samples are mixed with 200 μl of a mixture of 100 μl

Hoechst-33342 stock solution with 20 ml Ix TNE buffer (10 niM Tris, 1 mM EDTA, 0.2 M NaCl in 1000 ml ddH ₂ O, pH 7.4).

• The samples are incubated for 30 min with the exclusion of light and with shaking at 120 rpm.

• The fluorescence intensity of the samples is measured by excitation at 360 nm and detection at 460 nm in the fluorescence photometer.

Analogous to the description in the last section, the DNA-containing solutions were used to examine the DNA binding to the acid-activated bentonites in the dynamic systems. The previously created calibration line was used to determine the DNA content.

The invention is explained in more detail below with the aid of non-limiting examples.

Show it:

1 shows the DNA loading of the adsorbents A and B according to the invention at different pH values

2 shows the DNA loading of the adsorbents A and B according to the invention at different flow rates through a column packing. - -

Examples

1. Preparation of a sorbent

A raw clay with a montmorillonite content between 70 to 80% is slurried in water and purified by centrifugation. The slurry obtained is then subjected to acid activation. The concentrations are adjusted so that 56% bentonite is mixed with 44% 36% by weight hydrochloric acid and the mixture is boiled at a temperature of 95 to 99 ^° C. for 8 hours. The mixture is then washed with water until the residual chloride content is less than or equal to 5%, based on the solid. To analyze the residual chloride content, 10 g of solid is boiled in 100 ml of distilled water and filtered through a pleated filter. The filtrate is titrated with silver nitrate solution to determine the residual chloride content. Finally drying takes place to a residual moisture content of 8 to 10% by weight. The end product obtained has a weight of 430 to 520 g / l. Particularly preferred particle sizes can be set by sieving or additional grinding.

2. Characterization of the sorbent

The characteristic data of this sorbent (adsorbent 1) and the corresponding freeness are given in the following tables. The characterization of the surface shows that there is a negative zeta potential in solutions. However, the surface charge density is relatively small. Values above 200 μeq / g can be achieved with specially modified materials. - -

Table 1: Surface charge density and zeta potential

Table 2: Particle size distribution

Sample Particle Size Distribution Particle Size Distribution

in air in water

μm [%] μm [%]

Adsorbent 1> 25 4.13> 25 5.41

> 20 6.14> 20 9.15

> 10 18.56> 10 30.00

<5 57.42 <5 38.42

<2 27.57 <2 7.61

<1 10.79 <1 0.87

Adsorbent 1 was characterized according to the BJH method and BET method (DIN 66131) with regard to the average pore diameter and the BET surface area. The following values resulted:

- -

Table 3: BET surface area and pore diameter

According to the CCl ₄ method (see above), the following values resulted:

Table 4: Pore diameter and pore volume

In order to test the suitability of the new adsorbent type for binding DNA, adsorption experiments of herring sperm DNA (Aldrich) were carried out.

To determine the concentration in the adsorption experiments, the DNA concentration was determined photometrically. A wavelength of 260 nm was set for the measurement. In order to calibrate the method with the DNA salt used, a measurement was carried out using a series of concentrations. The calibration line obtained was used for the photometric determination of the DNA concentration in the adsorption experiments. - -

For the adsorption experiments, a herring sperm DNA solution with a concentration of 1 mg / ml, 2 mg / ml, 5.63 mg / ml and 9.9 mg / 1 was prepared and adjusted to pH 8 using 1OmM Tris / HCL and ImM EDTA . Then 0.1 g of the adsorbents were each mixed with 5 ml of the DNA solution and shaken for 1 hour at room temperature. The mixture was then centrifuged at 2500 rpm for 15 minutes and the supernatant was sterile filtered. Finally, the DNA concentration in the supernatant was measured and from this the binding capacity for DNA was calculated. The results are summarized in the following table and in the following graphic:

Table 5: DNA binding capacities

BK = binding capacity - ^ converted into mg DNA based on 1 g of the adsorbents

In order to recover the bound DNA from the adsorbents, elution is carried out with 1.5 molar sodium chloride solution in 10 mM Tris HCL pH 8.5 for 1 hour (elution volume: 100 ml), 15 min. Centrifuged at 2500 rpm, the supernatant sterile filtered and the absorption measured. Table 4: Elution of the bound DNA

It was found that the bound DNA can be recovered almost quantitatively from the adsorbents. This shows the potential use of the new adsorbents both for separating and for purifying DNA.

In order to be able to classify the binding capacity of the adsorbents according to the invention for DNA in comparison with the prior art, analogous binding tests were carried out using a commercially available anion exchanger (Quiagen®, genomic tip). The matrix was removed from the column and ground to a grain size comparable to that of the material according to the invention. The comparison results are listed in the following table.

Table 5: Comparative results on DNA binding capacity with commercially available adsorbent

As the comparison of Table 3 and Table 4 shows, the adsorbent type according to the invention has a significantly higher binding capacity than the comparison anion exchanger. Consequently _ _ Binding capacities of adsorbents that are commercially available according to the state of the art are reached or exceeded. In addition, the adsorbents according to the invention have a much faster DNA binding because the corresponding amounts of DNA are already bound after 1 hour compared to the adsorption time of 16 hours for the comparison material.

The data suggest that the binding sites of the adsorbents according to the invention are much more accessible, especially for large biomolecules, than for the comparative adsorbent.

2. Production of further sorbents (according to the invention)

Two further raw clays with a montmorillonite content between 70 to 80% were activated with acid analogously to the method described in Section 1. The end products were dried to a residual moisture content of 8 to 10% by weight. The end products obtained had bulk densities between 460 to 510 g / l. Particularly preferred particle sizes can be set by sieving or additional grinding. Materials with dry sieve residues of more than 90% at 5, 10 and 35 μm were used.

Characterization of the sorbents

The characteristic data of the above sorbents (adsorbent A or B) are given in the following tables. - -

Table 6: Surface charge density and zeta potential

Adsorbents A and B were characterized according to the BJH method and BET method (DIN 66131) with regard to the average pore diameter and the BET surface area. The following values resulted:

Table 7: BET surface area and pore diameter

According to the CClή method (see above), the following values were obtained: Table 8: Pore diameter and pore volume

The silcat analysis showed the following values:

Table 9: Silcat analysis

A comparable DNA binding capacity to that described above for adsorbent 1 could be demonstrated for both adsorbents A and B. Examination of the DNA binding capacity at various pH values (pH 3 to pH 8) showed good DNA loading over the entire pH range, as shown in FIG. 1. - -

Examination of the DNA binding to a layer of the sorbent (in the dynamic system)

In order to check the suitability of the sorbents for DNA binding or separation in the process according to the invention, the materials adsorbent A and adsorbent B were packed in a chromatography column (15 × 50 mm with 10 μm PTEE frits) and loaded with DNA at different flow rates (DNA sodium salt from herring sperm, Sigma D6898). The columns were packed in detail as follows: 1000 mg of the adsorbent (A or B) were slurried in a 15 ml falcon tube with 5 ml of 50 mM citrate buffer (pH 4.0) and into that with a stamp down completed column pipetted. The second column end piece is put on, the column is connected to an FPLC system so that the mobile phase flows through it from bottom to top. The FPLC pump is set to the flow rate for which the capacity of the adsorber is to be determined. The movable plunger of the column is slowly hand-tightened during equilibration with 50 ml 50 mM citrate buffer (pH 4.0) and then relieved by a quarter turn of the thread.

To load the column with DNA, 25 ml of a DNA solution with a DNA concentration of 1 mg / ml in 50 mM citrate buffer (pH 4) were packed with 1000 mg of adsorbent A or B using an FPLC pump Pumped columns.

Capacities for the flow rates 0.5, 1, 3, 5 and

10 ml / min determined. The DNA content of the run was determined by fluorescence photometry in accordance with the method described in the method section by labeling with the fluorescent dye Hoechst 33342 (Sigma, Steinheim). The difference between the amounts of DNA in the run and the solution applied is assumed to be bound to the adsorber. If you divide this difference by the mass of the adsorber used, you get its load at the associated flow rate. The measured values for adsor bens A and B are shown as mean values of a double determination in FIG. The flow rate was converted with the area of the column cross section into the flow speed with the unit cm / h. The capacities determined under the specified test conditions were both adsorbents above 12 μg / mg ¹ for a flow rate of 16.8 cm / h.

According to the manufacturer's instructions, the weakly basic anion exchanger (Genomic Tip) from Qiagen has a dynamic capacity of 0.2 μg-mg-1 for genomic DNA.

Elution of the bound DNA

The following buffer was used to elute DNA bound to the adsorbents. All chromatography steps were carried out at a flow rate of 1 ml / min.

Elution buffer: 50 mM Tris in ddH ₂ O, pH 8.0

1000 mg of adsorbent A were charged in flow mode (see above) with 25 ml of a DNA solution with a concentration of 1 mg / ml in 50 mM citrate buffer pH 4. The column was rinsed with 20 ml of 50 mM citrate buffer (pH 4). The DNA was eluted with 30 ml of elution buffer. The DNA content of the run, the wash and the elution fractions were determined. The results are shown in Table 10 below as the mean of a duplicate determination.

Table 10

This results in a recovery rate in the eluate, expressed as% of the DNA loading, of more than 85%.

Claims

- - PATENT CLAIMS

1. A method for the sorption of at least one nucleic acid molecule from a liquid medium, comprising the following steps:

a. Providing a liquid medium containing at least one nucleic acid molecule;

b. Providing a layer containing at least one acid-activated layered silicate, the layer being permeable to the liquid medium and the

Layer thickness is at least 1 mm;

2. The method according to claim 1, characterized in that the acid-activated layered silicate has an iron content, calculated as Fe ₂ Cb based on the total amount of acid-activated layered silicate used, of less than 6% by weight, preferably less than 4% by weight preferably has less than 3% by weight, in particular less than 2.5% by weight.

3. The method according to any one of the preceding claims, characterized in that the layer thickness of the layer according to claim 1, step b. is more than 0.3 cm, preferably more than 0.5 cm, in particular between 0.1 and 100 cm.

4. The method according to any one of the preceding claims, characterized in that the acid-activated layered silicate has a swellability of not more than 15 ml / 2g, in particular not - - Has more than 10 ml / 2g, more preferably from 1 to 15 ml / 2g, in particular from 2 to 10 ml / 2g.

5. The method according to any one of the preceding claims, characterized in that the acid-activated layered silicate in particulate form with a dry sieve residue of less than 10%, in particular less than 5%, more preferably less than 1% at 5 .mu.m, in particular at 10 .mu.m, more preferably at 35 µm.

6. The method according to any one of the preceding claims, characterized in that the liquid medium is an aqueous or alcoholic medium.

7. The method according to any one of the preceding claims, characterized in that the layered silicate is selected from the group of natural or synthetic layered silicates.

8. The method according to any one of the preceding claims, characterized in that the layered silicate is not treated with a cationic polymer or polycation.

9. The method according to any one of the preceding claims, characterized in that the cation exchange capacity of the acid-activated layered silicate is less than 70 meq / 100 g, in particular less than 50 meq / 100 g, more preferably less than 40 meq / 100 g.

10. The method according to any one of the preceding claims, characterized in that the acid-activated layered silicate has a BET surface area of at least 50 m ² / g, in particular at least 100 m ² / g, particularly preferably at least 130 m ² / g. - -

11. The method according to any one of the preceding claims, characterized in that the acid-activated layered silicate, preferably in granular form, has a grain size (D50) of 100 to 500 µm, in particular from 200 to 2,000 µm.

12. The method according to any one of the preceding claims, characterized in that the acid-activated layered silicate has a porosimetry as follows: pores up to 80 nm in diameter between about 0.15 and 0.80 rhl / g, in particular between about 0.2 and 0.7 ml / g; Pores up to 25 nm in diameter between about 0.15 and 0.45 ml / g, in particular 0.18 to 0.40 ml / g; Pores up to

14 nm between about 0.10 and 0.40 ml / g, in particular about 0.12 to 0.37 ml / g, each determined according to the CCl ₄ method.

13. The method according to any one of the preceding claims, characterized in that the average pore diameter according to the BJH method of the acid-activated layered silicate is between 2 and 25 nm, in particular between 4 and 20 nm.

14. The method according to any one of the preceding claims, characterized in that the at least one nucleic acid molecule is selected from mono-, oligo- and polynucleotides or mixtures thereof.

15. The method according to any one of the preceding claims, characterized in that the at least one nucleic acid molecule is selected from ribonucleic acids (RNA) and deoxyribonucleic acids (DNA) or mixtures thereof.

16. The method according to any one of the preceding claims, characterized in that the at least one nucleic acid molecule at least 10 nucleotide building blocks, preferably at least 100 nucleotide building blocks and particularly preferably at least 1000 - -

Has nucleotide building blocks.

17. The method according to any one of the preceding claims, characterized in that the preferably aqueous or alcoholic medium with the at least one nucleic acid molecule is a colloidal solution, suspension, dispersion, solution or emulsion.

18. The method according to any one of the preceding claims, characterized in that the layer with the sorbed nucleic acid molecule after step c. of claim 1 is washed with an aqueous or alcoholic buffer to remove contaminants.

19. The method according to any one of the preceding claims, characterized in that after step c. from at least one separation or desorption of the at least one nucleic acid molecule from the layer, whereby the nucleic acid molecule is obtained and the layer can optionally be used again for sorption of at least one nucleic acid molecule.

20. The method according to any one of claims 1 to 18, characterized in that after step c. of claim 1 in a further step the layer is disposed of together with the nucleic acid molecule.

21. The method according to any one of the preceding claims, characterized in that the layer consists essentially or completely of at least one acid-activated layered silicate.

22. The method according to claim 19, characterized in that the desorption or separation of the at least one nucleic acid molecule in an alkaline (pH 8 or more) buffer solution, in particular a high salt buffer solution, preferably with more than 1 M NaCl.

23. The method according to any one of the preceding claims, characterized in that the at least one acid-activated

Layered silicate is combined in the layer with a binder to form particle aggregates or moldings or is applied to a carrier.

24. The method according to any one of the preceding claims, characterized in that the binder is alginate, agar-agar, chitosans, pectins, gelatin, lupine protein isolates or gluten.

25. Layered composition containing at least one acid-activated layered silicate and at least one nucleic acid molecule, the layer thickness being at least 1 mm.

26. Use of a method according to any one of claims 1 to 24 or a layered composition according to claim 25 for the separation, recovery or purification of a nucleic acid molecule.

27. Use according to claim 26 for separating a nucleic acid molecule from protein components in a liquid medium.