EP1960519A1 - Procede de sorption d'au moins une molecule d'acide nucleique a l'aide de phyllosilicates d'activation acide - Google Patents

Procede de sorption d'au moins une molecule d'acide nucleique a l'aide de phyllosilicates d'activation acide

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Publication number
EP1960519A1
EP1960519A1 EP05819297A EP05819297A EP1960519A1 EP 1960519 A1 EP1960519 A1 EP 1960519A1 EP 05819297 A EP05819297 A EP 05819297A EP 05819297 A EP05819297 A EP 05819297A EP 1960519 A1 EP1960519 A1 EP 1960519A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
acid molecule
layered silicate
layer
acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05819297A
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German (de)
English (en)
Inventor
Ulrich Sohling
Thomas Scheper
Cornelia Kasper
Kirstin Suck
Daniel Riechers
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sued Chemie AG
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Sued Chemie AG
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Filing date
Publication date
Application filed by Sued Chemie AG filed Critical Sued Chemie AG
Publication of EP1960519A1 publication Critical patent/EP1960519A1/fr
Withdrawn legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B01J20/12—Naturally occurring clays or bleaching earth
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28004—Sorbent size or size distribution, e.g. particle size
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28016—Particle form
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/2803—Sorbents comprising a binder, e.g. for forming aggregated, agglomerated or granulated products
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28033—Membrane, sheet, cloth, pad, lamellar or mat
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28057—Surface area, e.g. B.E.T specific surface area
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28069—Pore volume, e.g. total pore volume, mesopore volume, micropore volume
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28078—Pore diameter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424—Elution mode

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • step 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • biomolecule is understood to be a molecule which has, as building blocks, nucleotides or nucleosides (nucleobases), amino acids, monosaccharides and / or fatty acids.
  • nucleic acid (molecule) also includes other biomolecules.
  • use for the sorption of nucleic acids is particularly preferred.
  • 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.
  • 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.
  • 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.
  • further constituents including, for example, non-acid-activated layered silicates
  • 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.
  • 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
  • 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.
  • Layer thickness 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.
  • 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 ⁇ m, determined on the basis of the dry sieve residue (as stated above).
  • 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.
  • the acid-activated layered silicate has a swellability of less than 15 ml / 2 g, in particular less than 10 ml / 2 g.
  • 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.
  • 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 .
  • 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.
  • 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 such as bentonite are particularly preferred.
  • 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.
  • 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.
  • the sorbent according to the invention or the layer consists essentially or completely of at least one _ I 2 - acid activated layered silicate.
  • 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.
  • 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.
  • 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.
  • the micropore volume of the layered silicate can be increased.
  • the cation exchange capacity is decreasing.
  • 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.
  • 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 are brought into contact with the acid (s).
  • the acid 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.
  • any organic or inorganic acids or mixtures thereof can be used.
  • acid can be sprayed on using a so-called SMBE process (Surface Modified Bleaching Earth).
  • SMBE process Surface Modified Bleaching Earth
  • the activation of the layered silicate is thus carried out in the aqueous phase.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the SMBE process is usually carried out at room temperature, with elevated temperatures allowing better acid activations in individual cases.
  • 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.
  • the HBPE process attacks the layer structure, which results in areas with silica, in addition to structurally largely unchanged areas.
  • 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.
  • the contact times with the acid are often around 15 minutes in the laboratory.
  • 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.
  • 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.
  • 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).
  • the exchangeable (metal) 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.
  • 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 can be used in the form of a powder, granulate or any shaped body.
  • the sorbents can be used in any form, including supported or immobilized forms.
  • 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.
  • 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.
  • these can be chromatography columns, including gravity or centrifugation columns, solid phase chromatography, filter cartridges or membranes.
  • 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.
  • 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 ⁇ m to 5,000 ⁇ m, in particular 200 to 2,000 ⁇ m particle size.
  • the above-mentioned dry sieve residues are particularly preferred for the layers or column packs used according to the invention.
  • 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,
  • the sorbent used according to the invention can be in immobilized form.
  • 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. _
  • 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.
  • the nucleic acids are DNA or RNA molecules in double-stranded or single-stranded form with one or more nucleotide units.
  • 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).
  • 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.
  • 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 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.
  • nucleic acids such as high-purity plasmid DNA
  • 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.
  • 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.
  • the particularly preferred aqueous or alcoholic media are understood to mean all media containing water or alcohol, including aqueous-alcoholic media.
  • all media are also included in which water is completely miscible or completely mixed with other solvents.
  • 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.
  • 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.
  • 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 ⁇ m, in particular more than 50 ⁇ m, 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.
  • the following can be used as binders: agar-agar, alginates, chitosans, pectins, gelatins, lupine protein isolates or gluten.
  • 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.
  • 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.
  • nucleic acids 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.
  • the depletion or removal of nucleic acid molecules from culture media can also be carried out.
  • 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 -
  • nucleic acid molecules 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.
  • the extraction or purification of desired nucleic acids from solutions is one of the standard procedures in biological and medical research.
  • 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.
  • 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 ⁇ m citrate buffer (pH 4.0).
  • the acid-activated phyllosilicates have a high binding capacity for nucleic acid molecules over a very wide pH range.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • such a layered composition can advantageously be used, for example, to separate, recover or purify a nucleic acid molecule from a liquid medium.
  • Another aspect of the present invention thus also relates to the use of an acid-activated
  • 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.
  • the sorbents according to the invention are also suitable for efficiently introducing these biomolecules into prokaryotic or eukaryotic cells.
  • 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.
  • 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).
  • 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.
  • 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.
  • the particle sizes (distribution) are determined using the Malvern method.
  • 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).
  • 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.
  • 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.
  • deionized water approximately 800 ml
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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
  • the SiO 2 is incinerated and weighed with the filter.
  • 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.
  • 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.
  • 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)
  • the fluorescence intensity of the samples is measured by excitation at 360 nm and detection at 460 nm in the fluorescence photometer.
  • 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.
  • 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.
  • Adsorbent 1 25 4.13> 25 5.41
  • 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:
  • 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. - -
  • 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:
  • BK binding capacity - ⁇ converted into mg DNA based on 1 g of the adsorbents
  • 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.
  • 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.
  • 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 ⁇ m were used.
  • 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:
  • the materials adsorbent A and adsorbent B were packed in a chromatography column (15 ⁇ 50 mm with 10 ⁇ m 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.
  • 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 ⁇ g / mg 1 for a flow rate of 16.8 cm / h.
  • the weakly basic anion exchanger (Genomic Tip) from Qiagen has a dynamic capacity of 0.2 ⁇ g-mg-1 for genomic DNA.
  • Elution buffer 50 mM Tris in ddH 2 O, pH 8.0
  • adsorbent A 1000 mg 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.

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Abstract

L'invention concerne un procédé de sorption d'au moins une molécule d'acide nucléique d'un agent liquide. Ce procédé consiste (a) à prendre un agent liquide contenant au moins une molécule d'acide nucléique; (b) à utiliser une couche contenant au moins un phyllosilicate d'activation acide, cette couche étant perméable à l'agent liquide et ayant une épaisseur de 1 mm minimum; (c) à faire passer l'agent contenant la ou les molécules d'acide nucléique conformément à l'étape (a) à travers la couche conforme à l'étape (b) pour la sorption de la ou des molécules d'acide nucléique dans la couche.
EP05819297A 2005-12-09 2005-12-09 Procede de sorption d'au moins une molecule d'acide nucleique a l'aide de phyllosilicates d'activation acide Withdrawn EP1960519A1 (fr)

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