WO2000049031A2

WO2000049031A2 - Method for isolating nucleic acids

Info

Publication number: WO2000049031A2
Application number: PCT/EP2000/001028
Authority: WO
Inventors: Wolfgang Podszun; Rainer Neumann
Original assignee: Bayer Aktiengesellschaft
Priority date: 1999-02-19
Filing date: 2000-02-09
Publication date: 2000-08-24
Also published as: WO2000049031A3; AU2547100A; DE19907023A1; CA2362979A1; EP1155026A2; JP2002537306A

Abstract

Nucleic acids can be efficiently adsorbed on specific water-insoluble pearl polymers with an average size of 3 to 100 mu m, at a neutral or acidic pH value. The acids can then be liberated at an alkaline pH with high yields.

Description

Methods for isolating nucleic acids

The invention relates to a method for separating and selectively releasing nucleic acids using special bead polymers.

In recent times, so-called gene diagnostics has become increasingly important. Genetic diagnostics has found its way into the diagnosis of human diseases (e.g. detection of infectious agents, detection of mutations in the genome, analysis of circulating tumor cells and identification of risk factors for the predisposition of a disease). But gene diagnostics is now also used in veterinary medicine, environmental analysis and food testing. Another area of application is investigations at pathological / cytological institutes or in the context of forensic questions. But also in the context of quality and process control (for example

Genetic diagnosis is now used and the legislator plans to expand the number of tests already prescribed by law in the future.

Methods that are used in gene diagnostics (such as hybridization or amplification techniques such as PCR, bDNA or NASBA, TMA technology) are also part of routine procedures for basic scientific work.

Due to the increasing spread of non-radioactive detection methods, which also play a role in the context of gene diagnostics, it is to be expected that gene diagnostics will be more widespread in the future than at present.

In gene diagnostics, the acquisition of gene samples from biological material such as cells, blood, sputum, cerebrospinal fluid, serum or urine is an important sub-step. The binding of nucleic acids to polyquaternary cationic polymers is known from US-A-4 046 750. However, the binding is irreversible, so that with this method the adsorbed nucleic acids cannot be released again.

US-A-4 055 469 discloses a method for purifying enzymes, wherein

Nucleic acids and unwanted proteins are precipitated using water-soluble cationic polymers.

US-A-4 839 231 discloses supports coated with vinylpyridine polymer which can adsorb proteins and nucleic acids. However, the capacity of

Carrier quite low and the adsorption of the nucleic acids not quantitative.

WO-A-91/05606 describes a silanized, porous support material with hydroxyalkylamino groups for the chromatographic separation of nucleic acids. However, this material is for quick and as quantitative as possible

Separation of nucleic acids from biological material is less suitable.

DE-A-4 139 664 describes a device and a method for isolating and purifying nucleic acids with the aid of anion exchangers. A disadvantage of this method is that the desorption of the nucleic acids from

Anion exchangers are only successful with buffer solutions of high ionic strength and the separation of the salts from the buffer solution requires additional preparation steps.

DE-A-4 333 805 claims the extraction of nucleic acids from a sample with the aid of water-soluble carriers such as dextran, acrylamide or carboxymethyl cellulose and other reagents, the nucleic acids being precipitated.

EP-A-0 707 077 (corresponds to US-A-5 582 988) describes a method for

Isolation of nucleic acids from biological material using soluble, weakly basic polymer is described. With this method, a precipitate is made from the soluble, weakly basic in an acidic pH range Polymer and the nucleic acid generated, the precipitate separated from the unprecipitated components of the biological material and washed and the nucleic acid released from the precipitate by adjusting a basic pH.

A disadvantage of the methods according to DE-A-4 333 805 and EP-A-0 707 077 is that handling, in particular the separation and purification of the precipitation product, is difficult and very time-consuming. These methods can also not be carried out or only under difficult conditions with the aid of automated analysis devices.

WO-A-96/18731 describes a method for isolating nucleic acids with the aid of a detergent and a solid support. Since solid supports without pores and without swellability are used, the binding capacity of the supports is relatively low.

WO-A-97/08547 describes a method for isolating nucleic acids in which the nucleic acids are bound to a solid hydrophilic organic polymer without an effective positive charge, for example to cellulose. With this method, the bond occurs via weak forces, such as Van der Waal's interactions.

WO-A-97/34909 describes a method for isolating nucleic acids using a special particulate polymer with a lower critical solubility temperature (LCST) of 25-45 ° C. A disadvantage of this method is that the release of

Nucleic acids Buffers with high ionic strengths, which can interfere with the further use of the nucleic acids, must be used. Because of the small size of the particles used, from 0.05 to 2 μm, processing is also time-consuming and difficult to automate. Despite the numerous previously described methods for isolating nucleic acids, there is still an urgent need for a simple method that can be automated as far as possible for the effective separation and re-release of nucleic acids from biological material.

Surprisingly, it has now been found that certain water-insoluble bead polymers of polymerized units of amino monomer, crosslinking agent and vinyl monomer are outstandingly suitable for isolating nucleic acids.

The invention relates to a method for isolating nucleic acids from a sample, comprising the following steps

A) mixing the sample with a water-insoluble, non-ionic polymer in the basic and neutral range at a pH of 7 or less, the nucleic acids being adsorbed,

B) separating the water-insoluble polymer and

C) mixing the water-insoluble polymer with an aqueous phase with a pH of greater than 7, the adsorbed nucleic acids being released,

characterized in that the water-insoluble polymer is a bead polymer with an average particle size of 3 to 100 microns and from polymerized units of

a) 5 to 98% by weight of amino monomer b) 0.3 to 30% by weight of crosslinking agent and c) 0 to 93% by weight of vinyl monomer consists.

If appropriate, the biological material is lysed in an intermediate step after process step A).

The present invention preferably relates to a method for isolating nucleic acids from a sample, comprising the steps

B) separating the water-insoluble polymer and

characterized in that the water-insoluble polymer is a bead polymer with an average particle size of 3 to 100 μm and a specific surface area measured according to BET of 5 to 500 m / g and composed of polymerized units of

a) 5 to 98% by weight of amino monomer b) 0.3 to 30% by weight of crosslinking agent and cl) 0 to 93% by weight of hydrophobic vinyl monomer

consists, or that the water-insoluble polymer from

bead polymer readily swellable in water with an average particle size of 3 to 100 μm, which consists of polymerized units of a) 5 to 79.7% by weight of amino monomer b) 0.3 to 10% by weight of crosslinking agent and c2) 10 to 93% by weight of hydrophilic vinyl monomer

consists.

The present invention particularly preferably relates to a method for isolating nucleic acids from a sample, comprising the steps A), B) and C) defined above, characterized in that the water-insoluble polymer is a macroporous bead polymer having a particle size of 3 to 100 μm, one

Has pore diameter in the range of 10 to 1000 nm, the specific surface determined according to BET 5 to 500 m ² / g (very particularly preferably 20 to 200 m ² / g) and from polymerized units of

a) 5 to 98% by weight of amino monomer b) 2 to 30% by weight of crosslinking agent cl) 0 to 93% by weight of hydrophobic vinyl monomer

consists, or that the water-insoluble polymer from

bead polymer readily swellable in water with an average particle size of 3 to 100 μm, which consists of polymerized units

a) 5 to 79.7% by weight of amino monomer b) 0.3 to 10% by weight of crosslinking agent and c2) 10 to 93% by weight of hydrophilic vinyl monomer

consists.

The method according to the invention is suitable for the isolation and / or purification of nucleic acids of different origins, for example from cells, tissue materials, blood or infectious agents. Before isolating the nucleic acids, this is The material to be examined is disrupted by techniques known per se, such as digestion by protease digestion, a sample suitable for the further steps A to C, a lysate, being obtained. The biological material is optionally lysed in an intermediate step after process step A). Further suitable digestion processes have been described in DE-A-4 333 805.

The sample is mixed with a water-insoluble polymer at a pH of 7 or less, preferably in the range 2-6, particularly preferably in the range 2-3, at room temperature. The water-insoluble polymer is separated off by e.g. Filtration or centrifugation. The complex of nucleic acid and polymer thus obtained can now be washed by washing with suitable buffers, e.g. TE can be cleaned.

To release the bound nucleic acids from the complex, the pH of the complex is now adjusted to pH values above 7, preferably from 8 -

14, particularly preferably in the range 12-14.

The bead polymers according to the invention provide higher adsorption and re-release rates than the soluble polymers according to EP-A-0 707 077. The isolation is easier, i.e. perform with fewer steps and in shorter times. The purity of the isolated nucleic acids is higher, in particular they receive less inhibiting by-products, so that amplification of the nucleic acids, for example by means of the so-called "PCR reaction" and "RT-PCR", is particularly successful. The method according to the invention is also superior to the method described in EP-A-0 707 077 with regard to digestion of the nucleic acids obtained by means of restriction enzymes.

The present invention furthermore relates to the macroporous bead polymers, characterized in that they have an average particle size of 3 to 100 μm, a pore diameter of 10 to 1,000 nm and a specific upper Area measured according to BET from 5 to 500 m ² / g and from polymerized units of

a) 5 to 98% by weight of amino monomer b) 2 to 30% by weight of crosslinking agent and cl) 0 to 93% by weight of hydrophobic vinyl monomer

exist, as well

the water-insoluble but swellable bead polymers, characterized in that they have an average particle size of 3 to 100 μm and from polymerized units of

a) 5 to 79.7% by weight of amino monomer b) 0.3 to 10% by weight of crosslinking agent and cl) 10 to 93% by weight of hydrophilic vinyl monomer

consist.

The present invention furthermore relates to a process for the preparation of water-insoluble, macroporous bead polymers having an average particle size of 3 to 100 μm, a pore diameter of 10 to 1000 nm and a specific surface area measured according to BET of 5 to 500 m ² / g, characterized in that one a mixture of

a) 5 to 98 parts by weight of amino monomer b) 2 to 30 parts by weight of crosslinking agent cl) 0 to 93 parts by weight of hydrophobic vinyl monomer d) 10 to 150 parts by weight of porogen and e) 0.1 to 2.5 Parts by weight radical generator dispersed in an aqueous medium using a protective colloid, then the dispersion obtained is polymerized by heating to the decomposition temperature of the radical generator and, after the polymerization, the porogen is removed by extraction and / or evaporation,

and a process for the preparation of water-insoluble but swellable bead polymers having an average particle size of 3 to 100 μm, characterized in that a mixture of

a) 5 to 79.7% by weight of amino monomer b) 0.3 to 10% by weight of crosslinking agent and c2) 10 to 93% by weight of hydrophilic vinyl monomer d) 10-150 parts by weight of solvent and e) 0, 1 - 2.5 parts by weight of radical generator

dispersed in an aqueous medium using a protective colloid, then the resulting dispersion polymerized by heating to the decomposition temperature of the radical generator and, after the polymerization, the solvent removed by extraction and / or evaporation.

Amino monomers (a) in the context of the invention are polymerizable, ethylenically unsaturated compounds having at least one primary, secondary or tertiary amino group. The secondary or tertiary amino group can also be part of a cycloaliphatic or aromatic ring. Examples include N-vinylimidazole, N-vinylbenzimidazole, 2-vinylpyridine and 4-vinylpyridine. Highly suitable amino monomers are also the derivatives of acrylic acid and methacrylic acid, such as, for example, 2-aminoethyl methacrylate, N, N-dimethylaminoethyl methacrylate, N, N-dimethylaminopropyl methacrylate, N, N-dimethylaminoethyl acrylate, N-tert-butylaminopropyl methacrylate, N- (3-aminopropyl) methacrylamide, N- (3-imidazoyl-propyl-methacrylamide, N- (2-imidazoylethyl) methacrylamide, N- (3-aminopropyl) -acrylamide, N- (3-imidazoyl-pi-opyl) acrylamide, N- (2-imidazoylethyl) acrylamide, N- (l, l - Dimethyl-3-imidazoylpropyl) methacrylamide, N- (l, l-dimethyl-3-imidazoylpropyl) acrylamide, N- (3-benzimidazoylpropyl) methacrylamide and (3-benzimidazoylpropyl) acrylamide.

Highly suitable amino monomers are also the reaction products of isocyanatoethyl (meth) acrylate and imidazoylalkylamines, such as, for example, the amino monomer of the formula (I) which is new in the context of the present invention.

Further suitable amino monomers according to the present invention are the pyridine derivatives of the formulas (II) and (III)

(II) (III).

Derivatives of styrene and α-methylstyrene with amino groups are also well suited. Examples include: 4-N, N-dimethylaminostyrene, 2-N, N-dimethylaminostyrene, 4-N, N-diethylaminostyrene and 4-N, N-bis (2-hydroethyl) aminostyrene.

Ethylene glycol distearate methacrylate, butanediol dimethacrylate, hexanediol dimethacrylate, acrylate Pentaerytritoldimeth-, 1, 2-glycerol dimethacrylate, 1, 3-glycerol dimethacrylate, Triethylenglycoldi- methacrylate, taerytritoltrimethacrylat Tetraetylenglycoldimethacrylat, trimethylolpropane trimethacrylate, pen, Pentaerytritoltetramethacrylat, ethylene glycol diacrylate: According to the present invention, suitable crosslinking agents (b) are , Butanediol diacrylate, pentaerytritol diacrylate, 1, 3-glycerol diacrylate, triethylene glycol diacrylate, trimethylolpropane triacrylate, pentaerytritol triacrylate, pentaerytritol tetraacrylate, allyl methacrylate, allyl acrylate, methylene-N, N'-bisacrylamide, p-divinylbenzene and m-divinylbenzene.

Hydrophobic vinyl monomers (c1) which can be present in the bead polymer according to the invention and are suitable for the purposes of the present invention are C 1 -C 6 -alkyl acrylate, C 1 -C 6 -alkyl methacrylate, such as methyl methacrylate or butyl acrylate, acrylonitrile, methacrylonitrile, Vinyl chloride, vinylidene chloride, vinyl acetate and aromatic vinyl monomers, such as Styrene, vinyl naphthalene, vinyl toluene, ethyl styrene, α-methyl styrene, chlorostyrenes and vinyl benzyl chloride.

Hydrophilic vinyl monomers (c2) which are suitable in the context of the present invention are, for example: 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, triethylene glycol monomethacrylate,

Tetraethylene glycol monomethacrylate, acrylamide, methacrylamide and N, N-dimethyl acrylamide.

Porogens are used for the process according to the invention for the production of water-insoluble macroporous bead polymers. For this are liquid with

Water-immiscible compounds which dissolve the monomers used and precipitate the polymer formed are suitable. Examples include aliphatic hydrocarbons such as hexane, heptane, octane, isooctane, isododecane and alcohols such as octanol. The porogen is used in amounts of 10 to 150% by weight, preferably 20 to 100% by weight, based on the sum of the monomers and crosslinking agents used.

Suitable radical formers in the context of the present invention are preferably oil-soluble initiators. Examples include: peroxy compounds such as dibenzoyl peroxide, dilauryl peroxide, bis (p-chlorobenzoyl peroxide), dicyclohexyl peroxydicarbonate, tert-butyl peroctoate, 2,5-bis (2-ethylhexanoylperoλy) -2,5-dimethylhexane and tert-amylperoxy-2-ethylhexane, further azo compounds such as 2,2'-azobis (isobutyronitrile), 2,2'-azobis (2,4-dimethylvalereonitrile) and 2,2'-azobis (2-methylisobutyronitrile) . The initiators are generally used in amounts of 0.05 to 2.5% by weight, preferably 0.2 to 1.5% by weight, based on the monomer mixture.

Protective colloids are optionally used in the aqueous phase in the preparation of the gel-shaped and macroporous bead polymers according to the invention. Suitable protective colloids according to the present invention are natural and synthetic water-soluble polymers, such as gelatin, starch,

Cellulose derivatives, in particular cellulose esters and cellulose ethers, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and copolymers of acrylic acid, methacrylic acid, methacrylic acid esters and / or acrylic acid esters. Copolymers of methacrylic acid and methacrylic acid ester neutralized with alkali metal hydroxide are particularly suitable. The amount of protective colloids used is generally 0.05 to 2%, based on the aqueous phase, preferably 0.1 to 1%.

The aqueous phase can optionally also contain a buffer system. Buffer systems are preferred which adjust the pH of the aqueous phase at the start of the polymerization to a value between 12 and 5, preferably between 10 and 6. Under these conditions, dispersants with carboxylic acid groups are wholly or partly present as salts. In this way, the effect of the protective colloids is influenced favorably. Buffer systems that are particularly suitable contain phosphate or borate salts.

The amount of the water phase is generally 75 to 1200% by weight, preferably 100 to 500% by weight, based on the sum of monomers, crosslinking agents and porogen. The stirring speed during the polymerization is important for the adjustment of the particle size. In the production process according to the invention, the size of the bead polymers obtained decreases with increasing stirrer speed. The exact stirring speed for setting a certain predetermined bead size depends in individual cases on the reactor size, the reactor geometry and the stirrer geometry. It has proven to be expedient to determine the necessary stirring speed experimentally. For laboratory reactors with a reaction volume of 3 liters, which are equipped with blade stirring, when using copolymers of acrylic acid, methacrylic acid, acrylic acid esters and / or methacrylic acid esters as dispersing agents, bead sizes of 6 to 30 μm are generally achieved at speeds of 300 to 500 rpm.

The polymerization temperature of the production process according to the invention depends on the decomposition temperature of the initiator used. It is generally between 50 and 150 ° C, preferably between 55 and 100 ° C. The polymerization takes 0.5 to a few hours. It has proven useful to use a temperature program in which the polymerization is started at a low temperature, for example 70 ° C., and the reaction temperature is increased as the polymerization conversion progresses.

After the polymerization, the polymer can be isolated using customary methods, for example by filtration or decanting, and optionally dried after one or more washes. The porogen can be removed during drying. In the case of low-boiling porogens, such as, for example, hexane, it is also possible to remove all or part of the porogen from the aqueous reaction mixture before the bead polymer is isolated by distillation.

The water-swellable bead polymers are prepared analogously to the preparation of the macroporous bead polymers, with hydrophilic vinyl monomers (c2) instead of the hydrophobic vinyl monomers (c2) and a instead of the porogen

Solvent is used. Suitable solvents are those which are immiscible with water, which dissolve the monomers and the crosslinking agent and which do not precipitate out, but rather dissolve or swell the polymer formed. Suitable solvents are toluene, xylene, tetrachloromethane, chloroform, methylene chloride, dichloroethane and. Ethyl acetate. The amount of

Auxiliary solvent is generally 10 to 200% by weight, preferably 10 to 150% by weight, particularly preferably 20 to 100% by weight, based on the sum of monomers and crosslinking agent. If desired, the auxiliary solvent can be separated off after the polymerization, for example by distillation. Toluene is particularly easy to remove by azeotropic distillation.

The water-swellable bead polymers according to the invention have swelling indices of 1.2 to 12, preferably 1.5 to 8, measured at 25 ° C. and pH 7. The swelling index is the quotient of the volume of the bead polymer swollen in water until saturation and the Volume of the anhydrous polymer beads defined.

The present application furthermore relates to compositions for isolating nucleic acids containing water-insoluble macroporous bead polymers having an average particle size of 3 to 100 μm and a pore diameter of 10 to

1,000 nm and a specific surface area measured according to BET of 5 to 500 m ² / g, consisting of polymerized units of

or of water-insoluble but swellable bead polymers with an average particle size of 3 to 100 μm, consisting of polymerized units of a) 5 to 79.7% by weight of amino monomer b) 0.3 to 10% by weight of crosslinking agent and c2) 10 to 93% by weight of hydrophilic vinyl monomer.

These agents are preferably in the form of aqueous dispersions with a solids content of 0.1-50%, particularly preferably 1-10%. The aqueous phase can optionally contain buffers which are preferably effective in the range 2-6.

Possible areas of application of an agent formulated, for example, as a test kit are the above-mentioned application examples, for example in the isolation of nucleic acids from cells, tissue materials, blood or infectious agents, all questions of diagnostics playing a role in particular. The test kits also include the polymers described for routine nucleic acid tests in microtiter plates and / or test tubes or other formats such as, for example, in the context of chip technology.

Examples

example 1

Preparation of the amino monomer of formula 1

To a stirred solution of 50.07 g (0.4 mol) of 3-aminopropylimidazole, 140 mg of 2,6-di-tert-butyl-4-methylphenol (stabilizer), 140 mg of dibutyltin dilaurate (catalyst) in 250 ml of chloroform were added 20 ° C. with cooling, 62.08 g (0.4 mol) of 2-isocyanatoethyl methacrylate were added dropwise within 60 minutes. The batch was then heated to 50 ° C. (about 5 h) until no NCO band was detectable in the IR spectrum. 112 g of amino monomer of the formula (I) were obtained.

Example 2

Production of a gel-like bead polymer

A solution of 5 g of polyvinyl alcohol (Mowiol® 40-88) and 1.5 g of disodium hydrogenphosphate in 130 ml was deionized in a 250 ml reaction vessel with a blade stirrer, reflux condenser, thermometer, gas inlet and gas outlet tube

Submitted water. An organic solution of 7.91 g of N, N-dimethylamino-ethyl methacrylate, 7 g of 2-hydroxyethyl methacrylate, 0.17 g of triethylene glycol dimethacrylate, 0.225 g of 2,2'-azobis was added to this aqueous solution at 20 ° C. with stirring at 450 rpm (2,4-dimethylvalereonitrile) and 37.5 g chloroform added within 15 min. It was flushed lightly with nitrogen and the temperature was raised to 67 ° C. and left at this temperature for 20 hours. After cooling, the bead polymer formed was separated from the reaction liquor by decanting and chloroform was removed in vacuo at 50 ° C. 13.5 g of bead polymer were obtained with an average particle size of 20 μm and a swelling index of 5.1 measured at 25 ° C. in water. Example 3

Production of a macroporous bead polymer

In a 2 liter reaction vessel with blade stirrer, reflux condenser, thermometer,

A solution of 42.5 g of polyvinyl alcohol (Moviol 40-88) and 12.75 g of sodium hydrogenphosphate in 1240 g of deionized water was placed in the gas inlet and gas outlet tube. An organic solution of 23.71 g of N, N-dimethylaminoethyl methacrylate, 13.04 g of styrene, 10.67 g of divinylbenzene, 0.71 g of 2 was added to this aqueous solution at 20 ° C. with stirring at 280 rpm (revolutions per minute). 2'-

Azobis (2,4-dimethylvalereonitrile) and 31.62 g of hexane were added within 30 min at 25 ° C. It was flushed slightly with nitrogen and the temperature was raised to 66 ° C. and left at this temperature for 20 hours. The hexane was then distilled off at an internal temperature of 70 to 98 ° C. After cooling, the bead polymer formed was separated from the reaction liquor by decanting and dried in vacuo at 50.degree. 38 g of bead polymer were obtained with an average particle size of 15 μm and a specific surface area of 62.3 m ² / g.

Example 4

Production of a macroporous bead polymer

Example 3 was repeated, an organic solution consisting of 23.71 g of amino monomer from Example 1, 13.04 g of styrene, 10.67 g of divinylbenzene, 0.71 g of 2,2'-azobis (2,4-dimethylvalereonitrile) and 38 g of hexane was used. 35 g of macroporous bead polymer with an average particle size of 25 μm and a specific surface area of 74 m ² / g were obtained. Biological result

100 μl of blood were mixed with 10 ⁵ Caski cells. These cells contain the human papillomavirus type 16 (HPV). 10 μl of a 2.4% particle dispersion were extracted from this mixture

Example 2 added

After the addition of 200 μl of a suitable buffer, for example TE, there was an incubation for 5 mm at room temperature (TE stands for 10 mmol of Tπs HC1 and 1 mmol of ethylenediaminetetraacetic acid pH 7 4 in the final concentration)

• The particles were then sedimented at 7,000 φM for 3 mm in an Eppendorf centrifuge

200 μl of a suitable lysis buffer, for example 0.5% IGEPAL CA-630® m TE (IGEPAL CA-630® is a non-ionic detergent, which can be obtained, for example, from Sigma, order number I 3021) were then added to the sediment. , given and incubated again for 5 mm at room temperature

Instead of IGEPAL CA-630®, other lysis buffers can also be used. Examples include classic methods such as with Protease K digestion and subsequent cleaning using phenol / chloroform or

Natπumlaurylsulfat solutions called (Sigma order number L 6026), for example as a 0.5% water solution

• The centrifugation and sedimentation were carried out again at 7000 φM for 3 mm in an Eppendorf centrifuge. • The particles were then washed twice with a suitable buffer (eg TE). After the second washing step, the supernatant was discarded and only the particles were worked on

• The nucleic acid bound to the particles was released by adjusting the pH to> 12 by adding 1 μl of 0.5 normal NaOH

• A 15 minute incubation at room temperature followed • After centrifugation (3 min, 7,000 φM in an Eppendorf centrifuge), the concentration of the nucleic acid obtained was determined using a suitable method, for example by analysis in a gel system, particularly preferably the "Submerged Gel Nucleic Acid Electrophoresis System", order no. 170 4406 from BIO-RAD (webpage: www.bio-rad.com).

In comparison to beads for nucleic acid extraction, as were known from the process of EP-A-0 707 077, surprisingly significantly better results were achieved with the processes according to the invention and the bead polymers used therein. • 15 μl of the supernatant obtained in this way were now specific to an HPV

PCR (polymerase chain reaction) was used, which means that primers were used in a PCR that are specific for human papilloma viruses (HPV). When analyzing the result on a gel system, the expected nucleic acid band could be clearly identified.

Claims

1. A method for isolating nucleic acids from a sample comprising the following steps

B) separating the water-insoluble polymer,

C) mixing the water-insoluble polymer with an aqueous phase with a pH greater than 7, the adsorbed nucleic acids being released,

a) 5 to 98 wt .-% amino monomer b) 0.3 to 30 wt .-% crosslinker and c) 0 to 93 wt .-% vinyl monomer

consists.

2. The method according to claim 1, characterized in that after step A) the lysis of the biological material takes place.

3. A method for isolating nucleic acids comprising steps A), B) and C) according to claims 1 and 2, characterized in that the polymer is a water-soluble, macroporous bead polymer with a medium Particle size of 3 to 100 microns and a specific surface measured according to BET of 5 to 500 m ² / g and from polymerized units of

consists, or that the water-insoluble polymer from

bead polymer readily swellable in water with an average particle size of 3 to 100 μm, which consists of polymerized units of

a) 5 to 79.5% by weight of amino monomer b) 0.3 to 10% by weight of crosslinking agent and c2) 10 to 93% by weight of hydrophilic vinyl monomer

consists.

4. Water-insoluble, macroporous polymer beads with an average particle size of 3 to 100 microns, a pore diameter of 10 to 1,000 nm and a specific surface area measured according to BET of 5 to 500 m ² / g, characterized in that they consist of polymerized units of

consist.

5. Water-insoluble but water-swellable bead polymers with an average particle size of 3 to 100 microns, characterized in that they consist of polymerized units of

consist.

6. A process for the preparation of water-insoluble, macroporous bead polymers with an average particle size of 3 to 100 microns, a pore diameter of 10 to 1,000 nm and a specific surface area measured according to BET of 5 to 500 m ² / g, characterized in that a Mix of

a) 5 to 98 parts by weight of amino monomer b) 2 to 30 parts by weight of crosslinking agent cl) 0 to 93 parts by weight of hydrophobic vinyl monomer d) 10-150 parts by weight of porogen and e) 0.1 to 2.5 parts by weight Radical generator

dispersed in an aqueous medium using a protective colloid, then the dispersion obtained is polymerized by heating to the decomposition temperature of the radical generator and, after the polymerization, the porogen is removed by extraction and / or evaporation.

7. A process for the preparation of water-insoluble but swellable bead polymers with an average particle size of 3 to 100 microns, characterized in that a mixture of a) 5 to 79.7% by weight of amino monomer b) 0.3 to 10% by weight of crosslinking agent and c2) 10 to 93% by weight of hydrophilic vinyl monomer d) 10 to 150 parts by weight of solvent and e) 0.1 to 2.5% by weight -Share radical generator

dispersed in an aqueous medium using a protective colloid, then the dispersion obtained is polymerized by heating to the decomposition temperature of the radical generator and, after polymerization, the solvent is removed by extraction and / or evaporation.

8. Amino monomer according to formula (I)

9. A process for the preparation of amino monomers of the formula (I) according to

Claim 8, characterized in that 2-isocyanatoethyl methacrylate is reacted with 3-aminopropylimidazole.

10. Use of water-insoluble, macroporous bead polymers with an average particle size of 3 to 100 microns, a pore diameter of 10 to 1000 nm and a specific surface area measured according to BET of 5 to 500 m ² / g, consisting of polymerized units of

a) 5 to 98% by weight of amino monomer b) 2 to 30% by weight of crosslinking agent cl) 0 to 93% by weight of hydrophobic vinyl monomer or of water-insoluble but swellable bead polymers with an average particle size of 3 to 100 μm, consisting of polymerized units of

for the isolation of nucleic acids from a sample.

11. Means for isolating nucleic acids from a sample containing water-insoluble macroporous bead polymers with an average particle size of 3 to 100 μm, a pore diameter of 10 to 1,000 nm and a specific surface area measured according to BET of 5 to 500 m ² / g, consisting of polymerized units of

a) 5 to 98% by weight> amino monomer b) 2 to 30% by weight crosslinker cl) 0 to 93% by weight hydrophobic vinyl monomer

or of water-insoluble but swellable bead polymers with an average particle size of 3 to 100 μm, consisting of polymerized units of

a) 5 to 79.7% by weight of amino monomer b) 0.3 to 10% by weight of crosslinking agent and c2) 10 to 93% by weight of hydrophilic vinyl monomer.