DE10103652A1 - Magnetic polyvinyl alcohol carrier, useful for isolating and purifying nucleic acid, is modified with germanium compound for improved reversible binding - Google Patents

Abstract

Magnetic or magnetizable poly(vinyl alcohol) (PVA) carrier material (A) for reversible binding of nucleic acid (NA) is modified by chemical reaction with a germanium-containing compound (I). Independent claims are also included for the following: (a) method for preparing (A); (b) method for isolating and/or purifying NA, or derivatives, using (A); and (c) kit for method (b).

Description

The invention relates to magnetic or magnetizable Po lyvinyl alcohol (PVA) carrier materials used for reversible Are capable of binding nucleic acids, and which for the Isolation and / or purification of nucleic acids used can be.

The invention further relates to methods of manufacture such carrier materials, and methods for cleaning of nucleic acids using such support materials s.

The development of new molecular biological methods and their establishment in the most diverse areas of academic as well as industrial research, as well as in Area of diagnostics, has crucial to rapid wax contributed to the so-called "life science" area. The application of many of these methods, such as. B. the polyme rase chain reaction (PCR), cloning or the sequences ornamentation of nucleic acids, places high demands on the Quality of the used in these reactions or methods th starting substrates. Especially the insulation and the Purification of the starting substrate in many reactions serving nucleic acids is therefore of great importance. Contamination of the starting substrate (e.g. nucleic acid ren, DNA, RNA) can the yield at the said metho or significantly reduce reactions, or even a result in complete failure, or for the occurrence be responsible for unwanted artifacts.

Because DNA and RNA are usually very complex, heterogeneous composite, biological sample materials as Aus materials (e.g. blood or tissue or bacterial lysates)  the insulation has to be won of nucleic acids with the required degree of purity often difficult. Such sample materials or starting materials materials often contain a large number of contaminants conditions such. B. proteins, polysaccharides, enzymes, but also Enzyme inhibitors, which are found in subsequent chemical or enzymatic reactions, can prove disruptive, if they cannot be cleaned by suitable cleaning methods Nucleic acids are separated.

The degree of purity of nucleic acid prep rations improve by the biological out nucleic acids obtained several times in a row subjected to various known cleaning processes become. However, this is very time consuming and leads to egg ner very low yield, which is why relatively large amounts of the biological starting material must be used what is often not possible.

In many areas of molecular biological research, Especially in diagnostics, nucleic acids are used rations with a high degree of purity are required, and also must these highly pure nucleic acid preparations often from egg a very large number of different, complex bio logical starting materials (sample materials) isolated and be cleaned.

Due to the constantly growing number of to be carried out investigations in which high-purity nucleic acids needed as the starting substrate is therefore the on management of suitable, preferably automated "high throughput "process for isolating and cleaning nu cleic acids, starting from various sample materials s and different starting quantities, urgently required Lich.

Previously known methods for isolating nucleic acids are either problematic in automated use or they do not provide quantitative yields, or the nucleic acids thus obtained are of insufficient purity. Conventional methods for the extraction of nucleic acids from complex mixtures include the lysis or the digestion of the biological starting material in the presence of proteolytic enzymes, a multiple extraction with organic solvents (e.g. phenol, chloroform) and the subsequent precipitation of the purified nucleic acids by adding an alcohol. To inactivate the nucleases released during cell lysis, e.g. B. guanidine hydrochloride or guanidine thiocyanate can be used (DDL BOWTELL: Rapid Isolation of Euka ryotic DNA; Analytical Biochemistry 162 ( 1987 ), 463-465). Complete removal of proteins from the nucleic acid preparation is e.g. B. achieved by the addition of sodium perchlorate (J. WILCOCRSON: The Use of Sodium Per chlorate in Deproteinization during the Preparation of Nucleic Acids; Biochem. Journal 135 ( 1973 ), 559-561).

However, with the cleaning methods mentioned above various complex work steps required that their use in automated cleaning processes for Prevent nucleic acids. An application in connection with High-throughput processes are therefore not possible.

Other purification or separation processes for nucleic acids are based on nucleic acid-binding, non-magnetic, fe Most support materials (matrices), for example in chromatographic separation columns can be filled. As a nuclein acid-binding carrier materials are mainly silica Materials such as silica gels or glass particles are used. These carrier materials are used in conjunction with hochkon centered, strongly chaotropic salt solutions (e.g. from Na trium iodide, sodium perchlorate, guanidinium thiocyanate) for Isolation or purification of DNA used. For example  is the use of diatomaceous earth in US 5,075,430 Carrier material for the purification of DNA described, the Binding takes place in the presence of a chaotropic salt. EP 0 389 063 B1 and US 5,234,809 disclose a method of Isolation of nucleic acids in which silicon dioxide Particles can be used as a DNA binding solid phase. Here too, the binding takes place in the presence of a chaotropic Substance.

Other methods of isolating or purifying nuclein acids are based on the use of detergents in com combination with a nucleic acid binding matrix (WO-A- 96/18731), or with the use of a solid support DNA-binding functional groups in combination with Po ethylene glycol and salts in high concentrations (WO 99/58664).

Automation of insulation or cleaning of Nu cleic acids using those described above non-magnetic substrates are generally extremely difficult because this would require centrifugation or vacuum steps in such an automatic separator driving be integrated. A complete separation of the solid carrier matrix by centrifugation is also often not possible. The use of ge with carrier material filled separation columns is disadvantageous because of this method due to the inevitable dead volumes of the columns and the consequent losses for small amounts of biological Sample material is not suitable. When using Separation columns also pose a mutual risk Contamination of different biological samples, esp especially when emptying adjacent columns by vacuum. However, it is just in terms of one High-throughput purification of nucleic acids in diagnostics area, e.g. B. for the detection of genetically determined Diseases absolutely necessary, even starting from  the smallest possible amounts or volumes of biological Reliable high-purity nucleus from the source or sample material Acid preparations can be obtained. With the übli Chen microtiter plates with 384 "wells" is the sample volume in the individual "wells", for example, a maximum of approx. 30 µl.

The use of magnetic solid substrates, which are increasingly used in biochemical or molecular biological working methods are used against via conventional (i.e. non-magnetic) separation processes decisive advantages. Leave these substrates easily with the help of magnetic forces separate the remaining components of the starting material. They are also suitable for smaller sample volumes, provided the size and magnetic properties of the used carrier material for the respective separation problem are suitable.

Magnetic carrier materials have been described and Z. T. commercially available for isolation or Purification of nucleic acids can be used. at are for example for the separation of nucleic acids specific sequences e.g. B. streptavidin-coated ma gnetic particles known after binding a biotiny oligonucleotide to the immobilized streptavidin for the sequence-specific separation of nucleic acids with complementary sequences can be used. A wich The example of this is the isolation of mRNA under Use of biotinylated oligo (dT).

In addition, magnetic particles are also known, which the Separation or isolation of nucleic acids, independently of their nucleotide sequence. Such particles can be based on silica by storing ma magnetic or magnetizable iron oxide particles  become; for example, they are in U.S. patents 4,395,271 or 4,297,337. Known disadvantages on the one hand, these particles are the often difficult or incomplete detachment of those bound to the particles Nucleic acids, and secondly a leakage of dissolved Iron salts from the encapsulated iron oxide that interferes rend affects other applications.

In addition, with the known magnetic particles quantitative, complete isolation of nucleic acids complex raw materials or the extraction of high-purity nucleic acid preparations usually do not possible. Another disadvantage of these particles is there too see that this usually only a small proportion of magne have tisizable material, which is why their complete magnetic separation is difficult.

The object of the present invention was therefore to ma be magnetic or magnetizable solid support materials which are for the reversible binding of nuclein Acids enabled and for the isolation or cleaning of nu cleic acids are suitable. Furthermore, such Trä germ materials both quickly and completely by means of magnetic force from which nucleic acids contain be starting material and they should be the quantitative isolation from high-purity nucleic acid enable complex biological starting materials. Of further demands that such carrier materials for use in automated high throughput processes suitable for isolating or purifying nucleic acids, and that they can also be used to isolate or clean Nu cleic acids from very small quantities of the starting material can be used.

Surprisingly, it was found that the ge named task by magnetic or magnetizable polyvinyl alcohol carrier materials  solved according to claim 1 becomes. The carrier materials according to the invention are thereby characterized by chemical reaction with a Germanium-containing compound are modified. Further advantageous embodiments of the invention germaterialien be in the dependent claims 2-10 wrote.

The magnetic or magnetizable according to the invention Polyvinyl alcohol substrates are capable of nuclein acids reversible and in a sequence-unspecific manner selectively bind high binding capacity.

As such, polyvinyl alcohol has little nucleic acid re-binding properties. Surprisingly, has demonstrated that the binding capacity of polyvinyl alcohol Backing materials is increased considerably if this Ma materials by chemical treatment with a germanium containing compound can be modified.

On the other hand, the polyvinyl according to the invention are distinguished alcohol carrier materials due to their highly hydrophilic Properties through a minimal non-specific protein bin dung out. This is in view of the use of this Support materials for the isolation or purification of nucleus acids a very important criterion because of this Binding behavior a high purity of the nucleus obtained acids is ensured.

Another advantage of the germanium modes according to the invention Defected polyvinyl alcohol particles is that these improved over unmodified PVA particles Have suspending properties so that they are Washing or elution steps after previous magneti shear separation again easier to bring into suspension sen. This has the advantage that the washing steps or Elution can be carried out more effectively.  

Magnetic or magnetizable polyvinyl alcohol carrier materials which can be modified according to claim 1 by reaction with a germanium-containing compound are preferably magnetic or magnetizable polyvinyl alcohol particles which are produced by the processes described in WO 97/04862. With regard to the production of these polyvinyl alcohol particles, reference is therefore made to WO 97/04862. The magnetic or magnetizable polyvinyl alcohol particles obtainable with the processes described there have a number of advantages:

• - You can use the procedures mentioned in different Size distributions, preferably with a diameter in Range from 0.5 to 15 µm;
• - With regard to the size distribution (diam ser) with regard to a specific application ad be accepted; For example, with that in WO 97/04862 described magnetic polyvinylal alcohol particles are obtained, the size of which range from 0.5 to 1 µm or in the range from 1 to 3 µm;
• - They can be with regard to their magnetite content certain applications are adapted, for example with a very high magnetite content of up to 90% by weight; such polyvinyl alcohol particles have, based on the particle volume, causing a large surface area high binding capacity when used in one Separation or purification procedures for nucleic acids can be enough;
• - The polyvinyl alcohol used as the polymer matrix due to certain properties, e.g. B. the hydrophilic Character, especially ge for biochemical separation processes suitable.

For the reasons mentioned are called polyvinyl alcohol germaterialien according to the invention by treatment with Germanium-containing compounds are modified, especially  preferably the magneti described in WO 97/04862 polymer particles used.

In principle, however, there are also other ways posed, magnetic or magnetizable polyvinyl alcohol Carrier materials for the modification according to the invention use reaction with a compound containing germanium.

The inventive, by modification with germaniumhal term connections produced magnetic or magneti sizable polyvinyl alcohol carrier materials, in particular in the form of polyvinyl alcohol particles, can quantita tive separation of nucleic acids from different Starting materials are used. The so won Nucleic acids have a high purity, so that they di rect as a substrate in other applications, e.g. B. in the PCR, can be used.

The magnetic or magnetizable polyvinyl carrier mat rial is according to claim 1 by chemical reaction with egg modified germanium-containing compound, wherein preferably a hydrolyzable germanium compound is applied.

This modification can be that the polyvinyl alcohol carrier materials covalently bound germanium or germanium (IV) dioxide or a covalently bound ger contain polymer containing manium, preferably on the upper area of the carrier materials.

In the case of using a tetraalkoxy germane (e.g. germanium tetraethoxide (germanium (IV) ethylate, Ge (OC ₂ H ₅ )) ₄ ) to modify the support materials, germanium dioxide is bound to the surface of the support materials, so that a coating is generated from germanium dioxide.

The germanium atoms, or at least some of them, are bound to the polyvinyl alcohol via an oxygen atom. They can also be connected to one another via oxygen bridges, so that a three-dimensional network can be formed (“polygermoxane”; cf. FIG. 1).

Consequently, by the modification reaction mentioned act that the polyvinyl alcohol carrier materials a surface of germanium (IV) oxide or Have polygermoxane, which is covalent to the polyvinyl alcohol carrier material is bound. However, the Modifi ornamental reaction not necessarily to the surface of the carrier material limited.

Nor is it necessary that the layer mentioned is fully trained; it can also be just one partial coating act. The layer can be mono be formed layer, but it can also consist of several La conditions exist.

The invention preferably relates to those magnetic or magnetizable carrier materials which, by treating polyvinyl alcohol carrier materials with germanium halides, particularly preferably germanium tetrachloride, or organic germanium compounds, preferably germanium alcoholates (alkoxy derivatives of germans), particularly preferably germanium tetraethoxide (germanium ( IV) ethylate, Ge (OC ₂ H ₅ )) ₄ ).

The carrier materials according to the invention have magnetic ones Properties on or are magnetizable; preferably this is achieved by being superparamagnetic or contain paramagnetic iron oxide particles, especially preferably magnetite.

So that the separation of the magnetic carrier materials from the biological starting material, after binding of the nucleus acids to the carrier material, as efficiently as possible  can, it is preferred that the carrier according to the invention materials a magnetite content of at least 50% by weight have, particularly preferably of at least 75 wt .-%.

Furthermore, he sees a preferred embodiment of the find that with a germanium-containing Ver bond modified magnetic polyvinyl alcohol carrier materials are available as a hydrogel.

The carrier materials according to the invention are preferably in Form of particles, which particle sizes of 0.2-50 microns, preferably have from 0.4 to 8 µm. Most before there are particles with a size of 0.4-4 µm, since this is a particularly favorable surface / volume ver have ratio, whereby a high binding capacity achieved the use for the separation of nucleic acids becomes.

The "diameter" of the particles is the mean diameter of the Understood particles. The sizes given above rich say that at least 70%, preferably at least 80% of the particles have a size within the respective size range. For example, the average diameter for particles with a size of 1-3 µm at approx. 2 µm.

With regard to the geometric shape of the particles mentioned it is preferred that they be essentially pearl or ku gel-like particles (so-called "beads") are present.

Best suited for nucleic acid isolation procedures Cleaning or cleaning are pearl or spherical, magneti cal or magnetizable polyvinyl alcohol particles, which according to the invention by reaction with a germaniumhal term connection have been modified, and the one ge rings size (0.4-4 µm) and a high magnatite content (min at least 50% by weight).  

The high magnetite content and the small size of the inventions Particles according to the invention enable nucleic acid isolation from both very small quantities of biological samples terials (e.g. 1 µl blood), as well as from very large samples amounts (e.g. 20 ml of blood), with the usual centri fugation steps can be omitted. centrifugation steps in the isolation of nucleic acids, e.g. B. from Blood, are often used to increase the purity of the blood Nucleic acid performed. Because of the possibility of using the particles according to the invention a highly pure preparation can also be obtained without centrifugation steps these time-consuming and difficult to automate Steps to be avoided.

The invention further relates to methods for the production of magnetic or magnetizable polyvinyl alcohol carrier materials containing by treatment with germanium the connections are modified.

The manufacturing method according to the invention is based on Implementation of a magnetic or magnetizable Polyvi nyl alcohol carrier material, with a germanium compound, preferably with an organic germanium compound or with a germanium halide. Suitable for this purpose such germanium compounds, the hydrolyzable alkoxy or halide functions ent hold.

In principle, germanium compounds with the general formula X _q -Ge- (Y) _4-q can be used for the reaction mentioned,

• - wherein X = hydrogen or alkyl, preferably with 1-6 C atoms, or X = aryl, preferably phenyl, or X = alkoxy (OR), where R is a hydrocarbon radical, preferably a methyl, ethyl, propyl or Isopropyl radical,
  or wherein X = halide, preferably chlorine or bromine, and
• wherein Y = alkoxy (OR '), where R' is a hydrocarbon radical, preferably methyl, ethyl, propyl or isopropyl,
  or wherein Y is a halide, preferably chlorine or bromine, and
• - where q is an integer from 0 to 3.

The hydrocarbon radical R or R 'can be branched be unbranched as well. The total number of carbon atoms in the residues R and R 'are in each case between 1 and 30 preferably between 1 and 12.

The germanium compound is preferably selected from the group consisting of germanium (IV) ethoxide (= germanium (IV) ethyl = tetraethyl germanium, Ge (OC ₂ H ₅ ) ₄ ), germanium (VI) methoxide (= germanium (IV) methylate = germanium acid tetramethyl ester, Ge (OCH ₃ ) ₄ ), germanium (IV) isopropoxide (= germanium (IV) isopropylate = germanium acid tetra-isopropyl ester, Ge [OCH (CH ₃ ) ₂ ] ₄ ), Germanium (IV) butoxide, and germanium tetrachloride (GeCl ₄ ), with germanium (IV) ethoxide and germanium tetrachloride being particularly preferred.

The process for the chemical modification of a magnetic or magnetizable polyvinyl alcohol carrier material by reaction with a germanium-containing compound is preferably carried out as follows (cf. Example 1):
The polyvinyl alcohol carrier material is suspended in an aqueous buffer at a pH of 2 to 7 and heated with stirring (to about 50 to 95 ° C.). Then one of the germanium compounds mentioned is added, and the reaction mixture is allowed to cool to room temperature (20-25 ° C.). After washing the modified support material at least once with water or an aqueous buffer, the support material can be used for the separation or purification of nucleic acids. The method described above is particularly suitable when germanium (IV) ethoxide or another alkoxy derivative is used as the germanium-containing compound.

Alternatively, the chemical modification method a magnetic or magnetizable polyvinyl alcohol Carrier material are carried out in such a way that the Polyvinyl alcohol carrier material in an organic solvent means, e.g. As acetone or toluene is suspended (see example 2). The suspension is then heated (approx. 50-150 ° C) and the germanium-containing compound puts. After the reaction has ended, the reak is left Cool the tion mixture and water will drop dropwise added. Finally, the modified carrier material washed with water or an aqueous buffer. The the latter method is particularly suitable if Germanium (IV) chloride or another germanium halide is used as a germanium-containing compound.

In addition to acetone or toluene, the organic solvent can be any other solvent or solvent mixture ver be used in which the PVA particles are stable; these include, for example, hydrocarbons, dimethyl formamide, dimethoxyethane, chloroform, dichloromethane or Tetrahydrofuran.

In the reaction reactions described above The method used is the respective germanium compound preferably in an amount of 0.1-10 mmol per gram of Po lyvinyl alcohol particles used, particularly preferably in a quantitative ratio of 0.7-2.5 mmol per gram of polyvinyl alcohol particles.  With the latter quantity ratio nis become germanium-modified particles with special good nucleic acid binding properties.

As magnetic or magnetizable polyvinyl alcohol Carrier materials are preferably spherical particles ("beads") used with those described in WO 97/04862 a process and are obtained from chemagen AG, D-52499 Baesweiler are available.

The present invention further relates to methods for Isolation or purification of nucleic acids from complex Starting materials or mixtures, using the according to the invention, by reaction with a germaniumhal term modified magnetic polyvinyl alcohol hol-carrier materials.

These procedures are based on the fact that he support material according to the invention with the nucleic acid Starting material or mixture brought into contact or there is mixed with, the nucleic acids reversibly the carrier material are bound. Then that will Carrier material with the help of magnetic force from separated from the starting material or mixture, and the bound nucleic acids become from the support material solved (eluted).

The "complex starting materials" from which nuclein acid (s) to be isolated or separated can for example from body fluids such as blood or Se rum, or fractions thereof, e.g. b. buffy coat, or made of plant or animal cells or tissues, fungi, Bacteria or virus or other biological mate rial can be won. If necessary, these will be off materials by suitable methods known to those skilled in the art  or reagents digested to the nucleic acids to be released from the cells or tissues. In any case the raw materials or the biological samples material is pretreated so that at least part of the Nucleic acids is released and is in solution. at for example, cells or tissues can be mechanically Shear forces, by the action of lytic enzymes or with Detergent can be digested or lysed.

In addition, the method according to the invention can also used to extract nucleic acids from mixtures, solutions or to separate suspensions that are chemically or enzyma table modified, synthesized or synthetically ampli contained nucleic acids (see Example 3). Under "nucleic acids" in particular DNA and RNA are ver stood, as well as oligonucleotides, DNA or RNA fragments, chemically or enzymatically modified nucleic acids cell or double stranded nucleic acids, genomic DNA (see Example 4) or RNA, total cellular DNA or RNA, Plasmids and viral DNA as well as viral RNA, where it is here are only examples.

In the methods of separation or Purification of nucleic acids is a first step a magnetic or magnetizable, nucleic acid binding, solid carrier material with the starting material or mixed or mixed with where at the nucleic acid (s) or nucleic acid derivative (s) rever is / are bound to the solid support material. As The carrier material becomes a magnetic or magneti settable, nucleic acid binding, solid polyvinyl alcohol Carrier material used, which by reaction with a Germanium-containing compound has been modified, such as described above. The carrier is preferred material around pearl or spherical, magnetic or magnetisable  Polyvinyl alcohol particles, which are low Size (0.4-4 µm) and a high proportion of magnetite (at least 50% by weight).

The addition of the carrier material or the particles to the nucle acidic starting material is preferably in Presence of a buffer that binds the nucleic acids supported on the carrier particles ("binding buffer"). For removing proteins from cell lysates, in particular for the separation of DNA-binding proteins, can during of this first step (binding step) preferably natri umperchlorate in a final concentration of 1-1.5 M added be set.

A solution containing water (40-70% by volume), ethanol, sodium acetate and sodium perchlorate is preferably used as the binding buffer, the order in which these components are added is not important. Alternatively, the following buffers, solutions or mixtures can be used as binding buffers:

• - 2.2 M NaClO ₄ , 0.3 M sodium acetate, in water, pH 5.5-6.5.
• - 40-60 vol.% Ethanol, 10-15 vol.% Dimethylformamide, 0.1- 0.5% NaCl, water ad 100%; pH 6-7. Optionally, instead Dimethylformamide also z. B. glycerin or a dimethoxy alkylene glycol can be used.
• - 60-90 vol .-% ethanol, 0.01-0.2 M MgCl ₂ , 20-100 mM Tris pH 6.5 (Tris = tris (hydroxymethyl) aminomethane).

Which buffer in a particular application, i. H. for a certain biological starting material, best is suitable, the person skilled in the art by means of corresponding preliminary ver search easily. It has z. B. ge shows that in some cases ethanol-free binding buffer on are best suited (e.g. nucleic acid isolation from Bacterial lysates), in other cases ethanol-containing ones Binding buffer (e.g. nucleic acid isolation from blood).  

The nucleic acids are bound to the support material relatively fast and usually requires only one incubation ontime from one to a few (five) minutes. If necessary Therefore, the incubation period for the first step (Binding step) can also be extended by 5 min. to about 2 hours or longer.

At the end of the binding step, the starting material can who are separated and discarded together with the binding buffer (e.g. suction or decanting), the carrier ma material (to which the nucleic acids are bound) with With the help of a magnet or other magnetic field will hold (= "magnetic separation").

In a second step, they are attached to the carrier mat rially bound nucleic acids using appropriate Wash buffer washed. As a washing buffer z. B. dilute ter alcohol (30-80 vol .-%) can be used, optional salts can also be dissolved in it (e.g. sodium perchlo rat, sodium acetate). Depending on the area of application, too other low or high salt buffers or solutions without Al alcohol additive can be used as a washing buffer. "Low salt" be indicates a salt concentration of 0.01-0.2 M, "high salt" means a salt concentration of 1-7 M.

Due to the magnetic properties of the carrier material it is very easy to change the washing solutions, because the carrier material held back with the help of a magnet can be removed while the washing solution is being removed. If required, this washing step can be done one or more times be repeated.

Alternatively, the following buffers, solutions or mixtures can be used as wash buffers:

• - 30-90 vol% ethanol, 0.05-0.2 M NaCl. Instead of NaCl can also z. B. sodium acetate, ammonium acetate, magnesium chloride rid or sodium perchlorate can be used.  
• - 30-90 vol .-% ethanol, 1-2 M sodium perchlorate (or, al ternatively, one of the salts mentioned above).
• - 1-4 M sodium perchlorate, in water; (or alternatively, one of the aforementioned salts).
• - 0.02-0.2 M annonium acetate, in water; (or alternatively, one of the aforementioned salts).

It is also possible to use water as a "washing buffer" the.

Instead of ethanol, the binding or washing buffer can be used another alcohol, or a mixture of alcohols, be used; for example, ethanol can be passed through Replace isopropanol.

In a third step (elution step) the the carrier material from reversibly bound nucleic acids released or eluted from the binding. This is done with Support material loaded with nucleic acids preferably with egg low salt buffer (0.01-0.2 M) or water (as Eluti buffer or solution), if necessary at an elevated temperature ture (preferably 40-70 ° C, more preferably 50-60 ° C) delt, thereby eluting the nucleic acid from the support material particles is caused. As salts in low salt Buffers come, for example, the above under "binding puff fer "or" washing buffer "salts into consideration. Optionally, the elution step can be omitted; in this case the one bound to the carrier material Nucleic acid used directly in subsequent reactions puts. For example, that of the invention Particle bound nucleic acid without previous elution di be used directly in a PCR.

The invention also includes isolation and / or kits Purification of nucleic acids, which are magnetic or contain magnetizable polyvinyl alcohol carrier material,  that according to the invention by reaction with a germaniumhal term connection has been modified, as well as solutions and / or reagents for performing the isolation and / or Cleaning. For example, as solutions or reagents binding buffer, washing buffer, elution buffer or solutions, or reagents or solutions for digestion (Ly se) of the nucleic acid-containing starting material (e.g. Zel len, tissue) may be included.

The invention is illustrated by the following examples purifies.

example 1 Modification of polyvinyl alcohol particles by implementation tongue with a germanium-containing compound in aqueous solution

500 mg magnetizable size polyvinyl alcohol particles 1-3 µm, with a magnetite content of 90% by weight twice with 20 ml 0.1 M sodium acetate buffer (pH 4,5), which was previously degassed, washed under argon, where with the separation of the particles with a manual measure gneten takes place, and then in 20 ml also degassed 0.1 M Resuspended sodium acetate buffer (pH 4.5). The suspension is heated to 60 ° C with stirring and then dropwise 200 μl of germanium tetraethoxide in an argon stream set. After two hours of reaction at this temperature the reaction mixture is allowed to cool to room temperature, separates the supernatant after magnetic separation and washes the particles three times with 20 ml distilled each Water; this can be done in the presence of air. subsequently, The particles are stored for storage in 0.1 M sodium ace tat buffer (pH 5.5) and the concentration of Particles adjusted to 25 mg / ml.  

Example 2 Modification of polyvinyl alcohol particles by implementation tongue with a germanium-containing compound in an orga African solvent

500 mg magnetizable size polyvinyl alcohol particles 1-3 microns, with a magnetite content of 90 wt .-%, are successively cessive dewatered by washing with acetone-water mixture and then by washing with absolute acetone. After the acetone supernatant has been separated off, the particles become under argon in 10 ml absolute toluene (purity min. 99.5%) and then in 20 ml of absolute toluene resus pendiert. The suspension is heated to 100 ° C heats, and then 100 µl Germa in the argon stream Added sodium tetrachloride dropwise. After four hours Reaction at this temperature is allowed to react Cool to 80 ° C and add 500 µl of what to this. After another two hours at this temperature the supernatant is separated off after magnetic separation and washes the particles in the air with 50 ml de still water. Then the particles become Storage resuspended in 0.1 M sodium acetate (pH <6).

Example 3 Purification of a PCR amplification product under Ver Use of the particles produced according to Example 1

A 595 bp DNA fragment of the malB region of genomic DNA isolated from E. coli (K12) using the "Bugs'n Beads Kit" (from Chemagen AG, D-52499 Baesweiler). is amplified in a PCR (primer and reaction conditions according to CANDRIAN et al., Int. J. Food Micro biol. 12 ( 1991 ) 339).

After the amplification, 40 μl of the PCR product are separated from the oil phase, 16 μl of the particle suspension from Example 1 are added, and 100 μl binding buffer (60 vol.% Ethanol, 2.2 M NaClO ₄ , 0, 3 M sodium ace tat) added. After five minutes of incubation, the mixture is magnetically separated and the supernatant is discarded. It is washed twice with in each case 400 μl of 60% ethanol and, after the washing solution has been completely separated off, the particles are dried for 8 minutes with the reaction vessel open. The particles are then suspended in 30 μl Tris-HCl (10 mM, pH 8.0). After 5 minutes of incubation at 55 ° C., the supernatant with the purified DNA amplification product is separated from the particles (by magnetic separation) and transferred to a new vessel.

Example 4 Isolation of genomic DNA from whole blood, using tion of the particles produced according to Example 1

10 ul whole blood (EDTA stabilized) are in a 1.5 ml reaction vessel with 65 ul lysis buffer (1.6 M guanidine hydrochloride; 30 mM Tris-HCl, pH7; 30 mM EDTA; 10 vol .-% Tween 20 ; 1 vol .-% Triton X-100) and mixed for 1 min at room temperature. 8 μl of the modified particles from example 1 and 110 μl of binding buffer (see example 3) are then added. After 5 minutes of incubation at room temperature, the supernatant is discarded after magnetic separation. The particles are then washed with 190 μl washing buffer A (30 vol.% Ethanol; 1.1 M NaClO ₄ ; 0.15 M sodium acetate) and then briefly with 300 μl water. After the last washing solution has been separated off, the particles are incubated in 30 μl elution buffer (10 mM Tris-HCl) at 55 ° C. for 5 min in order to elute the DNA. The genomic DNA isolated iso z. B. is set in a PCR.

Fig. 1 shows an example of the formation of a "Polygermoxan" - monolayer ( 2 ) on the surface of a polyvinyl alcohol carrier material ( 1 ); however, the modification according to the invention can also lead to different types of molecular structures which differ from the one shown here.

R stands for example for an alkyl radical or a free one (terminal) OH group.

( 1 ) denotes the surface of the polyvinyl alcohol carrier material to which the "polygermoxane" monolayer ( 2 ) is bonded via oxygen atoms.

Claims (24)

1. Magnetic or magnetizable polyvinyl alcohol carrier materials for the reversible binding of nucleic acids, characterized in that they are modified by chemical implementation with a germanium-containing compound. 2. Magnetic or magnetizable polyvinyl alcohol Support materials according to claim 1, characterized in that they are covalently bound germanium or germanium (IV) - oxide or a covalently bound germanium-containing polymer included, preferably on the surface of the carrier mat rials. 3. Support materials according to claim 1 or 2, characterized ge indicates that they have one or more on their surface Layer (s) of Germani connected via oxygen bridges have umatomen, with at least part of the germanium atoms covalently to the polyvinyl alcohol via oxygen atoms hol carrier material is bound. 4. carrier materials according to one of claims 1 to 3, characterized in that they are wholly or partly with Germanium (IV) oxide are coated. 5. carrier materials according to one of claims 1 to 4, characterized in that by treatment of poly vinyl alcohol carrier materials with germanium halides or organic germanium compounds, preferably Ger manium alcoholates or alkoxy derivatives of germanium are posed.   6. carrier materials according to one of claims 1 to 5, characterized in that they are superparamagnetic or contain paramagnetic iron oxide particles, preferably Magnetite. 7. carrier materials according to claim 6, characterized is characterized by a magnetite content of at least 50% by weight, preferably at least 75% by weight, most preferably have from 90 wt .-%. 8. Carrier materials according to one or more of the above going claims, characterized in that it as Hy there are drugs. 9. Carrier materials according to one or more of them going claims, characterized in that they as Particles are present, which have particle sizes of 0.2-50 μm, preferably 0.4-8 µm, most preferably 0.4-4 µm exhibit. 10. Support materials according to claim 9, characterized records that they are essentially pearl or spherical Particles are present. 11. Process for the production of a carrier material according to one of claims 1 to 10 by chemical modification a magnetic or magnetizable polyvinyl alcohol Carrier material by implementing this carrier material with a germanium compound, preferably with an organic Germanium compound or with a germanium halo halide. 12. The method according to claim 11, characterized in that the structure of the germanium compound corresponds to the general formula X _q -Ge- (Y) _4-q ,
wherein X = hydrogen or alkyl, preferably with 1-6 C atoms, or X = aryl, preferably phenyl, or X = alkoxy (OR), where R is a hydrocarbon radical, preferably a methyl, ethyl, propyl or isopropyl radical is
or wherein X = halide, preferably chlorine or bromine, and
where Y = alkoxy (OR '), where R' is a hydrocarbon radical, preferably methyl, ethyl, propyl or isopropyl,
or wherein Y is a halide, preferably chlorine or bromine, and
where q is an integer from 0 to 3. 13. The method according to claim 11 or 12, characterized indicates that the germanium used for the modification Compound is selected from the germanium (IV) ethoxide, Germanium (VI) methoxide, Germanium (IV) isopropoxide, Germani group comprising (IV) butoxide and germanium tetrachloride, with germanium (IV) ethoxide and germanium tetrachloride who are preferred. 14. The method according to any one of claims 11-13, characterized ge indicates that the implementation of magnetic or ma gnetizable polyvinyl alcohol carrier material with the or ganic germanium compound at temperatures of 20-150 ° C, preferably from 50-95 ° C. 15. The method according to one or more of claims 11- 14, characterized in that the polyvinyl alcohol Carrier material for reaction with the germanium compound, preferably a germanium alcoholate or an alkoxy Derivative, in an aqueous buffer at pH 2 to 7 is suspended.   16. The method according to one or more of claims 11- 14, characterized in that the polyvinyl alcohol Carrier material for reaction with the germanium compound, preferably a germanium halide, in an organic solvent is suspended. 17. Use of polyvinyl alcohol carrier materials according to one or more of claims 1 to 10, or of poly vinyl alcohol carrier materials, which are prepared by one or more of the methods 11-16 , as a nucleic acid-binding matrix for isolation and / or Purification of nucleic acids. 18. Use of polyvinyl alcohol carrier materials according to one or more of claims 1 to 10, or of polyvinyl alcohol carrier materials, which are prepared by one or more of the methods 11-16 , as a nucleic acid-binding matrix for isolation and / or for cleaning of nucleic acids from complex starting materials or mixtures, which are preferably obtained from blood, serum, buffy coat, plant or animal cells or tissues, fungi, bacteria or viruses or another biological material. 19. Use of polyvinyl alcohol carrier materials according to one or more of claims 1 to 10, or of polyvinyl alcohol carrier materials, which are prepared by one or more of the methods 11-16 , as nucleic acid-binding matrix for isolation and / or for cleaning of nucleic acids from mixtures, solutions or suspensions containing one or more chemically or enzymatically modified, synthesized or synthetically amplified nucleic acids. 20. A process for the isolation and / or purification of nucleic acids or nucleic acid derivatives from complex starting materials or mixtures, the nucleic acid (s) or nacleic acid derivative (s) being reversibly bound to a solid support material, characterized in that the solid support material a magnetic or magnetizable polyvinyl alcohol carrier material according to one or more of claims 1 to 10, or a polyvinyl alcohol carrier material produced according to one or more of methods 11-16 is used. 21. A method for the isolation and / or purification of nucleic acids or nucleic acid derivatives from complex starting materials or mixtures, wherein
in a first step, a magnetic or magnetizable, nucleic acid-binding, solid support material is brought into contact with or mixed with the starting material or mixture, the nucleic acid (s) or nucleic acid derivative (s) being reversibly bound to the solid support material / be, and
in a second step, the nucleic acids bound to the carrier material are washed, and
in a third step, the nucleic acid (s) bound to the solid support material are eluted or released from this support material,
characterized in that a magnetic or magnetizable polyvinyl alcohol carrier material according to one or more of claims 1 to 10 or a polyvinyl alcohol carrier material produced according to one or more of the methods 11-16 is used as the solid carrier material. 22. The method according to claim 20 or 21, characterized records that said nucleic acids or nucleic acid complex starting materials containing red derivatives or  Mixtures preferably from blood, serum, buffy coat, plants Lichen or animal cells or tissues, fungi, bacteria or viruses or other biological material can be obtained, said biological materia lien for nucleic acid isolation or purification be closed. 23. The method according to claim 20 or 21, characterized records that the starting materials mentioned or Gemi one or more chemically or enzymatically modified graced, synthesized or synthetically amplified Contain nucleic acids. 24. A kit for the isolation and / or purification of nucleic acids, which contains a carrier material for the reversible binding of nucleic acids and solutions and / or reagents for carrying out the isolation and / or purification, characterized in that the kit as a carrier material is a magnetic or contains magnetizable polyvinyl alcohol carrier material according to one or more of claims 1 to 10, or a polyvinyl alcohol carrier material produced according to one or more of methods 11-16 .