A method for carrying out an enzymatic reaction on nucleic acids

The invention relates to a method for isolating and enzymatically reacting to nucleic acids, comprising contacting a sample which contains the nucleic acids in a liquid with magnetic particles with glass surfaces under conditions in which the nucleic acids can bind to the glass surface in native form the nucleic acids in native form by adsorption on the glass surface of the magnetic particles, Separating the bound nucleic acids from the liquid and inserting the nucleic acids as a substrate in an enzymatic reaction.

Some biological materials, especially nucleic acids, have special requirements with regard to their isolation from the natural environment. On the one hand, they are often present in very low concentrations and, on the other hand, they are often in the vicinity of many other solid and dissolved substances that impair their isolation or determination.

There has been no shortage of attempts recently to propose methods and materials for isolating nucleic acids from their natural environment. <nplcit id="ncit0001" npl-type="s"><text>From proc. Natl. Acad. USA 76, 615-619 (1979</text></nplcit>) describes the binding of nucleic acids from agarose gels in the presence of sodium iodide in ground flint glass.

In <nplcit id="ncit0002" npl-type="s"><text>Anal. Biochem. 121, 382-387 (1982</text></nplcit>) the purification of plasmid DNA from bacteria on glass dust in the presence of sodium perchlorate is described.

In <patcit id="pcit0001" dnum="DE3734442A"><text>DE-A 37 34 442</text></patcit> describes the isolation of single-stranded M13 phage DNA on glass fiber filters by precipitation of the phage particles with the aid of acetic acid and lysis of the phage particles with perchlorate. The nucleic acids bound to the glass fiber filters are eluted after washing with a methanol-containing buffer in Tris / EDTA buffer.

In <nplcit id="ncit0003" npl-type="s"><text>Anal. Biochem. 175, 196-201 (1988</text></nplcit>) a similar method for the purification of DNA from Lambdaphagen is described.

In <patcit id="pcit0002" dnum="WO9504140A"><text>WO 95/04140</text></patcit> describes a method for the amplification of purified nucleic acids from a Gram-positive organism from a biological sample. The biological sample is mixed with lysozyme, a chaotropic substance and a solid phase, eg silica particles. The nucleic acids that bind to the solid phase are then separated from the rest of the sample. Then the silica-nucleic acid complexes are cleaned with the aid of buffer solutions, the nucleic acids are precipitated, eluted and amplified.

The previously known methods of the prior art have in common the selective binding of nucleic acids to glass surfaces in chaotropic salt solutions, the nucleic acid being separated from impurities such as agarose, proteins or cell debris. To separate the glass particles from the contaminants, either centrifugation of particles or suction of liquids through glass fiber filters is used according to the prior art. However, this is a limiting step that severely hinders the processing of large numbers of samples.

In <nplcit id="ncit0004" npl-type="s"><text>Anal. Biochem. 201, 166-169 (1992</text></nplcit>) or. <patcit id="pcit0003" dnum="GB9100212W"><text>PCT GB 91/00212</text></patcit> describes the use of magnetic particles for immobilizing nucleic acids after precipitation by adding salt and ethanol. Here, the nucleic acids are agglutinated, including the magnetic particles. The agglutinate is separated from the original solvent by applying a magnetic field and washing. After a washing step, the nucleic acids are dissolved in a Tris buffer. However, this method has the disadvantage that the precipitation is not selective for nucleic acids, but instead a large number of solid and dissolved substances are also agglutinated. It is therefore not possible with this method to remove any existing inhibitors for certain enzymatic reactions to a sufficient extent.

In <patcit id="pcit0004" dnum="US4233169A"><text>US-A-4,233,169</text></patcit> describes a porous glass that contains embedded magnetic particles.

There is currently also so-called magnetic, porous glass, which contains magnetic particles in a porous, particulate glass matrix, and the surface of which is coated with a streptavidin-containing layer. This product can be used to isolate biological materials, for example proteins or nucleic acids, if they are modified in a complex preparation step so that they are covalently bound to biotin.

The object of the invention was to provide a simple method for enzymatic reaction to nucleic acids which is suitable for routine diagnostics.

The invention relates to a method for isolating and enzymatically reacting to nucleic acids, comprising contacting a sample which contains the nucleic acids in a liquid with magnetic particles with glass surfaces under conditions in which the nucleic acids can bind to the glass surface in native form the nucleic acids in native form by adsorption on the glass surface of the magnetic particles, Separating the bound nucleic acids from the liquid and inserting the nucleic acids as a substrate in an enzymatic reaction.

The person skilled in the art refers to solid materials with a small diameter. Sometimes such particles are also called pigments. Particles which have an average grain size of less than 100 μm are particularly suitable for the purposes of the present invention. They particularly preferably have an average grain size of between 10 and 60 μm. The grain size distribution is preferably relatively homogeneous, in particular there are almost no particles &lt;10 μm or&gt; 60 μm.

Materials that can be attracted by a magnet, ie, for example, ferromagnetic or superparamagnetic materials, are referred to as magnetic. Magnetic are also understood to mean materials which are referred to as soft magnetic materials, e.g. B. ferrites. Ferromagnetic materials are particularly preferred in the sense of the invention, in particular if they have not yet been magnetized. In this context, premagnetization is to be understood as contacting a magnet, which increases the remanence. Ferromagnetic materials, such as. B. magnetite (Fe₃O₄) or Fe₂O₃.

An outer surface of a particle is understood to mean the coherent surface from which perpendiculars can be formed in the direction of the surroundings of the particle, which do not intersect the same particle again.

A pore is understood to mean a recess in the outer surface of the particle in which the surface extends so far into the particle that an imaginary perpendicular formed in the recess on the surface in the direction of the immediate vicinity of the particle intersects the particle at least once. Pores also extend deeper than a radius of the pore into the particle.

A glass in the sense of the present invention is understood to mean a silicon-containing amorphous material. The glass may contain other materials, e.g. B.<tables id="tabl0001" num="0001"><table frame="none"><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="17mm" /><colspec colnum="2" colname="col2" colwidth="20mm" /><tbody><row><entry>B₂O₃</entry><entry>(0-30%),</entry></row><row><entry>Al₂O₃</entry><entry>(0-20%),</entry></row><row><entry>CaO</entry><entry>(0-20%),</entry></row><row><entry>BaO</entry><entry>(0-10%),</entry></row><row><entry>K₂O</entry><entry>(0-20%),</entry></row><row><entry>N₂O</entry><entry>(0-20%),</entry></row><row><entry>MgO</entry><entry>(0-18%),</entry></row><row><entry>Pb₂O₃</entry><entry>(0-15%).</entry></row></tbody></tgroup></table></tables>

To a lesser extent, 0-5%, a variety of other oxides, such as. B. Mn₂O₃ TiO₂, As₂O₃, Fe₂O₃, CuO, CoO, etc. may be included. Surfaces with a composition of borosilicate glass, flint glass or silica have proven to be particularly effective. Borosilicate glasses which are particularly preferred from the point of view of the yield of nucleic acids have a boron oxide content of more than 25%; a glass with the composition SiO was found to be particularly valuable₂/ B₂O₃ 70/30 recognized. Glasses which are formed by the so-called gel-sol process and subsequent drying and compacting of the layer formed are particularly preferred in the sense of the invention. The main features of this process are known and have been described e.g. B. in<nplcit id="ncit0005" npl-type="b"><text>CJ Brinker, GW Scherer "Sol Gel science - The physics and chemistry of Sol Gel Processing", Academic Press Inc. 1990</text></nplcit> and <nplcit id="ncit0006" npl-type="b"><text>Sol-Gel Optics, Processing and Applications LisaC. Little Ed. KluwerAcademic Publishers 1994, page 450 ff</text></nplcit>. as in<patcit id="pcit0005" dnum="DE1941191A"><text>DE-A-1941191</text></patcit>, <patcit id="pcit0006" dnum="DE3719339A"><text>DE-A-3719339</text></patcit>, <patcit id="pcit0007" dnum="DE4117041A"><text>DE-A-4117041</text></patcit> and <patcit id="pcit0008" dnum="DE4217432A"><text>DE-A-4217432</text></patcit> described. However, it has not yet been described for magnetic particles. It was not to be expected that magnetic particles could be generated with this, which have very surprising properties when isolating nucleic acids. In the gel-sol process, alkoxides of network-forming components, e.g. B. SiO₂, B₂O₃, Al₂O₃, TiO₂, ZrO₂, GeO₂ together with oxides and salts of other components, e.g. B. in alcoholic solution, submitted and hydrolyzed. The equation shows the manufacture of sodium boron aluminum silicate glass.<chemistry id="chem0001" num="0001"><img file="EP1577389B2_D0001.tif" /></chemistry>

The hydrolysis process of the starting components is started by adding water. The reaction proceeds relatively quickly, since the alkali ions have a catalytic effect on the rate of hydrolysis of the silicic acid ester. After the gel formation has ended, the resulting gel can be dried and compressed into a glass by a thermal process.

The sol / pigment ratio has a significant influence on the yield of magnetic pigment. There are limits to the fact that the pigment content is so low that a mass that can still be pumped and sprayed is created. If the pigment content is too low, the fine content, e.g. B. of non-magnetic material too large and disturbing. 10 to 25 g pigment / 100 ml sol were found to be appropriate quantitative ratios with regard to the pigment yield.

The slurry is preferably sprayed through a nozzle to form a powder and the aerosol is dried on a falling section. The nozzle is preferably heated to accelerate drying of the slurry. Depending on the geometry of the nozzle, the nozzle temperature is preferably approx. 120 to 200 ° C. A compromise is found by sufficient evaporation rate, but avoiding splashing.

In view of the yield, the compression temperature should be as high as possible. However, if it is too high, the particles stick together and agglomerates form which should be screened out. The post-treatment in air leads to a loss of magnetic properties at too high temperatures, which is why excessive temperatures should be avoided.

An essentially pore-free surface is understood to mean a surface that is permeated with less than 5%, preferably less than 2%, particularly preferably less than 0.1%, with pores as defined above. If pores are present, they preferably have a diameter of less than 10, particularly preferably 1 nm.

In the context of the invention, particular preference is given to using particles which have a core composed of TiO₂ contain coated mica and magnetite particles immobilized thereon, the composite thus formed being enclosed by the glass layer. Both the core and the magnetite particles are crystalline and not porous. The spaces on the surface of the mica, which are not occupied by the magnetite particles, are covered by a thicker glass layer than the tips of the magnetite particles, so that an essentially non-porous glass surface results.

The non-porosity of the magnetic particles refers only to the outer surface, not to the inside of the particle, so that the particle can be porous inside if the surface is only of substantially non-porous glass or a glass surface with pores of less than a diameter 10 nm is enclosed.

Surprisingly, the magnetic particles are particularly advantageously suitable for isolating nucleic acids from samples. In particular, long nucleic acids are destroyed very little or not at all when they are immobilized on them. The material of the core is also a natural resource and therefore ecologically unproblematic. The production of the particles is also very inexpensive and inexpensive.

Ferromagnetic particles with a glass surface are preferably used in the method according to the invention. Superparamagnetic particles are described in the prior art. It has now been found that ferromagnetic particles, when coated with a glass surface, have considerable advantages in the isolation of nucleic acids. As long as the ferromagnetic particles were not yet exposed to a magnetic field, they sediment only under the influence of gravity. They can be easily and quickly suspended again by shaking. The process of deposition without the influence of a magnetic field is preferably slower than the immobilization of nucleic acids on their surface. The ferromagnetic particles can be collected in a simple manner by means of a magnet at a specific point in the sample liquid in order to separate the liquid from the particles and thus the immobilized nucleic acids.

The glass surface of the ferromagnetic particles according to the invention can be pore-free or contain pores. For the reasons mentioned above, it is preferred that the outer surface of the ferromagnetic particles is also essentially pore-free or has pores with a diameter of less than 10 nm. The ferromagnetic particles also preferably have a grain size between 10 and 60 μm, particularly preferably 20 and 50 μm. Particularly preferred are particles in which pores that may be present in the surface have a diameter of less than 10, particularly preferably 1 nm. An example of a ferromagnetic particle is the above-mentioned composite of mica and magnetite particles, enclosed by a glass layer.

The method according to the invention comprises the steps<ul id="ul0001" list-style="bullet"><li>Bringing a sample containing the nucleic acids in a liquid into contact with the magnetic particles with glass surfaces under conditions in which the nucleic acids can bind to the glass surface in native form,</li><li>Binding the nucleic acids in native form by adsorption on the glass surface of the magnetic particles,</li><li>Separation of the nucleic acids from the liquid and</li><li>Use of the nucleic acids as a substrate in an enzymatic reaction.</li></ul>

Biological materials are understood to mean nucleic acids, for example DNA or RNA.

Samples within the meaning of the invention are, for example, clinical samples such as blood, serum, mouthwash, urine, cerebral fluid, sputum, stool, punctate and bone marrow samples. The sample can also be used in the field of environmental analysis, food analysis or molecular biological research, e.g. B. from bacterial cultures, phage lysates and products of amplification processes, for. B. PCR.

The magnetic particles described have an inner core to which the outer glass surface is applied. The core can be a composite, but also simple iron cores. The core can also consist of a crystalline or ceramic or glass-like structure in which iron oxide is embedded.

With the described method, native or modified nucleic acids can be isolated. Native nucleic acids are understood to mean those whose structure has not been irreversibly changed compared to the naturally occurring nucleic acids. However, this does not exclude the modification of other components of the sample. The medium surrounding the cells can be modified, but not the nucleic acids as such. The nucleic acids should be in their native form, ie not be denatured, cut or modified by coupling reactive groups. The term native nucleic acids therefore does not include biotinylated nucleic acids. Examples of native nucleic acids are phage DNA or cellular nucleic acids from blood.

Modified nucleic acids include those that do not occur in nature, for example nucleic acids which are modified by attachment of reactive, detectable or immobilizing groups, for example biotinylated nucleic acids.

In certain cases, the sample can be used in the isolation procedure without pretreatment. In many cases, however, the sample should be digested using a suitable method and the nucleic acids contained in the sample released. Methods for the digestion of samples are known to the person skilled in the art and can be chemical, enzymatic or physical in nature. A combination of these methods is also possible. Examples include lysis by ultrasound, high pressure or by shear, by alkali, detergents or chaotropic salt solutions, or by the action of proteinases or lipases. Especially with regard to the digestion process for obtaining nucleic acids<nplcit id="ncit0007" npl-type="b"><text>Sambrook et al .: Molecular Cloning, A Laboratory Manual, 2nd Addition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY</text></nplcit> and <nplcit id="ncit0008" npl-type="b"><text>Ausubel et al .: Current Protocols in Molecular Biology 1987, J. Viley and Sons, NY</text></nplcit>, referred.

In addition to the nucleic acids to be isolated, the sample can contain further constituents, e.g. B. cell debris, proteins, salts and other substances not to be isolated in a liquid. This sample, which contains the nucleic acids in native form, is brought into contact with the particles under conditions in which the desired nucleic acids bind to the particle surface. The conditions for this are known in principle. They depend on the type of binding via which the nucleic acids are bound to the surface. In the case of the modified nucleic acids, binding via the groups of the nucleic acids which represent the modification is possible, e.g. B. Biotin via binding to surfaces coated with streptavidin. According to the invention, however, the case is the direct binding of nucleic acids to glass. The binding of native nucleic acids to glass particles can be carried out analogously to methods of the prior art. It is preferably carried out in the presence of chaotropic salts, the concentration of these salts being between 2 and 8 mol / l, preferably 4 to 6 mol / l. Chaotropic salts are e.g. B. sodium iodite, sodium perchlorate, guanidininium thiocyanate, guanidinium isothiocyanate or guanidinium hydrochlorite, but is not limited to these compounds.

To bring the sample into contact with the particles, the sample is mixed with the particles and incubated for a time sufficient for the binding. The length of the incubation is generally known to the person skilled in the art from treatment with non-magnetic particles. Optimization is possible by carrying out a determination of the amount of immobilized nucleic acids on the surface at different times. Incubation times between 10 seconds and 30 minutes can be expedient.

Depending on the size and type of magnetic particles, the particles are separated from the liquid during the incubation period or the suspension is retained over a longer period. If the particles have a very small particle size and are superparamagnetic, the suspension is preserved over a longer period. If the particles are larger, the particles are slowly separated from the liquid during the incubation. Such aggregates form in particular when ferromagnetic particles are involved. In the preferred case that the ferromagnetic particles are not premagnetized, a particularly gentle separation is guaranteed.

The immobilization does not take place through precipitation by lowering the solubility of the nucleic acids to be immobilized. Instead, the immobilization is based on adsorption. This largely avoids non-specific inclusions of impurities.

After the incubation, the nucleic acids are separated from the liquid. This is generally achieved by separating the nucleic acids bound to the magnetic particles using a magnetic field. For example, the magnetic particles can be drawn to the wall of the vessel in which the incubation took place. The liquid containing the sample ingredients that were not bound to the magnetic particles can then be removed. This distance depends on the type of vessel in which the incubation took place. Suitable process steps are pipetting off or suctioning off the liquid.

The magnetic particles can then, if desired, be cleaned one or more times with a washing solution. The washing solution is chosen so that the nucleic acids are not detached from the particle surface as far as possible, but impurities that are not to be isolated are washed away as well as possible. This washing step preferably takes place by incubating the washing solution with the particles, with a resuspension of the particles preferably being carried out, e.g. B. by shaking or applying a magnetic field that is not identical to the first magnetic field. The contaminated washing solution is preferably removed in the same way as the sample in the above-mentioned step for binding the nucleic acids.

After the last washing step, a short drying step of the magnetic particles can be carried out in a vacuum or by evaporating (letting) the liquid, whereby a pretreatment with acetone is also possible.

The nucleic acids thus purified can, if desired, be removed from the magnetic particles. Since the nucleic acids are native nucleic acids and the magnetic particles are glass-coated particles, the nucleic acid can be removed from the particles by means of an elution buffer with a low salt content. Such buffers are out<patcit id="pcit0009" dnum="DE3724442"><text>DE 3724442</text></patcit> and <nplcit id="ncit0009" npl-type="s"><text>Analytical Biochemistry 175, 196-201 (1988</text></nplcit>) known. Buffers with a content of less than 0.2 mol / l are used in particular as elution buffers with a low salt content. In a particularly preferred embodiment, the elution buffer contains Tris. In another particular embodiment, the elution buffer is demineralized water.

In a further embodiment, the cleaning and isolation method described can be carried out following an immunomagnetic separation of cells (e.g. viral particles or prokaryotic or eukaryotic cells) from a body fluid or a tissue. For this purpose, the sample with magnetic particles, to which an antibody against an antigen is immobilized on the cell, e.g. B. incubated with shaking. Such particles can be the particles described above, but also commercially available (e.g. MACS microbeads from Miltenyi Biotec GmbH, Bergisch Gladbach, FRG). After applying a magnetic field, one or more washing steps take place with a saline washing solution. Particles are obtained to which the desired cells are bound. To isolate the nucleic acids, the bound cells are resuspended in a saline buffer. In a preferred embodiment, this salt-containing buffer is a chaotropic salt solution, so that the nucleic acids present in the cell are released from the cells.

Combining the isolation of cells described above with the isolation of nucleic acids in their native form on the magnetic particles, likewise described, results in a particularly advantageous method for isolating nucleic acids from cell-containing samples for the subsequent enzymatic reaction. Advantages of this embodiment are the possible simplicity (single-tube method), high sensitivity (particularly important in medical microbiology and oncology) and the ease of automation.

The isolated nucleic acids can now be used in any way as a substrate for various enzymatic reactions. Examples include sequencing, radioactive or non-radioactive labeling, amplification of one or more sequences contained therein, transcription, hybridization with labeled probe nucleic acids, translation or ligation. An advantage of the method according to the invention is that the separation of the nucleic acids from the liquid is very simple. In the prior art, either a centrifugation step was used to separate glass particles from impurities or, in the case of binding of the nucleic acids to glass fiber filters, the liquid was sucked through this liquid. This is a limiting step that hinders the processing of large numbers of samples.

With the described particles, a more effective separation of the nucleic acids from impurities is possible. In particular, inhibitors for certain enzymatic reactions can be removed to a particularly good extent. The yield of nucleic acids is comparatively high. Fractionation of long nucleic acids was not observed. The particles described can preferably be magnetized more quickly.<ul id="ul0002" list-style="none"><li>In <figref idref="f0001">Figure 1</figref> isolation of nucleic acids from a cell-containing sample is shown schematically.</li><li>In <figref idref="f0002">Figure 2</figref> the separation of isolated nucleic acids in an agarose gel is shown.</li><li>In <figref idref="f0002">Figure 3</figref> the separation of reaction products after isolation and PCR amplification according to the invention is shown.</li><li>In <figref idref="f0003">Figure 4</figref> a gel of the results from Example 4 is shown.</li></ul>

in <figref idref="f0001">Figure 1</figref> isolation from nucleic acids from a cell-containing sample is shown schematically. The sample (specimen), which contains cells, is pretreated sample-specifically so that the cells in which the nucleic acids are to be detected are in a suitable form. This includes e.g. B. in samples from which body fluids have been removed, the addition of reagents, for. B. for the liquefaction of viscous samples, for. B. Saliva samples. An antibody bound to a solid phase, preferably to a bead (bead), which can recognize and bind the cell, is added to the sample prepared in this way in a vessel. For example, antigens on the cell surface have proven to be suitable partners for the antibody. The specificity of the antibody can depend on the specificity of the analysis task to be solved. If the solid phase is the wall of the vessel, the cells are bound directly to the wall. In the event that the solid phases are pearls, they are separated from the liquid by suitable separation methods. This can be done, for example, by filtration. In the case of magnetic beads, separation is possible by applying a magnetic field to the outer wall of the vessel. The separated cells are washed with a liquid to remove contaminants which would interfere with the detection with the medium surrounding the cells. Conditions are preferably used in which the cells are neither detached from the solid phase nor destroyed. Then the cells are destroyed, the so-called lysis. One possibility is to treat the cells with chaotropic salts. Other options are the action of proteinases and detergents.

In the preferred embodiment, the particles are added to the lysis mixture. After a suitable exposure time, which can be optimized by loading the surface with nucleic acids, the particles are separated from the liquid surrounding them, which contains further and undetectable cell components. Again, this is preferably done by applying a magnetic field to the vessel wall using a magnet.

In order to remove any impurities that may still be adhering, washing is preferably carried out with a liquid which is selected such that the nucleic acids to be determined do not detach from the glass surface.

To remove the nucleic acids from the glass surface, a so-called elution buffer is added, which has reagent conditions under which the nucleic acids detach from the glass surface. These are especially low salt conditions. Depending on the intended further treatment of the nucleic acids, the liquid can now be separated from the particles and processed further. It is preferred to carry out this separation with an applied magnetic field so that the particles are present separately.

The following examples illustrate the invention.

example 1

Production of the magnetic particles

6 different brines were used. The brine was produced according to the following schemes:         Sol 1 (SiO₂: B₂O₃ = 7:3):

The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 86.6 ml tetraethyl orthosilicate<ul id="ul0003" list-style="none" compact="compact"><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+ 14.1 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. Then add dropwise<ul id="ul0004" list-style="none" compact="compact"><li>+ 37.8 ml trimethyl borate.</li><li>The sol is then kept at 50 ° C. for 2 hours. Then add</li><li>+ 14.1 ml 0.15 M HCl</li></ul>        Sol 2 (SiO₂: B₂O₃=4:1):

The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 100.5 ml tetraethyl orthosilicate<ul id="ul0005" list-style="none" compact="compact"><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+ 16.3 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. This is followed by the dropwise addition of + 25.6 ml trimethyl borate. The sol is then kept at 50 ° C. for 2 hours. Then + 16.3 ml 0.15 M HCl is added         Sol 3 (SiO₂: B₂O₃ = 85:15):

The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 107.8 ml tetraethyl orthosilicate<ul id="ul0006" list-style="none" compact="compact"><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+ 17.5 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. Then add 19.4 ml of trimethylborate dropwise. The sol is then kept at 50 ° C. for 2 hours. This is followed by the addition of + 17.5 ml 0.15 M HCl         Sol 4 (SiO₂: B₂O₃ = 4: 1; 2 mol% P₂O₅):

The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 100.5 ml tetraethyl orthosilicate<ul id="ul0007" list-style="none" compact="compact"><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+ 16.3 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. Then add dropwise<ul id="ul0008" list-style="none" compact="compact"><li>+ 25.6 ml trimethyl borate</li><li>The sol is then kept at 50 ° C. for 2 hours. Then + 16.3 ml 0.15 M HCl is added</li><li>+ 1.63 g P₂O₅</li></ul>        Sol 5 (SiO₂: B₂O₃ = 4: 1 mol% of Al₂O₃):

The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 100.5 ml tetraethyl orthosilicate<ul id="ul0009" list-style="none" compact="compact"><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+ 16.3 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. Then add dropwise<ul id="ul0010" list-style="none" compact="compact"><li>+ 25.6 ml trimethyl borate.</li><li>The sol is then kept at 50 ° C. for 2 hours. Then + 16.3 ml 0.15 M HCl is added</li><li>+ 3.06 g AlCl₃</li></ul>        Sol 6 (SiO₂: B₂O₃ = 4: 1 mol of 1% ZrO₂):

The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 100.5 ml tetraethyl orthosilicate<ul id="ul0011" list-style="none" compact="compact"><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+ 16.3 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. Then add dropwise<ul id="ul0012" list-style="none" compact="compact"><li>+ 25.6 ml trimethyl borate</li><li>+ 5.15 ml of zirconium (IV) propylate, 70% by weight solution in 1-propanol</li><li>The sol is then kept at 50 ° C. for 2 hours. Then + 16.3 ml 0.15 M HCl is added</li></ul>

After a further 2 hours at 50 ° C., 22.5 g of Iriodin 600 (Black Mica) were stirred into 150 ml of the brine and then coated with a spray dryer (Büchi 190, Mini Spray Dryer). The nozzle temperature of the spray dryer was 134 ° C.

The powder obtained by the spray drying process was then subjected to a temperature treatment under a nitrogen atmosphere (90 l / h). The heating rate was 1 k / min and the holding time was 2 hours at the compression temperature. This temperature was 750 ° C for coating with Sol 1, 860 ° C for coating with Sol 2 and 800 ° C for the other coatings. After the sintering process, the furnace was switched off and the powder was cooled to room temperature. Any agglomerates formed were screened with a 50 µm sieve.

Example 2

Manufacture of GMP1, GMP2, GMP3 and GMP4

GMP1, GMP2, GMP3 and GMP4 are pigments from different production batches, which were obtained from sol 1 from example 1 in a process according to example 1 under the following conditions:<tables id="tabl0002" num="0002"><table frame="all"><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="52mm" /><colspec colnum="2" colname="col2" colwidth="17mm" /><colspec colnum="3" colname="col3" colwidth="17mm" /><colspec colnum="4" colname="col4" colwidth="17mm" /><colspec colnum="5" colname="col5" colwidth="17mm" /><thead><row><entry align="center" valign="top"><b>parameter</b></entry><entry align="center" valign="top"><b>GMP1</b></entry><entry align="center" valign="top"><b>GMP2</b></entry><entry align="center" valign="top"><b>GMP3</b></entry><entry align="center" valign="top"><b>GMP4</b></entry></row></thead><tbody><row><entry>Aging of the sol (h) (30 ° C)</entry><entry>36</entry><entry>36</entry><entry>36</entry><entry>36</entry></row><row><entry>Pigment fraction of the sol (g / 100 ml)</entry><entry>5</entry><entry>15</entry><entry>8</entry><entry>20</entry></row><row><entry>Airflow from the nozzle (%)</entry><entry>100</entry><entry>100</entry><entry>100</entry><entry>100</entry></row><row><entry>Air pressure (bar)</entry><entry>6</entry><entry>6</entry><entry>6</entry><entry>3</entry></row><row><entry>Nozzle temperature (° C)</entry><entry>135</entry><entry>120</entry><entry>130</entry><entry>143</entry></row><row><entry>Compression temperature (° C)</entry><entry>534</entry><entry>534</entry><entry>534</entry><entry>615</entry></row><row><entry>02 post-treatment (1 hour)</entry><entry>(300 ° C)</entry><entry>(300 ° C)</entry><entry>300 ° C)</entry><entry>(400 ° C)</entry></row><row><entry>Yield of pigment</entry><entry>low</entry><entry>high</entry><entry>medium</entry><entry>high</entry></row><row><entry>DNA yield</entry><entry>low</entry><entry>high</entry><entry>high</entry><entry>high</entry></row></tbody></tgroup></table></tables>

Example 3

PCR sample preparation from whole human blood with magnetic glass particles

Isolation of the nucleic acid

Approx. 10 mg of three glass magnetic particle batches (GMP2 - 4) were placed in Eppendorf reaction vessels. The exact weights are given in Table 1, triplicate determinations were carried out.

40 µl of Proteinase K (20 mg / ml, made from lyophilisate) were pipetted into 200 µl of thawed whole blood and mixed immediately. Then 200 μl binding buffer (6 M guanidine-HCl, 10 mM Tris-HCl, 10 mM urea, 30% Triton X-100, pH 4.4) were added, mixed and incubated at 70 ° C. for 10 minutes. After adding 200 .mu.l of i-propanol was mixed for 10 seconds on the vortex mixer, the sample was incubated for 20 minutes at room temperature and mixed again for 10 seconds as before. The magnetic separation was carried out for at least 30 seconds in the Boehringer Mannheim magnetic particle separator (ID No. 1 641 794). The supernatant was removed and analyzed as described below.

The magnetic particles were each washed with 500 μl washing buffer (20 mM NaCl, 10 mM Tris-HCl, pH 7.5 (25 ° C.), 80% ethanol) by mixing for 10 seconds, incubating for 1 minute at room temperature and mixing for 10 seconds and drawn to the vessel wall with the magnetic particle separator. The supernatant was removed and discarded. The washing procedure was repeated until the washing supernatant was colorless (4 x washing in total). The nucleic acids were then eluted 3 times with elution buffer (10 mM Tris-HCl, pH 8.5) preheated to 70 ° C. in each case by mixing for 10 seconds, incubating for 10 minutes at room temperature and mixing for 10 minutes.

Processing of the supernatant

The supernatant after the first binding to the magnetic glass particles was checked for the content of nucleic acids as follows: The supernatant was placed in a filter tube (Boehringer-Mannheim, Id.No. 1744003, e.g. contained in High Pure PCR Product Purification Kit) given and centrifuged for 1 minute at 8000 rpm in an Eppendorf table centrifuge. The run is discarded and the filter tube is washed twice with 500 μl washing buffer (centrifugation as before). The filter tube is briefly centrifuged dry and then eluted with 2 x 200 µl of 1 x elution buffer preheated to 70 ° C by renewed centrifugation.

Analysis of the eluate and the sample supernatant

50 µl of the eluates or the supernatants worked out with a filter tube were mixed with 10 µl of sample buffer and 45 µl of this was separated electrophoretically in a 0.8% agarose gel at 120 V for 90 minutes.

Different dilutions of the eluates or the processed supernatants were measured spectroscopically at 260 and 280 nm in a Uvikon 710 (Kontron).

Two 5 µl aliquots of the eluates were checked in duplicate by Expand ™ Long Template PCR (Boehringer Mannheim, Id. No. 1681834) with specific primers for the human tPA gene (expected product length 15 kb).<tables id="tabl0003" num="0003"><table frame="none"><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="32mm" /><colspec colnum="2" colname="col2" colwidth="21mm" /><colspec colnum="3" colname="col3" colwidth="37mm" /><colspec colnum="4" colname="col4" colwidth="21mm" /><thead><row><entry valign="top">Mix I</entry><entry valign="top">per approach</entry><entry valign="top">Mix II</entry><entry valign="top">per approach</entry></row></thead><tbody><row rowsep="0"><entry>dNTP, 100 mM each</entry><entry align="right">1 µl</entry><entry>Expand ™ buffer, 10 x</entry><entry align="right">5 µl</entry></row><row rowsep="0"><entry>Primer 1, 200 ng / µl</entry><entry align="right">1 µl</entry><entry>Expand ™ polymerase</entry><entry align="right">0.75 µl</entry></row><row rowsep="0"><entry>Primer 2, 225 ng / µl</entry><entry align="right">1 µl</entry><entry>H₂O_bidest</entry><entry align="right">19.25 µl</entry></row><row><entry rowsep="0">H₂O_bidest.</entry><entry align="right">17th µl</entry><entry rowsep="0" /><entry align="right" /></row><row rowsep="0">&lt;entry /&gt;<entry align="right">20 µl</entry>&lt;entry /&gt;<entry align="right">25th µl</entry></row></tbody></tgroup></table></tables>

Mix I is placed in a thin-walled PCR tube with 5 μl of eluate and Mix II is added. The mixture is mixed briefly and covered with 30 µl mineral oil. The approaches are amplified in a Perkin-Elmer Thermocycler 9600 with the following program:<tables id="tabl0004" num="0004"><table frame="none"><tgroup cols="3" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="41mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="19mm" /><tbody><row><entry>2nd Minutes</entry><entry>92 ° C</entry>&lt;entry /&gt;</row><row>&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;</row><row><entry>10th Seconds</entry><entry>92 ° C</entry>&lt;entry /&gt;</row><row><entry>30th Seconds</entry><entry>65 ° C</entry><entry>10th Cycles</entry></row><row><entry>12th Minutes</entry><entry>68 ° C</entry>&lt;entry /&gt;</row><row>&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;</row><row><entry>10th Seconds</entry><entry>92 ° C</entry>&lt;entry /&gt;</row><row><entry>30th Seconds</entry><entry>65 ° C</entry><entry>20 Cycles</entry></row><row><entry>12th Minutes</entry><entry>68 ° C</entry>&lt;entry /&gt;</row><row><entry>+20 seconds per cycle</entry>&lt;entry /&gt;&lt;entry /&gt;</row><row>&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;</row><row><entry>7 Minutes</entry><entry>68 ° C</entry>&lt;entry /&gt;</row><row><entry>subsequently</entry><entry>7 ° C</entry>&lt;entry /&gt;</row></tbody></tgroup></table></tables>

The 50 μl PCR batches were mixed with 10 μl sample buffer and 45 μl thereof were separated electrophoretically in a 0.8% agarose gel at 120 V for 90 minutes.

Results

<tables id="tabl0005" num="0005"><table frame="bottom"><title>Table 1: Yield of nucleic acids with magnetic glass particles from 200 µl blood</title><tgroup cols="11" colsep="0"><colspec colnum="1" colname="col1" colwidth="15mm" /><colspec colnum="2" colname="col2" colwidth="8mm" /><colspec colnum="3" colname="col3" colwidth="15mm" /><colspec colnum="4" colname="col4" colwidth="15mm" /><colspec colnum="5" colname="col5" colwidth="19mm" /><colspec colnum="6" colname="col6" colwidth="17mm" /><colspec colnum="7" colname="col7" colwidth="15mm" /><colspec colnum="8" colname="col8" colwidth="15mm" /><colspec colnum="9" colname="col9" colwidth="19mm" /><colspec colnum="10" colname="col10" colwidth="17mm" /><colspec colnum="11" colname="col11" colwidth="16mm" /><thead><row><entry valign="top" /><entry valign="top" /><entry namest="col3" nameend="col6" align="center" valign="top">Overlap 1: 8</entry><entry align="center" valign="top" /><entry namest="col8" nameend="col10" align="center" valign="top">1. Eluate 1: 8</entry><entry valign="top" /></row><row><entry valign="top" /><entry valign="top" /><entry valign="top">260 nm</entry><entry valign="top">280 nm</entry><entry valign="top">yield</entry><entry valign="top">260/280</entry><entry valign="top">260 nm</entry><entry valign="top">280 nm</entry><entry valign="top">yield</entry><entry valign="top">260/280</entry><entry valign="top" /></row></thead><tbody><row rowsep="0"><entry align="right">GMP / 2</entry><entry align="center">1</entry><entry align="right">0,021</entry><entry align="right">0,013</entry><entry align="right">1.7 µg</entry><entry align="right">1,6</entry><entry align="right">0,171</entry><entry align="right">0,164</entry><entry align="right">13.7 µg</entry><entry align="right">1,0</entry><entry align="center" /></row><row rowsep="0"><entry align="right">12th mg</entry><entry align="center" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="center" /></row><row rowsep="0"><entry align="right">10th mg</entry><entry align="center">2</entry><entry align="right">0,045</entry><entry align="right">0,035</entry><entry align="right">3.7 µg</entry><entry align="right">1,3</entry><entry align="right">0,137</entry><entry align="right">0,138</entry><entry align="right">11.0 µg</entry><entry align="right">1,0</entry><entry align="center" /></row><row><entry align="right">9 mg</entry><entry align="center">3</entry><entry align="right">0,036</entry><entry align="right">0,027</entry><entry align="right">2.9 µg</entry><entry align="right">1,3</entry><entry align="right">0,153</entry><entry align="right">0,164</entry><entry align="right">12.2 µg</entry><entry align="right">0,9</entry><entry align="center" /></row><row rowsep="0"><entry align="right">GMP / 3</entry><entry align="center">1</entry><entry align="right">0,050</entry><entry align="right">0,042</entry><entry align="right">4.0 µg</entry><entry align="right">1,2</entry><entry align="right">0,245</entry><entry align="right">0,246</entry><entry align="right">19.6 µg</entry><entry align="right">0,9</entry><entry align="center" /></row><row rowsep="0"><entry align="right">10mg</entry><entry align="center" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="center" /></row><row rowsep="0"><entry align="right">10th mg</entry><entry align="center">2</entry><entry align="right">0,033</entry><entry align="right">0,022</entry><entry align="right">2.6 µg</entry><entry align="right">1,5</entry><entry align="right">0,397</entry><entry align="right">0,398</entry><entry align="right">31.8 µg</entry><entry align="right">1,0</entry><entry align="center" /></row><row><entry align="right">10th mg</entry><entry align="center">3</entry><entry align="right">0,042</entry><entry align="right">0,030</entry><entry align="right">3.4 µg</entry><entry align="right">1,4</entry><entry align="right">0,278</entry><entry align="right">0,282</entry><entry align="right">22.2 µg</entry><entry align="right">0,9</entry><entry align="center" /></row><row rowsep="0"><entry align="right">GMP / 4</entry><entry align="center">1</entry><entry align="right">0,065</entry><entry align="right">0,056</entry><entry align="right">0.7 µg</entry><entry align="right">1,2</entry><entry align="right">0,135</entry><entry align="right">0,142</entry><entry align="right">11.0 µg</entry><entry align="right">1,0</entry><entry align="center" /></row><row rowsep="0"><entry align="right">10th mg</entry><entry align="center" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /><entry align="center" /></row><row rowsep="0"><entry align="right">11 mg 2</entry><entry align="center">2</entry><entry align="right">0,071</entry><entry align="right">0,142</entry><entry align="right">2.4 µg</entry><entry align="right">0,5</entry><entry align="right">0,140</entry><entry align="right">0,142</entry><entry align="right">11.2 µg</entry><entry align="right">1,0</entry><entry align="center" /></row><row><entry align="right">10mg</entry><entry align="center">3</entry><entry align="right">0,066</entry><entry align="right">0,051</entry><entry align="right">1.7 µg</entry><entry align="right">1,3</entry><entry align="right">0,130</entry><entry align="right">0,130</entry><entry align="right">10.4 µg</entry><entry align="right">1,0</entry><entry align="center" /></row></tbody></tgroup><tgroup cols="11" colsep="0"><colspec colnum="1" colname="col1" colwidth="15mm" /><colspec colnum="2" colname="col2" colwidth="8mm" /><colspec colnum="3" colname="col3" colwidth="15mm" /><colspec colnum="4" colname="col4" colwidth="15mm" /><colspec colnum="5" colname="col5" colwidth="19mm" /><colspec colnum="6" colname="col6" colwidth="17mm" /><colspec colnum="7" colname="col7" colwidth="15mm" /><colspec colnum="8" colname="col8" colwidth="15mm" /><colspec colnum="9" colname="col9" colwidth="19mm" /><colspec colnum="10" colname="col10" colwidth="17mm" /><colspec colnum="11" colname="col11" colwidth="16mm" /><thead><row><entry align="right" valign="top" /><entry namest="col2" nameend="col4" align="center" valign="top">2nd Eluate 1: 8</entry><entry align="center" valign="top" /><entry namest="col6" nameend="col8" align="center" valign="top">3rd Eluate 1: 4</entry><entry align="center" valign="top" /><entry align="center" valign="top" /><entry align="center" valign="top" /></row><row><entry align="right" valign="top" /><entry align="center" valign="top" /><entry align="center" valign="top">260 nm</entry><entry align="center" valign="top">280 nm</entry><entry align="center" valign="top">yield</entry><entry align="center" valign="top">260/280</entry><entry align="center" valign="top">260 nm</entry><entry align="center" valign="top">280 nm</entry><entry align="center" valign="top">yield</entry><entry align="center" valign="top">260/280</entry><entry align="center" valign="top">∑Eluate</entry></row></thead><tbody><row rowsep="0"><entry align="right">GMP / 2</entry><entry align="center">1</entry><entry align="right">0,099</entry><entry align="right">0,101</entry><entry align="right">7.9 µg</entry><entry align="right">1,0</entry><entry align="right">0,057</entry><entry align="right">0,062</entry><entry align="right">2.3 µg</entry><entry align="right">0,9</entry><entry align="right">23.9 µg</entry></row><row rowsep="0"><entry align="right" /><entry align="center">2</entry><entry align="right">0,078</entry><entry align="right">0,076</entry><entry align="right">6.2 µg</entry><entry align="right">1,0</entry><entry align="right">0,041</entry><entry align="right">0,049</entry><entry align="right">1.6 µg</entry><entry align="right">0,8</entry><entry align="right">18.8 µg</entry></row><row><entry align="right" /><entry align="center">3</entry><entry align="right">0,103</entry><entry align="right">0,112</entry><entry align="right">8.2 µg</entry><entry align="right">0,9</entry><entry align="right">is missing</entry><entry align="right" /><entry align="right" /><entry align="right" /><entry align="right" /></row><row rowsep="0"><entry align="right">GMP / 3</entry><entry align="center">1</entry><entry align="right">0,147</entry><entry align="right">0,147</entry><entry align="right">11.8 µg</entry><entry align="right">1,0</entry><entry align="right">0,084</entry><entry align="right">0,098</entry><entry align="right">3.4 µg</entry><entry align="right">0,9</entry><entry align="right">34.8 µg</entry></row><row rowsep="0"><entry align="right" /><entry align="center">2</entry><entry align="right">0,256</entry><entry align="right">0,252</entry><entry align="right">20.5 µg</entry><entry align="right">1,0</entry><entry align="right">0,042</entry><entry align="right">0,043</entry><entry align="right">1.7 µg</entry><entry align="right">1,0</entry><entry align="right">54.0 µg</entry></row><row><entry align="right" /><entry align="center">3</entry><entry align="right">0,147</entry><entry align="right">0,143</entry><entry align="right">11.8 µg</entry><entry align="right">1,0</entry><entry align="right">0,073</entry><entry align="right">0,093</entry><entry align="right">2.9 µg</entry><entry align="right">0,8</entry><entry align="right">36.9 µg</entry></row><row rowsep="0"><entry align="right">GMP / 4</entry><entry align="center">1</entry><entry align="right">0,106</entry><entry align="right">0,108</entry><entry align="right">8.5 µg</entry><entry align="right">1,0</entry><entry align="right">0,083</entry><entry align="right">0,098</entry><entry align="right">3.3 µg</entry><entry align="right">0,8</entry><entry align="right">22.8 µg</entry></row><row rowsep="0"><entry align="right" /><entry align="center">2</entry><entry align="right">0,111</entry><entry align="right">0,114</entry><entry align="right">8.9 µg</entry><entry align="right">1,0</entry><entry align="right">0,054</entry><entry align="right">0,063</entry><entry align="right">2.2 µg</entry><entry align="right">0,9</entry><entry align="right">22.3 µg</entry></row><row><entry align="right" /><entry align="center">3</entry><entry align="right">0,135</entry><entry align="right">0,141</entry><entry align="right">10.8 µg</entry><entry align="right">1,0</entry><entry align="right">0,077</entry><entry align="right">0,095</entry><entry align="right">3.1 µg</entry><entry align="right">0,8</entry><entry align="right">24.3 µg</entry></row></tbody></tgroup></table></tables>

The first eluates were still slightly yellow and partially contaminated with fine magnetic particles.

Analysis of the eluates in the agarose gel (<figref idref="f0002">FIG. 2nd</figref>) shows a good reproducibility of the yield. The magnetic particles GMP / 2 - 4 show no significant differences. Eluates 1 (top) and 2 (bottom) have approximately the same nucleic acid concentration (estimated by the gel). Eluate 3 shows only a low nucleic acid concentration. Only a low nucleic acid concentration can also be observed in the supernatants.

The Expand ™ PCR delivers consistently good and specific amplification products with all samples except a few outliers (Table 2). The magnetic glass beads were used to isolate nucleic acids from human blood samples, which in a subsequent PCR provided specific amplicons.<tables id="tabl0006" num="0006"><table frame="bottom"><title>Table 2. Results of Expand ™ PCR</title><tgroup cols="5" colsep="0"><colspec colnum="1" colname="col1" colwidth="18mm" /><colspec colnum="2" colname="col2" colwidth="18mm" /><colspec colnum="3" colname="col3" colwidth="17mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><colspec colnum="5" colname="col5" colwidth="16mm" /><thead><row><entry align="right" valign="top" /><entry namest="col2" nameend="col3" align="center" valign="top">15 kb Expand ™ PCR</entry><entry namest="col4" nameend="col5" align="center" valign="top">human tPA gene</entry></row><row><entry align="right" valign="top" /><entry namest="col2" nameend="col3" align="center" valign="top">1. Eluate</entry><entry namest="col4" nameend="col5" align="center" valign="top">2nd Eluate</entry></row></thead><tbody><row rowsep="0"><entry align="right">GMP / 2 1</entry><entry namest="col2" nameend="col3" align="center">is missing</entry><entry align="center">+</entry><entry align="center">+</entry></row><row rowsep="0"><entry align="right">2</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry></row><row><entry align="right">3</entry><entry align="center">+</entry><entry align="center">+</entry><entry namest="col4" nameend="col5" align="center">is missing</entry></row><row rowsep="0"><entry align="right">GMP / 3 1</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry></row><row rowsep="0"><entry align="right">2</entry><entry align="center">(+)</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry></row><row rowsep="0"><entry align="right">3</entry><entry align="center">-</entry><entry align="center">(+)</entry><entry align="center">+</entry><entry align="center">+</entry></row><row rowsep="0"><entry align="right">GMP / 4 1</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry></row><row rowsep="0"><entry align="right">2</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">(+)*</entry></row><row><entry align="right">3</entry><entry align="center">+</entry><entry align="center">+</entry><entry namest="col4" nameend="col5" align="center">is missing</entry></row><row><entry namest="col1" nameend="col2" align="center">K, BM control DNA</entry>&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;</row><row><entry align="right">* 3. Eluate</entry><entry align="right" />&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;</row></tbody></tgroup></table></tables>

In <figref idref="f0002">FIG. 3rd</figref> a gel with the reaction products after PCR amplification is shown. MWM III is a molecular weight marker (eluate 1, top; eluate 2, bottom).

Example 4

Binding of DNA length standard to magnetic glass particles

1. Preparation of the magnetic glass particles

12 mg of the glass magnet batch GMP4 were placed in Eppendorf reaction vessels.

2nd Lysis and binding

In a 1.5 ml Eppendorf tube with 12 mg magnetic glass particles, 900 µl lysis buffer (4.6 M GuSCN, 45 mM Tris, 20 mM EDTA, pH 7.3) and 100 µl DNA sample, in the model DNA Length standard III from Boehringer Mannheim (catalog no. 528552) was used, mixed for 2 to 10 seconds until a homogeneous suspension is formed. The solution is incubated for 20 minutes at room temperature, with mixing every 5 minutes.

Magnetic separation takes place in a magnetic particle separator for at least 15 seconds. The supernatant is pipetted off.

3rd Washing and drying

The magnetic glass particles are washed twice with washing buffer (5.2 M GuSCN, 50 mM Tris, pH 6.5) twice with 70% pre-cooled ethanol and once with acetone by removing the magnetic field, pipetting in 800 μl solution, mixing for 2 seconds, incubating for 1 min at RT , the magnetic field is applied and the supernatant is finally pipetted off.

After removal of the acetone, the particles are dried for 10 min at 56 ° C. in a heating block with the lid open.

4th Elution of the DNA

The DNA is eluted with 4x 50 μl elution buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) by incubating for 10 min with repeated shaking at 56 ° C. and finally transferring the supernatant containing DNA into a new Eppendorf tube .

5. Analysis of the eluate

A fifth of the eluate volume was mixed with sample buffer and the DNA was separated on a 1% agarose gel at 90 V. To determine recovery, a dilution series of DNA length standard III was applied to the same gel, which contains the DNA amounts to be expected in the samples.

The quantitative evaluation was carried out by scanning a polaroid photo of the agarose gel. The standard dilution series was used as the calibrator.

The yield of DNA with magnetic glass particles is shown in Table 1<tables id="tabl0007" num="0007"><table frame="all"><title>Table 1. Yield of DNA length standard III with magnetic glass particles</title><tgroup cols="9"><colspec colnum="1" colname="col1" colwidth="17mm" colsep="0" /><colspec colnum="2" colname="col2" colwidth="23mm" colsep="0" /><colspec colnum="3" colname="col3" colwidth="42mm" /><colspec colnum="4" colname="col4" colwidth="14mm" colsep="0" /><colspec colnum="5" colname="col5" colwidth="27mm" colsep="0" /><colspec colnum="6" colname="col6" colwidth="43mm" colsep="0" /><colspec colnum="7" colname="col7" colwidth="25mm" colsep="0" /><colspec colnum="8" colname="col8" colwidth="34mm" colsep="0" /><colspec colnum="9" colname="col9" colwidth="19mm" /><thead><row><entry align="center" valign="top">Standard No.</entry><entry align="center" valign="top">Standard DNA amount [ng]</entry><entry align="center" valign="top">Brightness standard (measured value) [rel. Units]</entry><entry align="center" valign="top">Sample No.</entry><entry align="center" valign="top">Pigment / bead type</entry><entry align="center" valign="top">Brightness intensity, sample (measured value) [rel. Units]</entry><entry align="center" valign="top">calculated amount of DNA on gel [ng]</entry><entry align="center" valign="top">calculated amount of DNA in sample [ng]</entry><entry align="center" valign="top">Recovery [%]</entry></row></thead><tbody><row rowsep="0"><entry align="center">1</entry><entry align="center">200</entry><entry align="center">65</entry><entry align="center">1</entry><entry align="center">GMP4</entry><entry align="center">45</entry><entry align="center">139</entry><entry align="center">695</entry><entry align="center">69,5</entry></row><row rowsep="0"><entry align="center">2</entry><entry align="center">175</entry><entry align="center">56</entry><entry align="center">2</entry><entry align="center">GMP4</entry><entry align="center">39</entry><entry align="center">120</entry><entry align="center">600</entry><entry align="center">60,0</entry></row><row rowsep="0"><entry align="center">3</entry><entry align="center">150</entry><entry align="center">51</entry><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /></row><row rowsep="0"><entry align="center">4</entry><entry align="center">125</entry><entry align="center">44</entry><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /></row><row rowsep="0"><entry align="center">5</entry><entry align="center">100</entry><entry align="center">37</entry><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /></row><row rowsep="0"><entry align="center">6</entry><entry align="center">75</entry><entry align="center">25</entry><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /></row><row rowsep="0"><entry align="center">7</entry><entry align="center">50</entry><entry align="center">17</entry><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /></row><row rowsep="0"><entry align="center">8</entry><entry align="center">25</entry><entry align="center">9</entry><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /></row><row><entry align="center">9</entry><entry align="center">10</entry><entry align="center">4</entry><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /></row></tbody></tgroup></table></tables>

The agarose gel, which served as the basis for the quantitative evaluation, is in <figref idref="f0003">FIG. 4th</figref> shown. It is a 1% ethidium bromide stained agarose gel. Lanes 1 through 10 correspond to a dilution series of DNA length standard III. 1: 1 µg DNA, 2: 200 ng DNA, 3: 175 ng DNA, 4: 150 ng DNA, 5: 125 ng DNA, 6: 100 ng DNA, 7: 75 ng DNA, 8: 50 ng NDA, 9: 25 ng DNA, 10:10 ng DNA.

Lanes 11 and 12 correspond to the DNA eluted from the magnetic glass particles when using 200 ng DNA length standard.

SEQUENCE LOG

<ul id="ul0013" list-style="none"><li>&lt;110&gt; Roche Diagnostics GmbH</li><li>&lt;120&gt; Magnetic pigment</li><li>&lt;130&gt; 28921 PEP-WO_WWHC</li><li>&lt;140&gt; &lt;141&gt; &gt;</li><li>&lt;150&gt; <patcit id="pcit0010" dnum="DE1952098"><text>DE1952098.4</text></patcit> &lt;151&gt; 1995-06-08</li><li>&lt;150&gt; <patcit id="pcit0011" dnum="DE19537985"><text>DE19537985.3</text></patcit> &lt;151&gt; 1995-10-12</li><li>&lt;150&gt; <patcit id="pcit0012" dnum="EP96921935A"><text>EP96921935.1</text></patcit> &lt;151&gt; 1996-06-06</li><li>&lt;160&gt; 2</li><li>&lt;170&gt; Patent In Ver. 2.1</li><li>&lt;210&gt; 1 &lt;211&gt; 34 &lt;212&gt; DNA &lt;213&gt; Artificial sequence</li><li>&lt;220&gt; &lt;223&gt; Description of the artificial sequence: oligodeoxyribonucleotide</li><li>&lt;400&gt; 1 actgtgcttc ttgacccatg gcagaagcgc cttc 34</li><li>&lt;210&gt; 2 &lt;211&gt; 34 &lt;212&gt; DNA &lt;213&gt; Artificial sequence</li><li>&lt;220&gt; &lt;223&gt; Description of the artificial sequence: oligodeoxyribonucleotide</li><li>&lt;400&gt; 2 ccttcactgt ctgcctaact ccttcgtgtg ttcc PNTo &lt;0: 3&gt;, PNTe &lt;0: 3&gt; 34</li></ul>