HK1051048B - Magnetic pigment - Google Patents

Description

The invention relates to methods for the isolation of nucleic acids using particles with a surface of silicon-containing amorphous material in the presence of chaotropic salts.

Some biological materials, particularly nucleic acids, have special requirements for their isolation from the natural environment: on the one hand, they are often present in very low concentrations and, on the other, they are often in the vicinity of many other solid and soluble substances, which affect their isolation or determination.

Recently, therefore, there has been no shortage of attempts to propose methods and materials for isolating nucleic acids from their natural environment.

In Anal. Biochem. 121, 382 - 387 (1982) the purification of plasmid DNA from bacteria from glass dust in the presence of sodium perchlorate is described.

DE-A 37 34 442 describes the isolation of single-stranded M13 phage DNA in glass fibre filters by precipitation of the phage particles with acetic acid and lysis of the phage particles with perchlorate. The nucleic acids bound to the glass fibre filters are eluted in Tris/EDTA buffer after washing with a methanol-containing buffer.

The methods described above are similar to the state-of-the-art methods of selective binding of nucleic acids to glass surfaces in chaotropic saline solutions, whereby the nucleic acid is separated from impurities such as agarose, proteins or cell debris.

Analytical Biochemistry 201, 166 - 169 (1992) and PCT GB 91/00212 respectively describe the use of magnetic particles to immobilize nucleic acids after precipitation by the addition of salt and ethanol. This involves agglutination of the nucleic acids 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 trisspuffer.

US-A-4,233,169 describes a porous glass containing magnetic particles in storage.

The so-called magnetic porous glass, which contains magnetic particles in a porous, particulate glass matrix and is coated with a streptavidin layer on the surface, is also currently on the market and can be used to isolate biological materials, such as proteins or nucleic acids, if they are modified in an elaborate preparation step to bind covalently to biotin.

The purpose of the invention was to provide a simple method for the isolation of nucleic acids suitable for routine diagnosis.

The object of the invention is the isolation of nucleic acids by the use of magnetic particles with an external glass surface in the presence of chaotropic salts.

The term "particle" is used to describe solid materials with a small diameter. Sometimes such particles are also called pigments. For the purposes of the present invention, particles with an average grain size of less than 100 μm are particularly suitable. They are particularly preferred for having an average grain size of between 10 and 60 μm. The grain size distribution is relatively homogeneous, in particular there are almost no particles < 10 μm or > 60 μm.

Magnetic materials are materials that can be attracted by a magnet, i.e. ferromagnetic or superparamagnetic materials. Magnetic materials are also materials that are called soft magnetic materials, e.g. ferrite.

An outer surface of a particle is the contiguous surface from which vertical lines can be formed in the direction of the particle's surroundings, without cutting the same particle again.

A pore is a gap in the outer surface of the particle, where the surface reaches so far into the particle that an imaginary vertical line formed in the gap on the surface cuts the particle at least once in the direction of the particle's nearest surroundings.

The glass, as defined in the present invention, is an amorphous material containing silicon.

	(0 - 30 %),
	(0 - 20 %),
CaO	(0 - 20 %),
BaO	(0 - 10 %),
	(0 - 20 %),
	(0 - 20 %),
MgO	(0 - 18 %),
	(0 - 15 %).

A variety of other oxides, such as Mn2O3, TiO2, As2O3, Fe2O3, CuO, CoO, etc., may also be present in a smaller proportion than 0-5%. Surfaces composed of borosilicate glass, flint glass or silica have been shown to be particularly effective. Borosilicate glasses particularly suitable for the yield of nucleic acids have a borosilicate content of more than 25%; a glass composed of SiO2/B2O3 70/30 has been identified as particularly valuable.Err1:Expecting ',' delimiter: line 1 column 77 (char 76)The equation shows the production of a sodium boron aluminium silicate glass.

The reaction is relatively fast, as the alkalions catalytically affect the rate of hydrolysis of the silica ester. After the gel formation, the resulting gel can be dried and condensed into a glass by a thermal process.

The ratio of sol to pigment has a considerable influence on the yield of magnetic pigment. Limits are given by the fact that the pigment content is so low that a mass that can still be pumped and sprayed is produced. If the pigment content is too low, the fine content, e.g. of non-magnetic material, becomes too large and disturbing.

The sludge is preferably sprayed through a nozzle to form a powder and the aerosol is dried on a dropline. The nozzle is preferably heated to speed up the drying of the sludge. Depending on the geometry of the nozzle, the nozzle temperature is preferably about 120 to 200°C. A compromise is found by sufficient evaporation rate, but avoiding spraying.

The compression temperature should be as high as possible in terms of yield, but if it is too high, the particles will stick together and aggregates will form which should be sifted out.

The method of the invention may be used with magnetic particles with an outer glass surface which is essentially pore-free (S. 3, S. 1-2).

A surface which is essentially pore-free is defined as one which is less than 5%, preferably less than 2%, preferably less than 0.1% permeated by pores as defined above; if pores are present, they are preferably less than 10 nm in diameter, preferably 1 nm.

For the purposes of the invention, particles may also be used which contain a core of TiO2-coated glue and magnetite particles immobilized on it, the composite material thus formed being enclosed by the glass layer. Both the core and the magnetite particles are crystalline and non-porous. The spaces on the surface of the glue which are not occupied by the magnetite particles are covered by a thicker glass layer than the tips of the magnetite particles, resulting in a substantially non-porous glass surface.

The non-porousness of the magnetic particles refers only to the outer surface, not to the inner surface of the particle, so that the particle may be porous in its interior if the surface is only surrounded by essentially porous glass or a glass surface with pores of less than 10 nm in diameter.

Surprisingly, the method according to the present invention is particularly advantageous for the isolation of nucleic acids from samples. In particular, long nucleic acids are very little or not destroyed when immobilized by the magnetic particles. The material of the nucleus is also a natural resource and therefore not environmentally sensitive.

The use of ferromagnetic particles with a glass surface is preferred. The state of the art describes superparamagnetic particles. It has now been shown that ferromagnetic particles, when coated with a glass surface, have considerable advantages in isolating nucleic acids. As long as the ferromagnetic particles have not been exposed to a magnetic field, they only sediment under the influence of gravity. They are easily and quickly resuspended by shaking. The process of decomposition without magnetic field influence is preferably longer than the immobilization of nucleic acids on their surface.

The glass surface of ferromagnetic particles may be porous or porous. For the reasons mentioned above, it is preferable that the outer surface of the ferromagnetic particle is also essentially porous or has pores of less than 10 nm in diameter. The ferromagnetic particles have a preferred grain size between 10 and 60 μm, preferably 20 and 50 μm. Particularly preferred are particles, for particles where pores may be present on the surface, with a diameter of less than 10 μm, preferably 1 nm. An example of a ferromagnetic particle is the above-mentioned composite of glimmer and glass particles, surrounded by a glass particle.

The object of the invention is a method for the isolation of nucleic acids from a sample containing the nucleic acids, e.g. DNA or RNA.

Samples within the meaning of the invention include, for example, clinical samples such as blood, serum, mouthwash, urine, cerebrospinal fluid, sputum, stool, sputum and bone marrow samples, or samples from environmental analysis, food analysis or molecular biology research, such as bacterial cultures, phagenlysates and products of amplification techniques such as PCR.

The invention uses magnetic particles with an inner core on which the outer glass surface is applied. The core may be a composite material, but it may also be simple iron cores. The core may also consist of a crystalline or ceramic or glass-like structure in which iron oxide is deposited.

The method described above allows the isolation of native nucleic acids. Native nucleic acids are those whose structure has not been irreversibly altered compared to that of naturally occurring nucleic acids. This does not, however, preclude the modification of other components of the sample.

Modified biological materials include materials that do not occur in nature, such as nucleic acids modified by attachment of reactive, detectable or immobilizable groups, such as biotinyl nucleic acids.

In certain cases, the sample may be used in the isolation process of the invention without pre-treatment. In many cases, however, the sample should be opened by a suitable method and the nucleic acid contained in the sample released. The methods of separation of samples are known to the expert and may be chemical, enzymatic or physical in nature. A combination of these methods is also possible. Lysis by ultrasound, high pressure or shearing, by alkali, detergents or chaotrope saline solutions, or by action of proteinases or lipases, are examples.

The sample may contain other constituents in addition to the nucleic acids to be isolated, e.g. cell debris, proteins, salts and other substances not to be isolated in a liquid. This sample, which preferably contains the nucleic acids in their native form, is brought into contact with the particles under conditions where the nucleic acid binds to the particle surface. The conditions are known in principle. They also depend on the type of bond by which the nucleic acids are bound to the surface. In the present invention, nucleic acids in their n-modifying form are bound directly to the glass surface. A chaotropic chaining of the nucleic acid is achieved. This reduces the binding of the nucleic acids to the glass particle before the process. This technique is performed in a concentrated state, but it is not expected to be between 4 and 6 mol/mol of sodium guanothiazide or sodium bicarbonate.

To bring the sample into contact with the particles, the sample is mixed with the particles and incubated for a period of time sufficient for binding. The incubation period is usually known to the professional from the treatment with non-magnetic particles, and can be optimized by determining the amount of immobilized nucleic acids on the surface at different times.

Depending on the size and type of the magnetic particles, the particles are separated from the liquid during the incubation period or the suspension is preserved for a longer period. If the particles have a very small grain size and are superparamagnetic, the suspension is preserved for a longer period. If the particles are larger in size, a slow separation of the particles from the liquid takes place during the incubation.

Immobilization is preferably not by precipitation by reducing the solubility of the nucleic acids to be immobilized.

After incubation, the bound 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 pulled to the wall of the vessel in which the incubation took place. Then the liquid with the sample ingredients that have not been bound to the magnetic particles can be removed. This distance depends on the type of vessel in which the incubation took place.

The washing solution is chosen in such a way that the bound nucleic acids are not removed from the particle surface as far as possible, but the impurities which cannot be isolated are washed away as far as possible. This washing step is preferably carried out by incubating the washing solution with the particles, preferably resuspension of the particles, e.g. by shaking or applying a magnetic field which is not identical to the first magnetic field.

After the final washing step, a short drying step of the magnetic particles can be carried out in vacuum or by evaporation (letting) of the liquid, with the possibility of pre-treatment with acetone.

The purified nucleic acids can be removed from the magnetic particles if desired. This step also depends on the type of binding of the nucleic acids to the magnetic particles. In the case of native nucleic acids and glass-coated magnetic particles, the nucleic acid can be removed from the particles of the invention by means of an elution buffer with a low salt content. Such buffers are known from DE 3724442 and Analytical Biochemistry 175, 196-201 (1988).

In another embodiment, the purification and isolation procedure described may be carried out after an immunomagnetic separation of cells (e.g. viral particles or prokaryotic or eukaryotic cells) from a body fluid or tissue. The sample is then incubated with magnetic particles to which an antibody against an antigen is immobilized on the cell, e.g. by shaking. Such particles may be particles of the invention, but also be purchased (e.g. MACSyi Microbeads of the company Salten Biotec GmbH, Bergisch Gladbach, BRD). A magnetic field is applied or a solution of saline is obtained in several steps.

The above described isolation of cells combined with the also described isolation of nucleic acids, preferably in their native form, on the magnetic particles of the invention, results in a particularly advantageous method for the isolation of nucleic acids from cell-containing samples, the advantages of which are its possible simplicity (single-tube method), high sensitivity (particularly important in medical microbiology and oncology) and easy automatability.

The nucleic acids isolated by the methods of the present invention can now be used in any way, for example as substrates for various enzymatic reactions, e.g. for sequencing, radioactive or non-radioactive labelling, amplification of one or more of the sequences contained in them, transcription, hybridization with labelled probes, translation or ligation. One advantage of the method of the present invention is that the separation of the nucleic acids from the liquid is very simple.

The method of the present invention allows for a more efficient separation of nucleic acids from impurities. In particular, inhibitors for certain enzymatic reactions can be removed to a particularly good extent. The yield on nucleic acids is comparatively high. No fractionation of long nucleic acids has been observed. Particles that are more rapidly magnetisable are preferred.

Figure 1 shows a schematic isolation of nucleic acids from a cell-containing sample.

The separation of nucleic acids isolated in an agarose gel according to the invention is shown in Figure 2.

The separation of reaction products following the isolation and PCR amplification of the invention is shown in Figure 3.

Figure 4 shows a gel of the results from example 4.

Figure 1 shows a schematic of nucleic acid isolation from a cell-containing sample. The sample (sample) containing cells is pre-treated in a sample-specific manner so that the cells in which the nucleic acids are to be detected are present in an appropriate form. This includes, for example, the addition of reagents, e.g. for the liquefaction of viscous samples, e.g. saliva samples, to samples from which body fluids have been taken. The sample so prepared is placed in a solid phase in a vessel, preferably to a pearly (bead) antibody which can detect and bind the cell.The specificity of the antibody may depend on the specificity of the analytical task to be solved. If the solid phase is the wall of the vessel, the cells are bound directly to the wall. If the solid phase is a bead, 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 impurities that would interfere with the detection of the surrounding medium.The preferred conditions are that the cells are neither dissolved from the solid phase nor destroyed, and then the destruction of the cells, called lysis, occurs.

The lysemixing is carried out by adding the magnetic particles in the preferred embodiment, and after an appropriate exposure time, which can be optimized by loading the surface with nucleic acids, the particles are separated from the surrounding liquid containing additional and undetectable cell components, preferably by applying a magnetic field to the vessel wall by means of a magnet.

To remove any remaining impurities, it is preferable to wash with a liquid selected so that the nucleic acids to be determined do not separate from the glass surface. To remove the nucleic acids from the glass surface, an elution buffer is added, which has reaction conditions under which the nucleic acids dissolve from the glass surface. These are particularly low salinity conditions. Depending on the intended further treatment of the nucleic acids, the liquid can now be separated from the particles and further processed.

The following examples explain the invention in more detail.

Example 1 Manufacture of magnetic particles

Six different salts were used and the production of the salt was carried out according to the following schemes: The following shall be added to the list of active substances: The synthesis was carried out in a 250 ml round flask under constant agitation. + 7 ml of anhydrous uncoated ethanol The total volume of the product shall be calculated as follows: Other The mixture is then mixed in two phases, stirred at room temperature until it becomes monophase, and then added by dripping. + 37,8 ml of trimethyl borate The sol is then kept at 50 °C for 2 hours, after which the sol is added to the solution. The total volume of the product shall be calculated as follows: The following shall be added to the list of active substances: The synthesis was carried out in a 250 ml round flask, stirring continuously.100,5 ml of tetraethyl orthosilicate + 7 ml of anhydrous uncoated ethanol 16, 3 ml 0,15 M + HCl is added Other A two-phase mixture is formed, stirred at room temperature until it becomes a single phase, after which + 25.6 ml trimethylborate is added by drip. Then the sol is kept at 50 °C for 2 hours, after which + 16.3 ml 0.15 M HCl is added. The following shall be added to the list of active substances: The synthesis was carried out in a 250 ml round flask under constant agitation. + 7 ml of anhydrous uncoated ethanol The total volume of the product shall be calculated as follows: Other A two-phase mixture is formed, which is stirred at room temperature until it becomes a one-phase mixture.Then add + 19.4 ml trimethyl borate by drip. Then the sol is kept at 50 °C for 2 hours, after which + 17.5 ml 0.15 M HCl is added. The following shall be added to the list of active substances: The synthesis was carried out in a 250 ml round flask under constant agitation. + 7 ml of anhydrous uncoated ethanol The total volume of the product shall be calculated as follows: Other The mixture is then mixed in two phases, stirred at room temperature until it becomes monophase, and then added by dripping. The following shall be added to the list of active substances: The sol is then kept at 50 °C for 2 hours, after which +16 °C is added.3 ml of 0,15 M HCl The following shall be added to the list of active substances: The synthesis was carried out in a 250 ml round flask under constant agitation. + 7 ml of anhydrous uncoated ethanol 16,3 ml 0.15 M HCl + other Other The mixture is then mixed in two phases, stirred at room temperature until it becomes monophase, and then added by dripping. + 25,6 ml of trimethyl borate Then the sol is kept at 50 °C for 2 hours, after which + 16.3 ml 0.15 M HCl is added. The following shall be added to the list of active substances: The synthesis was carried out in a 250 ml round flask, stirring continuously.100,5 ml of tetraethyl orthosilicate + 7 ml of anhydrous uncoated ethanol The total volume of the product shall be calculated as follows: Other The mixture is then mixed in two phases, stirred at room temperature until it becomes monophase, and then added by dripping. + 25,6 ml of trimethyl borate 5,15 ml of zirconia (IV) proylate, 70% by weight of Lsg in 1-propanol Then the sol is kept at 50 °C for 2 hours, after which + 16.3 ml 0 is added.

After another 2 hours at 50 °C, 22.5 g of black mica were added to 150 ml of brine and then coated with a spray dryer (book 190, mini spray dryer).

The powder obtained by the spray drying process was then subjected to a temperature treatment under 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 the Sol 1 coating, 860 °C for the Sol 2 coating and 800 °C for the other coatings. After the sintering process, the furnace was switched off and the powder cooled to room temperature.

Example 2 Manufacture of GMP1, GMP2, GMP3 and GMP4

GMP1, GMP2, GMP3 and GMP4 are pigments from different production batches obtained from Sol 1 from Example 1 in a process as described in Example 1 under the following conditions:

Alterung des Sols (h) (30°C)	36	36	36	36
Pigmentanteil des Sols (g/100 ml)	5	15	8	20
Luftstrom der Düse (%)	100	100	100	100
Luftdruck (bar)	6	6	6	3
Düsentemperatur (°C)	135	120	130	143
Verdichtungstemperatur (°C)	534	534	534	615
O2-Nachbehandlung (1 Stunde)	(300°C)	(300°C)	300°C)	(400°C)
Ausbeute an Pigment	niedrig	hoch	mittel	hoch
DNA-Ausbeute	niedrig	hoch	hoch	hoch

Example 3 PCR sample preparation from human whole blood with magnetic glass particles Isolation of nucleic acid

Three batches of glass magnetic particles (GMP2 - 4) were submitted in Eppendorf reaction vessels at approximately 10 mg each, the exact weights being given in Table 1, and three-fold determinations were performed.

40 μl of proteinase K (20 mg/ml, made from lyophilisate) were pipeted and immediately mixed with each 200 μl of whole blood dissolved, then 200 μl of binding buffers (6 M guanidin HCl, 10 mM Tris HCl, 10 mM urea, 30 % Triton X-100, pH 4.4), added, mixed and incubated for 10 minutes at 70°C. After adding 200 μl of i-propanol, the sample was mixed on the vortex mixer for 10 seconds, incubated at room temperature for 20 minutes and then mixed again for 10 seconds as before. The magnetic separation was carried out for at least 30 seconds in the magnetic particle separator of Boehringerheim (Id. No. 641 794). The surplus was removed and analysed as described below.

The magnetic particles were mixed for 10 seconds with 500 μl of washing buffer (20 mM NaCl, 10 mM Tris-HCl, pH 7.5 (25°C, 80 % ethanol), washed for 1 minute at room temperature and mixed for 10 seconds, and drawn to the vessel wall with the magnetic particle separator. The coating was removed and discarded. The washing procedure was repeated until the washing coating was colourless (a total of 4 washes). The nucleic acids were then mixed 3 times with 200 μl of elution buffer (10 mM Tris-HCl, pH 8.5) preheated at 70°C for 10 seconds, coated for 10 minutes at room temperature and eluted for 10 minutes.

The Commission shall adopt implementing acts laying down the rules for the application of this Regulation.

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

Analysis of eluate and sample survival

50 μl of eluate or filter tube-treated residues were added to 10 μl of sample buffer and 45 μl of this was separated electrophoretically at 120 V in a 0.8% agarose seal for 90 minutes.

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

Two 5 μl aliquots of eluate were double-checked by ExpandTM Long Template PCR (Boehringer Mannheim, ID No 1681834) with human tPA gene specific primers (expected product length 15 kb). Other

Mix I	pro Ansatz	Mix II	pro Ansatz
dNTP, je 100 mM	1 µl	Expand™ Puffer, 10 x	5 µl
Primer 1, 200 ng/nl	1 µl	Expand™ Polymerase	0,75 µl
Primer 2, 225 ng/µl	1 µl		19,25 µl
	17 µl
	20 µl		25 µl

Mix I is introduced into a thin-walled PCR tube with 5 μl eluate and Mix II is added. The approach is briefly mixed and coated with 30 μl mineral oil. The approaches are amplified in a Perkin-Elmer thermocycler 9600 using the following program: Other

2 Minuten	92°C
10 Sekunden	92°C
30 Sekunden	65°C	10 Zyklen
12 Minuten	68°C
10 Sekunden	92°C
30 Sekunden	65°C	20 Zyklen
12 Minuten + 20 Sekunden pro Zyklus	68°C
7 Minuten	68°C
anschließend	7°C

The 50 μl PCR approaches were replaced with 10 μl sample buffer and 45 μl of this was separated electrophoretically at 120 V in a 0.8% agarose seal for 90 minutes.

Results

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

Analysis of the eluates in the agarose gel (Fig. 2) shows good reproducibility of the yields. 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.

The ExpandTM PCR provides consistently good and specific amplification products with all samples except a few outliers (Table 2). Tabelle 2.

Ergebnisse Expand™ PCR
	15 kb Expand™ PCR			humanes tPA Gen
		1. Eluat		2. Eluat
GMP/2	1	fehlt		+	+
	2	+	+	+	+
	3	+	+	fehlt
GMP/3	1	+	+	+	+
	2	(+)	+	+	+
	3	-	(+)	+	+
GMP/4	1	+	+	+	+
	2	+	+	+	(+)*
	3	+	+	fehlt
K, BM Kontroll DNA

Tabelle 2.

* 3. Eluat

In Fig. 3 a gel with the reaction products after PCR amplification is shown.

Example 4 Binding of DNA length standard to magnetic glass particles 1. preparation of the magnetic glass particles

12 mg of the glass magnetic batch GMP4 were submitted in Eppendorf reaction vessels.

Lyse and binding

In a 1.5 ml Eppendorf vessel containing 12 mg of magnetic glass particles, mix 900 μl of lysis buffer (4.6 M GuSCN, 45 mM Tris, 20 mM EDTA, pH 7.3) and 100 μl of DNA sample using the DNA length standard III of Boehringer Mannheim (catalogue No 528552) for 2 to 10 sec until a homogeneous suspension is obtained.

The magnetic separation shall be carried out for at least 15 sec in a magnetic particle separator.

Washing and drying

Wash the magnetic glass particles twice with a 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, pipet 800 μl of solution, mix for 2 sec, incubate for 1 min at RT, apply the magnetic field and finally pipet the supernatant.

After removal of the acetone, dry the particles for 10 min at 56 °C in the heating unit with the lid open.

4. Elution of DNA

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

The analysis of the eluates

A fifth of the eluate volume was filled with sample buffer and the DNA 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 containing the DNA quantities expected in the samples.

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

The yield of DNA with magnetic glass particles is shown in Table 1 . Other Tabellel.

Ausbeute an DNA-Längenstandard III mit magnetischen Glaspartikeln
Standar Nr.	DNA - Menge im Stan dard [ng]	Helligkeitsintens. Standard (Meßwert) [rel.Einheiten]	Probe Nr	Pigment/ Bead-Typ	Helligkeitsintens. Probe (Meßwert) [rel.Einheiten]	errechnete DNA-Menge auf Gel [ng]	errechnete DNA-Menge in Probe [ng]	Recovery [%]
1	200	65	1	GMP4	45	139	695	69,5
2	175	56	2	GMP4	39	120	600	60,0
3	150	51
4	125	44
5	100	37
6	75	25
7	50	17
8	25	9
9	10	4

The agarose seal used as the basis for the quantitative evaluation is shown in Fig. 4. It is a 1% ethidium bromide-stained agarose seal. Traces 1 to 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.

Traces 11 and 12 correspond to the DNA eluted by the magnetic glass particles using the 200 ng DNA length standard.

The following shall be added:

The following table shows the total number of samples of the product: The following table shows the information to be provided by the Member States: The Commission has also been consulted on the draft directive. The Commission has also adopted a proposal for a Directive on the approximation of the laws of the Member States relating to the labelling of foodstuffs. The Commission has decided to initiate the procedure provided for in Article 93 (2) of the Treaty. The number of employees The following are the types of products: The following is the list of the main components of the test chemical: The following is added to the list of active substances: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: The following information is provided for the purpose of the analysis:

Claims (26)

1. Method for isolating nucleic acids by

   - contacting a sample which contains the nucleic acid in a liquid, with magnetic particles which have a surface made of a silicon-containing amorphous material, in the presence of chaotropic salts under conditions that enable the nucleic acids to directly bind in a native form to the surface and

   - separating the bound nucleic acids from the liquid.
2. Method as claimed in claim 1, characterized in that the particles have an average particle size of less than 100 µm.
3. Method as claimed in claim 1 or 2, characterized in that the particles contain an inner core made of magnetic material to which the outer surface is applied.
4. Method as claimed in claim 3, characterized in that the magnetic material is magnetite (Fe₃O₄) or Fe₂O₃.
5. Method as claimed in one of the claims 1 to 4, characterized in that the magnetic particles have a glass surface.
6. Method as claimed in one of the claims 1 to 5, characterized in that the surface of the magnetic particles optionally contains other materials.
7. Method as claimed in claim 6, characterized in that the other materials are selected from (0 - 30 %) (0 - 20 %) CaO (0 - 20 %) BaO (0 - 10 %) (0 - 20 %) (0 - 20 %) MgO (0 - 18 %) (0 - 15 %)
8. Method as claimed in one of the claims 1 to 7, characterized in that a clinical sample, a sample from the field of environmental analysis, food analysis or molecular biological research is examined.
9. Method as claimed in claim 8, characterized in that a sample selected from blood, serum, mouth rinse liquid, urine, cerebral fluid, sputum, stool, puncture biopsy and bone marrow samples is examined.
10. Method as claimed in claim 8, characterized in that a sample from bacterial cultures, phage lysates or products of amplification procedures is examined.
11. Method as claimed in one of the claims 1 to 10, characterized in that DNA is isolated.
12. Method as claimed in one of the claims 1 to 10, characterized in that RNA is isolated.
13. Method as claimed in one of the claims 1 to 29, characterized in that the sample is used without pre-treatment.
14. Method as claimed in one of the claims 1 to 12, characterized in that the sample is lysed by a suitable method and the nucleic acids contained therein are released.
15. Method as claimed in one of the claims 1 to 14, characterized in that the concentration of the chaotropic salts is 2 - 8 mol/l, preferably 4 - 6 mol/l.
16. Method as claimed in one of the claims 1 to 15, characterized in that the chaotropic salts are selected from sodium iodide, sodium perchlorate, guanidinium thiocyanate, guanidinium isothiocyanate and guanidinium hydrochloride.
17. Method as claimed in one of the claims 1 to 16, characterized in that the sample is incubated with the particles for a period between 10 seconds and 30 minutes.
18. Method as claimed in one of the claims 1 to 17, characterized in that the separation is carried out by separating the nucleic acids bound to the magnetic particles with the aid of a magnetic field.
19. Method as claimed in one of the claims 1 to 18, characterized in that the magnetic particles are washed once or several times with a wash solution.
20. Method as claimed in claim 19, characterized in that the washing step comprises an incubation of the wash solution with the particles during which the particles are resuspended.
21. Method as claimed in claim 20, characterized in that the resuspension is carried out by applying a magnetic field that is not identical to the first magnetic field.
22. Method as claimed in one of the claims 19 to 21, characterized in that a drying step is carried out after the last washing step.
23. Method as claimed in one of the claims 1 to 22, characterized in that the purified nucleic acids are removed from the magnetic particles.
24. Method as claimed in claim 23, characterized in that an elution buffer having a salt content of less than 0.2 mol/l is used.
25. Method as claimed in one of the claims 1 to 24, characterized in that it is carried out as a single-tube procedure.
26. Method as claimed in one of the claims 1 to 25, characterized in that it is carried out in an automated manner.