DE19854973B4 - Process for the purification of nucleic acids - Google Patents

Abstract

Preparation containing particles with a glass surface, wherein more than 75 wt.% Of these particles have a particle size of between 0.5 and 15 microns, characterized in that the glass surface of SiO ₂ , B ₂ O ₃ , K ₂ O, CaO, Al ₂ O. ₃ and ZnO.

Description

object The application is a preparation of particles with a glass surface, a Process for the preparation of such a preparation and a process for the purification of nucleic acids with the help of this preparation.

Nucleic acids have recently become more and more the focus of interest in medical diagnostics. In the meantime, a large number of detection methods have been developed in which the presence or absence of certain nucleic acids is assessed as an indication of a disease. These include z. B. Evidence of infectious organisms, eg. As viruses or bacteria in body fluids, but also the detection of mutations in genomic nucleic acids, eg. In oncology. However, nucleic acids are present in the commonly used sample material in very low concentrations. For this reason, various methods have been developed for isolating the nucleic acids from other sample constituents, such as proteins or other cellular constituents, which in part disturb the subsequent detection methods. Some of these methods use solid-phase capture probes that can hybridize to the nucleic acids to be separated and retain them on the solid phase while removing the remaining sample components. Such a method is for example in EP-B-0 305 399 described. However, these methods have the disadvantage that they are only suitable for the purification of nucleic acids with a very specific nucleotide sequence.

In WO 91/12079 describes a method for the isolation of nucleic acids with the aid of magnetic particles of cellulose and iron oxide, wherein the particle size is specified with between 1 and 10 microns. These particles contain no glass surface and are only suitable for isolation with precipitation of nucleic acids. Aggregation, however, involves a variety of sample components that interfere with later process steps.

In EP-B-0 389 063 For example, a method is proposed in which the sample is mixed with a mixture of a chaotropic guanidinium salt and silica particles. Under these conditions, nucleic acids bind to the silica surface relatively independently of the sequence. The remaining sample components can be washed off and the nucleic acids subsequently eluted.

In WO 96/41811 respectively. DE 195 20 398 A1 and DE 195 37 985 A1 Magnetic particles are described with a substantially non-porous glass surface for the sequence-independent purification of nucleic acids. The particles used there have a preferred particle size of between 10 and 60 microns.

In WO 96/41840 describes pigments which have a glass surface of a thickness of at least 0.8 microns. As a glass-forming component, compounds of zinc are also proposed. This results in pigment particles having a particle size of preferably 2 to 20 microns.

DE 689 20 778 T2A1 describes magnetically attractable particles and an associated manufacturing method.

DE 43 07 262 A1 involves a magnetic polymeric silica.

EP 0 757 106 A2 discloses nucleic acid-binding magnetic carriers in the form of superparamagnetic silica particles having a specific surface area of about 100 to 800 m ² / g.

GB 2 116 206 A deals with microcarriers with a glass surface for cell cultivation.

It has now been found that at the previously described method for the production of particles after the sol-gel process, at the core particle of a predetermined size are coated with a gel and subsequently a compression to a glass surface takes place, a high Particle is formed of particles containing no core particles. this leads to either to that at Nucleic acid detection methods performed with such preparations size Loss of nucleic acids take place or the fines to increase the yield consuming must be disconnected. Object of the present invention was the present state to improve the technique in whole or in part, especially particles with a relatively narrow particle size distribution and to further increase the yield in the case of nucleic acid purification.

The invention relates to a preparation containing particles with a glass surface, wherein more than 75% by weight of these particles have a particle size of between 0.5 and 15 μm.

Further objects The invention relates to a process for the preparation of a preparation of particles containing a core coated with a gel layer or a glass layer and a method for purifying nucleic acids with Help of the preparation according to the invention.

As a person skilled in the art refers to solid materials with a small diameter. Preferably, these particles have a substantially spherical surface. In order to be particularly suitable for the purification of nucleic acids, it is desirable that the particle has a core (pigment portion), which is preferably magnetic and which is coated with a glass layer. Such cores preferably contain metal oxides such as alumina, iron oxide, chromium oxide, copper oxide, manganese oxide, lead oxide, tin oxide, titanium oxide, zinc oxide and zirconium oxide. The composition of this core is less essential to the particles of the invention because the core is coated with a glass surface so that the core does not come into direct contact with the sample from which the nucleic acid is to be isolated. Such cores are commercially available. If the nucleus contains Fe ₃ O ₄ (magnetite) or Fe ₂ O ₃ (maghemite), these nuclei are magnetic.

A glass surface according to the present invention consists of a silicon-containing amorphous material. The glass preferably contains, in addition to silicon oxide, one or more of the following components (in mol%):
B ₂ O ₃ (0-30%), Al ₂ O ₃ (0-20%), CaO (0-20%), BaO (0-10%), K ₂ O (0-20%), Na ₂ O (0-20%), MgO (0-18%), Pb ₂ O ₃ (0-15%), ZnO (0-6%).

To a lesser extent from 0-5% can also be a variety of other oxides such. As Na ₂ O, Mn ₂ O ₃ , TiO ₂ , As ₂ O ₃ , Fe ₂ O ₃ , CuO, ZrO ₂ , CoO, etc. may be included. Surfaces of a composition of SiO ₂ , B ₂ O ₃ , Al ₂ O ₃ , CaO, K ₂ O, and ZnO have proved to be particularly effective. Particularly preferred borosilicate glasses from the viewpoint of the yield of nucleic acids have a zinc oxide content of 2-6, preferably about 4 mol%. Particularly preferably, the glass layer consists of 68-79 mol% SoO ₂ , 15-5 mol% B ₂ O ₃ , 6-2.5 mol% total amount of K ₂ O and Na ₂ O, 4-1 mol% CaO, 8-2 mol % Al ₂ O ₃ , 6-2 mol% ZnO. Particularly preferred according to the invention are glasses which are formed by the so-called gel-sol process and subsequent drying and compression of the layer formed. This process is known in its basic features and was z. In CJ Brinker, GW Scherer "Sol Gel Science - The Physics and Chemistry of Sol Gel Processing", Academic Press Inc. 1990 and Sol-Gel Optics, Processing and Applications Lisa C. Klein Ed. Kluwer Academic Publishers 1994, page 450 ff. As well as in DE-A-1941191 . DE-A-3719339 . DE-A-4117041 and DE-A-4217432 described. In the gel sol process alkoxides of network-forming components, eg. As SiO ₂ , B ₂ O ₃ , Al ₂ O ₃ , TiO ₂ , ZrO ₂ and ZnO together with oxides and salts of other components, eg. B. in alcoholic solution, submitted and hydrolyzed.

By the addition of water is the hydrolysis process of the starting components in Gear set. The reaction is ongoing relatively quickly, since the alkali metal ions catalytically on the hydrolysis rate of the silicic acid ester act. After gelation, the resulting gel can be dried and compacted by a thermal process into a glass become.

The ratio Sol / pigment has a significant influence on the yield of magnetic according to the invention Pigment. Limits are given by the fact that the pigment content is so low is that one more pumpable and sprayable mass arises. If the pigment content is too low, the fine fraction, eg. B. of non-magnetic material too big and disturbs. As for the Pigment yield appropriate proportions 10 to 45 g of pigment / 100 ml of sol were found.

The slurry is sprayed to form a powder preferably through a nozzle and the aerosol dried on a fall course. The nozzle is preferably heated, to the drying of the slurry to accelerate. Dependent from the geometry of the nozzle is the nozzle temperature preferably about 120 to 250 ° C. A compromise will be found by sufficient evaporation rate, however Avoid splattering.

With regard to 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. However, addition of zinc in the layer surprisingly increases the melting point, so that a higher compression temperature (between 710 and 800 ° C) is possible. The aftertreatment under air leads to a loss of magnetic properties at high temperatures, which is why too high temperatures should be avoided. Again, other temperatures are possible with the addition of zinc (preferred between 150 and 250 ° C).

to Preparation of the preparation according to the invention becomes a preparation of core particles in which more than 75% by weight the core particles have a grain size of between slightly less than 0.5 and slightly less than 15 microns have been used in the sol / gel process. The core particles must to be so much smaller than the glass coated particles, like the Thickness of the glass layer makes up. The glass layer is prepared by the process according to the invention between 5 nm and 1 μm be great dependent from the chosen one circumstances, like relationship Gel to core particles. On average, the glass layer between 0.2 and 0.3 μm be fat.

Especially preferred is a preparation containing particles with a glass surface wherein more than 75% by weight of these particles have a particle size of between 2 and 15 μm to have. Particularly preferred is the proportion of the particles with the particular Grain size greater than 90% by weight.

Especially Preferably, magnetic core particles are used. The preparation according to the invention has the advantage that preferred more than 95 wt.% Of the particles having a particle size of between 0.5 and 15 microns, preferably between 2 and 15 μm are magnetic. This means that over the known methods the proportion of non-nucleated particles is drastically reduced is. This can be seen from this, only a few non-magnetic particles are included. this leads to to that it practically no longer necessary, the formed non-magnetic Separate particles from the magnetic particles before preparing used in methods for purifying nucleic acids. This means a simplification in the manufacturing process.

In addition, can the preparation according to the invention characterized in that preferably less than 50% the particle has a grain size of less than 2 μm to have. This has the consequence that the non-magnetic Fine fraction, which has a high relative proportion for small grain sizes which is greatly reduced. Especially preferred are less than 2% of the particles have a particle size of less than 0.5 μm.

Prefers are not more than 10%, more preferably between 10 and 40% the particle of the preparation particles with a grain size of more than 10 μm.

The inventive preparation can in addition to the particles of the invention contain other non-glass containing ingredients such. B. Buffer substances or a suspending agent, for. As water or alcoholic solutions of water.

The Glass layer of the particles of the preparation according to the invention preferably contains a proportion of between 2 and 6 mol%, particularly preferably 4 mol% Zinc oxide. This can be achieved by the proportion of zinc oxide the solid mass of the sol, compared with the proportions of the other solid Components, in this order of magnitude lies. The proportion of zinc oxide increases with the reduction of Proportion of boron oxide, especially during prolonged heating, since boron oxide already volatile under the conditions of manufacture.

Particle, which have a glass layer in which the proportion of zinc oxide between 2 and 6 mol% have been found to be particularly effective in cleaning of nucleic acids exposed. The yield of nucleic acids could be compared with the same glass layer without zinc oxide, partially increased by 50%.

Also The invention relates to a process for the preparation of a Preparation of particles with a core coated with a gel layer containing less than 5% by weight of coreless particles containing the Steps suspending core particles in a sol using a core particle preparation and spray-drying the suspension below Gelation, wherein the core particle preparation to 75 wt.% Of particles with a grain size of between 0.5 and 15 μm, preferably between 2 and 15 microns consists.

To carry out the gel / sol process, which uses the production method according to the invention, reference is made to the description in the prior art. The essential difference of the invention to the above is the use of a particular core particle preparation by which a formulation with less than 5% by weight of coreless particles can be made. A process has proven particularly advantageous in which a sol of tetraalkyl orthosilicates, alkyl borates, aluminum alcoholates and alkali alcoholates is first prepared in ethanol and this mixture is heated with calcium. Subsequently, the mixture is hydrolyzed by the addition of water. The core particles are added in solid form to the sol thus formed and suspended, preferably with ultrasound. Subsequently, the suspension is subjected to gelation in a spray-drying process in which the nozzle is heated, and in the substantially Particles are formed in which 1 to only a few core particles per particle are contained (preferably less than 1% of the particles contain more than 10 core particles), sprayed. The spray product is then heated to densify the gel into a glass. Again, the addition of zinc oxide to the gel has a significant advantage. The compaction can be carried out at higher temperatures than those without zinc addition, since the softening point of the resulting glass is higher. As a result, organic residual constituents from the materials used can be driven out more easily.

There according to the inventive method a preparation with a very low content of coreless particles generally, there is no subsequent fractionation after coreless / nucleated Particles more needed.

Also The invention relates to a method for purifying nucleic acids by non-covalent Binding of the nucleic acids from a sample of particles with a glass surface, removing unbound Sample components and elution of the bound nucleic acids of the glass surface, where a preparation according to the invention is used. The process becomes particularly easy when the particles are magnetic.

Methods for purifying nucleic acids using magnetic particles having a glass surface are disclosed in U.S. Pat WO 96/41811 described. This disclosure is here fully incorporated by reference. In particular, clinical samples, such as blood, serum, mouthwash, urine, cerebral fluid, sputum, stool, plasma, punctates or bone marrow samples come into question as samples for the purification method according to the invention. Preferred sample material is serum. To clean the nucleic acids, the sample, if necessary, after lysis of any cellular structures containing and digestion of interfering sample components, with the preparation of the invention, for. B. in the form of a certain amount of a suspension of the particles. After an incubation period during which nucleic acids bind sequence-nonspecifically to the glass surface, the liquid is removed along with the unbound sample components and, if desired, the particles are washed to remove residues. The still bound nucleic acids are removed from the surface by elution with a liquid in which the nucleic acids dissolve well. The resulting liquid can now be processed as desired, in particular be used in amplification, such. As the PCR, since most enzyme inhibitors were separated during the purification process.

Provided the particles are magnetic, is the removal of the liquid of particles with the nucleic acids especially easy as the particles are collected with the help of a magnet and can be held while the liquid Will get removed. If the particles are not magnetic, they can from the liquid be separated by filtration with a suitable filter.

The The present invention is further illustrated by the following examples.

example 1

Sol for producing a zinc-free layer (74 SiO ₂ × 15 B ₂ O ₃ × 4 K ₂ O × 2 CaO × 5 Al ₂ O ₃ )

1750 ml of tetraethyl orthosilicate (manufacturer: Wacker, Burghausen) are placed in a 5 liter round bottom flask and rapidly added at room temperature with stirring (500 rpm):
541 ml of triethyl borate (manufacturer: Aldrich, Steinheim)
250 ml of potassium methoxide (25% strength in methanol (manufacturer: Fluka, Deisenhofen))
261 g of aluminum sec-butylate (manufacturer: Aldrich, Steinheim)
292 ml of ethanol and
8.49 g of calcium (manufacturer: Fluka, Deisenhofen)

The Mixture will follow with stirring heated to high reflux. About 30 minutes is a mixture of a total of 583 ml of ethanol and 233 ml of water dropwise. After cooling at <50 ° C will that Sol transfilled in an open container and 1200 g of pigment IRIODIN 600 Black Mica (manufacturer: Merck, Darmstadt) added. The sol is still 1 after complete addition of pigment Stirred at 500 rev / min and subsequently Sonicated for 5 minutes. After the ultrasound treatment the sol-pigment mixture is added with a dissolver stirrer stirred at about 500 rpm, until the entire amount is used up.

Example 2

Preparation of glass-coated pigment (MGP)

Spraying takes place in a spray tower from Nubilosa, Constance, with a diameter of 0.75 m, a height of 2.5 m and an evaporation capacity (based on water) of 1-3 liters / hour. The air inlet temperature is 270 ° C, the outlet temperature about 130 ° C. The throughput of air is 7.2 m ³ / min. For spraying a two-fluid nozzle is used with a spray pressure of 2 bar. The capacity of the ball valve diaphragm pump used is 60 g sol / min.

The spray product is trapped in a cyclone, precompressed in air at 250 ° C for 1 hour and subsequently in a nitrogen furnace at a heating rate of 1 K / min to one Temperature of 675 ° C brought, held there for 1 hour and cooled to 300 ° C. At 300 ° C, oxygen is added, Held for 1 hour, then cooled to room temperature. After cooling sieving is done with a sieve with a mesh size of 50 μm for removal of any existing aggregates. This is the production completed.

Example 3

Sol for producing a zinc-containing layer (70.67 SiO ₂ × 14.33 B ₂ O ₃ × 4 K ₂ O × 2 CaO × 5 Al ₂ O ₃ × 4 ZnO)

In the same manner as in Example 1, a zinc-containing sol is prepared. For this purpose, the following adducts are weighed and treated analogously:
1258 ml tetraethyl orthosilicate (manufacturer: Wacker, Burghausen)
387 ml of triethylborate (manufacturer: Aldrich, Steinheim)
188 ml potassium methanolate 25% in methanol (manufacturer: Fluka, Deisenhofen)
196 g of aluminum sec-butylate (manufacturer: Aldrich, Steinheim)
1285 ml of ethanol
6.39 g calcium (manufacturer: Fluka, Deisenhofen)
58.5 g of zinc acetate dehydrated dihydrate (manufacturer: Fluka, Deisenhofen)

After boiling under reflux, 178 ml of H ₂ O are added dropwise together with 444 ml of ethanol within 30 minutes. After cooling, 1200 g of pigment are added. Otherwise see example 1.

Example 4

Production of zinc-containing glass-coated pigment

The Pigment-containing sol from Example 3 is processed analogously to Example 2. The compression temperature is however 750 ° C.

Example 5

Production of zinc-containing glass-coated Modified post-treatment pigment (MGP)

The Pigment-containing sol from Example 3 is processed analogously to Example 2. The compression temperature is however 750 ° C and the temperature in the treatment in oxygen is 200 ° C.

Example 6

Determination of the yield of DNA or RNA with radioactive ³² P

For the direct detection of bound or non-bound DNA or RNA, ³² P-labeled HIVgag RNA standard with 1.4 kb or ³² P-labeled lambda amplicons with 3 kb are used. The sample used is negative plasma (human) containing 10 ⁹ copies each.

Carrying out the sample preparation

500 μl of negative plasma with 10 ⁹ copies of ³⁷ P labeled lambda amplicons are placed in a 2 ml Eppendorf tube. For this purpose, 480 μl of binding buffer / proteinase K solution (5: 1) are pipetted in, vortexed and at 70 ° C for 10 minutes. After cooling to room temperature, 400 μl of isopropanolic MGP suspension with a total content of 3 mg of MGP are pipetted in. Immediately afterwards it is mixed by vortexing. The sample is then left on a mixer for 15 minutes, e.g. B. Thermomixer 5436 from Eppendorf, incubated.

The MGP will be replaced by the transfer of Sample concentrated in a magnetic separator. After 1 minute will be the supernatant Completely pipetted.

5 ml of washing buffer are pipetted to the MGPs. The sample is vortexed and then transferred to the magnetic separator. The supernatant is abpipttiert after 1 minute. The washing procedure is repeated 2 more times.

To 200 ml of elution buffer are added to the MGP. At 80 ° C becomes 10 Minutes incubated on a thermo-mixer at 1400 RPM. The sample is transferred to the magnetic separator and after 1 minute, the entire eluate is removed. The eluate will then transferred to a new vessel and in a scintillation counter measured.

Out the relationship the radioactivity of the eluate to the radioactivity the sample before the cleaning procedure, the yield can be determined become.

Results with MGPs of different coating: Yield DNA Yield RNA MGP according to Example 2 (without zinc) 44% 44% MGP according to Example 4 (with zinc) 62% 59% MGP according to Example 6 (with zinc, modified aftertreatment) 66% 70%

Example 7

Black mica as a pigment base

According to example 1, a batch is made using the pigment Black Mica (BM) is.

Example 8

Black mica as a pigment base

According to example 1, a batch is made in which the pigment MMB (Microna Matte Black (manufacturer: Merck, Darmstadt)) is.

Example 9

Signal level after amplification during sample preparation with MGP on different pigment basis

MGP according to Examples 7 and 8 is used in a sample preparation. The sample is human plasma with 100 copies / ml of HCV virus. The sample preparation eluate is subjected to amplification and the amplification result is detected by an electroluminescent method. In another experiment, the sample was human plasma with 600 copies / ml of HBV virus. HCV ECL Counts HBV ECL Counts MGP according to example 7 (BM) 97000 25000 MGP according to example 8 (MMB) 127000 43000

Claims (13)

Preparation containing particles with a glass surface, wherein more than 75 wt.% Of these particles have a particle size of between 0.5 and 15 microns, characterized in that the glass surface of SiO ₂ , B ₂ O ₃ , K ₂ O, CaO, Al ₂ O. ₃ and ZnO. Preparation according to claim 1 characterized in that more as 95 wt.% Of the particles having a particle size of between 0.5 and 15 microns magnetic are. Preparation according to claim 1 or 2, characterized in that less than half of Particle a grain size of less than 2 μm to have. Preparation according to one of Claims 1, 2 and 3, characterized that less than 2% of the particles have a particle size of less than 0.5 μm to have. Preparation according to claim 2, characterized in that the magnetic particles having a magnetic core with Glass is sheathed. Preparation according to claim 1, characterized in that not more than 10% of these particles have particles with a grain size of more than 10 μm are. Preparation according to one of the preceding claims, characterized in that the Particles have a glass jacket, a proportion of between Contains 2 and 6 mol% of zinc oxide. A process for the preparation of a preparation of particles having a core coated with a gel layer containing less than 5% by weight of coreless particles and wherein the gel layer is composed of SiO ₂ , B ₂ O ₃ , K ₂ O, CaO, Al ₂ O ₃ and ZnO comprising the steps of - suspending core particles in a sol using a core particle preparation - spray-drying the suspension to gelation, characterized in that the core particle preparation consists of 75% by weight of particles having a particle size of between 0.5 and 15 μm. Process for purifying nucleic acids Non-covalent binding of nucleic acids from a sample to particles with a glass surface containing ZnO, removing unbound sample components and Elution of the bound nucleic acids from the glass surface. Method according to claim 9, characterized in that the Sample with a preparation according to a the claims 1 to 7 is brought into contact. Method according to one the claims 9 or 10, characterized in that the particles are magnetic and held by a magnet when the sample components are removed become. Use of zinc oxide in the sol / gel process Glass layers to increase the binding ability the glass surface for nucleic acids. A process for the preparation of a preparation of particles having a core coated with a glass layer containing less than 5 wt.% Of coreless particles and wherein the glass surface is composed of SiO ₂ , B ₂ O ₃ , K ₂ O, CaO, Al ₂ O ₃ and ZnO comprising the steps of suspending core particles in a sol using a core particle preparation, spray-drying the suspension under gelation, compacting the gel into the glass, characterized in that the core particle preparation consists of 75% by weight of particles having a particle size of between 0.5 and 15 μm ,