Magnetic particles for purifying nucleic acids

Details Magnetic particles for purifying nucleic acids Magnetic particles for purifying nucleic acids

2002-01-09

2003-04-08

Ketter, James (Department: 1636)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C106S426000, C428S402000, C428S404000, C428S406000

Reexamination Certificate

active

06545143

ABSTRACT:

The application concerns a preparation of particles having a glass surface, a process for producing such a preparation and a process for purifying nucleic acids with the aid of this preparation.
Nucleic acids have recently become more and more the focus of interest for medical diagnostics. Numerous detection methods have now been developed in which the presence or absence of certain nucleic acids is used as an indication for a disease. These include for example tests for infectious organisms e.g. for viruses or bacteria in body fluids and also the detection of mutations in genomic nucleic acids e.g. in oncology. However, nucleic acids are usually present at very low concentrations in the sample material. Hence various methods have been developed for isolating nucleic acids from other sample components such as proteins or other cellular components some of which interfere with the subsequent detection methods. Some of these methods utilize capture probes bound to solid phases that can hybridize with the nucleic acids to be separated and retain these on the solid phase while the other sample components are removed. Such a method is described for example in EP-B-0 305 399. However, a disadvantage of these methods is that they are each only suitable for purifying nucleic acids having a very special nucleotide sequence.
A process for isolating nucleic acids with the aid of magnetic particles composed of cellulose and iron oxide is described in WO 91/12079 in which the particle size is stated to be between 1 and 10 &mgr;m. These particles do not contain a glass surface and are only suitable for an isolation in which the nucleic acids are precipitated. However, the aggregation process also entraps many sample components which interfere with subsequent process steps.
EP-B-0389 063 proposes a process in which the sample is mixed with a mixture of a chaotropic guanidinium salt and silica particles. Under these conditions the binding of the nucleic acids to the silica surface is relatively independent of the sequence. The other sample components can be removed by washing and the nucleic acids can be subsequently eluted.
Magnetic particles having an essentially pore-free glass surface are described in WO 96/41811 for the sequence-independent purification of nucleic acids. The particles used in this case have a core which preferably contains magnetite as a magnetic material and they preferably have a particle size between 10 and 60 &mgr;m. Magnetite exhibits hard magnetic properties in crystals larger than ca. 30 to 50 nm. Permanent magnetism is induced by an external magnetic field. Particles having such hard magnetic cores have the properties of a small permanent magnet after their first exposure to an external magnetic field. In suspensions such particles attract one another and form larger units. Under the influence of an external field of gravity these large units sediment more rapidly than the individual particles. This is disadvantageous since long periods of incubation require frequent redispersing.
Pigments are described in WO 96/41840 which have a glass surface with a thickness of at least 0.8 &mgr;m. Zinc compounds are also proposed as a glass forming component. Pigment particles are formed in this process having a particle size of preferably 2 to 20 &mgr;m.
It has now turned out that in the previously described processes for the production of particles using a sol-gel process in which core particles having a specified size are coated with a gel and subsequently a compression takes place to form a glass surface, a large proportion of particles are formed which do not contain core particles. Nucleic acid detection methods carried out using such preparations either result in large losses of nucleic acids or the fines have to be laboriously separated in order to increase the yield. The object of the present invention was to completely or partially improve the present state of the art and in particular to produce particles having a relatively narrow particle size distribution and to further increase the yield in nucleic acid purifications, or/and to provide particles for nucleic acid purification which, even after exposure to an external magnetic field, only have a very low tendency to aggregate and sediment in a gravitational field just as slowly as particles that have never been exposed to a magnetic field.
The invention concerns a preparation containing particles having a glass surface wherein more than 75% by weight of these particles have a particle size between 0.5 and 15 &mgr;m.
Further subject matters of the invention are a process for producing a preparation of particles containing a core coated with a gel layer or a glass layer and a process for purifying nucleic acids with the aid of the preparation according to the invention.
A further subject matter of the invention is a process for producing particles and a preparation of particles having a superparamagnetic core.
The invention additionally concerns a process for producing particles and a preparation of particles having a magnetic and preferably a soft magnetic metallic core.
Solid materials having a small diameter are referred to as particles by a person skilled in the art. These particles preferably have an essentially spherical surface. However, platelet-shaped and filamentary particles having the dimensions stated below are also to be understood as particles. In order to be particularly suitable for purifying nucleic acids, it is desirable that the particles have a core (pigment part) which is preferably magnetic and is coated with a layer of glass. Such cores preferably contain metal oxides such as aluminium oxide, iron oxide, chromium oxide, copper oxide, manganese oxide, lead oxide, tin oxide, titanium oxide, zinc oxide and zirconium oxide or metals such as Fe, Cr, Ni or magnetic alloys. The composition of this core is less important for the function of the particles according to the invention since the core is coated with a glass surface and hence the core does not come into direct contact with the sample from which it is intended to isolate the nucleic acid. Such cores are commercially available. If the core contains Fe
3
O
4
(magnetite) or Fe
2
O
3
(maghemite) or Fe or Cr or Ni or magnetic alloys, then these cores are magnetic.
Suitable materials referred to as being soft magnetic are metals based on the pure elements Fe, Ni, Cr and alloys thereof preferably based on Ni. Examples of such alloys are known under the name permalloy. They are composed of 70 to 80% Ni with additives of Cr, Cu and Mo. Particles consisting of magnetically soft material do not attract one another or only to a negligible extent in the absence of an external magnetic field.
Finely-dispersed metal powders are very reactive. There is a risk of self-ignition in air, they are pyrophoric. Hence it was very surprising that such finely dispersed metal particles could be coated with a glass layer by a sol-gel process without significantly changing the magnetic properties. Carbonyl iron powder is particularly preferably used as a metal powder and types thereof that have been reduced in H
2
have particularly favourable magnetic properties. Carbonyl iron whiskers have particularly favourable properties.
Metal powders preferably have a particle size between 10 nm and 100 &mgr;m and particularly preferably between 200 nm and 8 &mgr;m.
A glass surface in the sense of the present invention is composed of an amorphous material containing silicon. In addition to silicon oxide the glass preferably contains one or several of the following components (in mole %):
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%).
A number of other oxides can also be present in small amounts of 0-5% such as e.g. Na
2
O, Mn
2
O
3
, TiO
2
, As
2
O
3
, Fe
2
O
3
, CuO, ZrO
2
, CoO etc. Surfaces having a composition of SiO
2
, B
2
O
3
, Al
2
O
3
, CaO, K
2
O and ZnO have proven to be particularly effective. Boron silicate glasses that are particularly advantageous with regard to the yield

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