Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-07-11
2003-07-08
Siew, Jeffrey (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C436S006000, C436S007000, C436S091000, C436S091000, C436S169000, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330, C526S080000
Reexamination Certificate
active
06590094
ABSTRACT:
The invention relates to crosslinked bead polymers doped with superparamagnetic iron oxide, to a process for the preparation of the bead polymers, and to the use thereof in nucleic acid diagnosis.
So-called genetic diagnosis has become increasingly important recently.
Genetic diagnosis has become involved in the diagnosis of human diseases (inter alia detection of pathogens, detection of genome mutations, discovery of circulating tumour cells and identification of risk factors for predisposition to a disease). However, genetic diagnosis is also now finding applications in veterinary medicine, environmental analysis and food testing. A further area of application comprises investigations in institutes of pathology/cytology or within the framework of forensic problems. However, genetic diagnosis is now employed also for the purposes of quality control (for example investigations of blood samples for freedom from pathogens), and legislation is planned to regulate such tests by law in future. Methods also employed in genetic diagnosis (such as, for example, hybridization and amplification techniques such as PCR, bDNA or NASBA technology) are also among the routine methods in fundamental scientific studies.
An important step in genetic diagnosis is the obtaining of gene samples from biological material such as cells, blood, serum or urine.
EP 0 707 077 describes a method for isolating nucleic acids from biological material using soluble, weakly basic polymer. In this method, a precipitation product is generated from the soluble, weakly basic polymer and the nucleic acid in an acidic pH range, the precipitation product is separated from the unprecipitated constituents of the biological material, and washed, and the nucleic acid is liberated again from the precipitation product by adjusting a basic pH.
One disadvantage of the method in EP 0 707 077 is that the manipulation, in particular the separation and purification of the precipitation product, is difficult and very time consuming. This method can moreover be carried out using automatic analysers only under difficult conditions or not at all.
U.S. Pat. No. 4,339,337 and U.S. Pat. No. 5,356,713 describe methods for preparing magnetic beads of vinylaromatic polymer using magnetic particles. These bead polymers do not, however, contain any functional groups for attaching nucleic acids. In addition, the beads show a marked residual magnetism (remanence), which impedes dispersability thereof.
WO 8303920 describes a method for preparing magnetic polymer particles in which polymer particles are treated with solutions of, for example, iron salt, the iron being precipitated in the form of iron hydroxide. In this method, the precipitated iron compound is present both in the polymer particles and on the surface of the polymer particles. The iron compound on the surface may interfere with some applications, for example amplification of nucleic acids by Taqman PCR.
U.S. Pat. No. 5,206,159 discloses a process for preparing superparamagnetic polyacrylamide carriers. These carriers are, however, unsuitable for removing nucleic acids.
U.S. Pat. No. 5,705,628 discloses a method for binding DNA to magnetic microparticles. The magnetic microparticles preferably have a particle size of 1 &mgr;m and have a surface coated with carboxyl groups. In order to achieve binding of the DNA to the particles it is necessary to use a specific salt concentration and add polyethylene glycol in a defined concentration and with specific molecular weight.
It has now been found that certain crosslinked bead polymers doped with superparamagnetic iron oxide and containing basic amino groups are outstandingly suitable for direct and automated isolation of nucleic acids.
The invention relates to crosslinked bead polymers doped with superparamagnetic iron oxide and containing basic amino groups, which are characterized in that the bead polymers contain copolymerized units of hydrophilic (meth)acrylate and amino (meth)acrylates.
The term (meth)acrylate means the derivatives of acrylic acid and methacrylic acid.
Hydrophilic (meth)acrylates are those whose homopolymers have a solubility of more than 2.5% in water at 25° C. Examples which may be mentioned are: 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, triethylene glycol monomethacrylate, tetraethylene glycol monomethacrylate, glycerol monomethacrylate, acrylamide, methacrylamide and N,N-dimethylacrylamide. Acrylamide is preferred.
Amino (meth)acrylates for the purpose of the present invention are derivatives of acrylic acid and methacrylic acid with, preferably, secondary and tertiary amino groups. The amino groups may also be part of a cycloaliphatic or aromatic ring. Suitable amino (meth)acrylates are, for example, N-(3-aminopropyl)methacrylamide, N-(3-imidazolylpropyl)methacrylamide, N-(2-imidazolylethyl)methacrylamide, N-(3-aminopropyl)acrylamide, N-(3-imidazolylpropyl)acrylamide, N-(2-imidazolylethyl)acrylamide, N-(1,1-dimethyl-3-imidazolylpropyl)methacrylamide, N-(1,1-dimethyl-3-imidazoylpropyl)acrylamide, N-(3-benzimidazolylpropyl)-methacrylamide and (3-benzimidazolylpropyl)acrylamide. Preferred amino (meth)acrylates are aminoalkyl (meth)acrylates such as, for example, N,N-dimethylaminoethyl methacrylate, N,N-dimethylaminopropyl methacrylate, N,N-dimethylaminoethyl acrylate, and N-tert-butylaminopropyl methacrylate. N,N-Dimethylaminoethyl methacrylate and N,N-dimethylaminopropyl methacrylate are particularly preferred. The amino groups in the bead polymers according to the invention may be wholly or partly in protonated form, for example as hydrochlorides.
Suitable crosslinkers are: ethylene glycol dimethacrylate, butanediol dimethacrylate, hexanediol dimethacrylate, pentaerythritol dimethacrylate, glycerol 1,2-dimethacrylate, glycerol 1,3-dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol trimethacrylate, pentaerythritol tetramethacrylate, ethylene glycol diacrylate, butanediol diacrylate, pentaerythritol diacrylate, glycerol 1,3-diacrylate, triethylene glycol diacrylate, trimethylolpropane triacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, allyl methacrylate, allyl acrylate, diethylene glycol divinyl ether and methylene-N,N′-bisacrylamide. Methylene-N,N′-bisacrylamide is preferred.
The amount of hydrophilic (meth)acrylate is 30 to 89% by weight, preferably 40 to 75% by weight, the amount of amino (meth)acrylates is 10 to 69% by weight, preferably 20 to 50% by weight, and the amount of crosslinker is 1 to 25% by weight, in each case based on the total of hydrophilic (meth)acrylate, amino (meth)acrylates and crosslinker.
The content of iron oxide in the superparamagnetic bead polymers according to the invention is 2 to 80% by weight, preferably 4 to 50% by weight, particularly preferably 5 to 35% by weight, based on the weight of the unswollen bead polymers.
The bead polymers according to the invention are superparamagnetic, that is to say they have a low residual magnetization (remanence) and a small coercivity. Their magnetic saturation is high, and they are strongly attracted by an inhomogeneous magnetic field. After the magnetic field has been switched off, they can be dispersed easily and completely in water or aqueous buffer solutions.
The particle size of these superparamagnetic bead polymers according to the invention is 1 to 200 &mgr;m, preferably 5 to 100, particularly preferably 10 to 50 &mgr;m. Microscopic image analysis is very suitable for determining the average particle size (Ø) and the particle size distribution.
As a measure of the breadth of the particle size distribution of the bead polymers, the ratio is formed from the average of the volume distribution (D
v
) and the average of the number distribution (D
z
). Narrow particle size distributions for the purpose of the invention mean D
v
/D
z
≦2.5, preferably D
v
/D
z
≦2, particularly preferably D
v
/D
z
≦1.5. It has been found that bead polymers accordi
Karlou-Eyrisch Kamelia
Neumann Rainer
Podszun Wolfgang
Bayer Aktiengesellschaft
Greenman Jeffrey M.
Klawitter Andrew L.
Siew Jeffrey
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