MXPA98007681A - Methods of isolating biological target materials using silica magnetic particles - Google Patents

Abstract

The present invention provides methods for isolating biological target materials, particularly nucleic acids, such as DNA or RNA or hybrid molecules of DNA and RNA, from other substances in a medium using silica magnetic particles. The methods of the present invention involve forming a complex of the silica magnetic particles and the biological target material in a mixture of the medium and particles, separating the complex from the mixture using external magnetic force, and eluting the biological target material from the complex. The preferred embodiments of magnetic silica particles used in the methods and kits of the present invention are capable of forming a complex with at least 2&mgr;g of biological target material per milligram of particle, and of releasing at least 60%of the material from the complex in the elution step of the method. The methods of the present invention produce isolated biological target material which is substantially free of contaminants, such as metals or macromolecular substances, which can interfere with further processing or analysis, if present.

Description

P T "METHODS OF ISOLATING BIOLOGICAL WHITE MATERIALS USING SILICONE SILICONE PARTICLES" REFERENCE TO RELATED REQUESTS Reference is made to the North American patent application filed simultaneously entitled "Adsorbent Silica in Magnetic Substrate", the application of which is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION The present invention relates to methods for separating or isolating a biological blank material from other substances in a medium to produce an isolated material of sufficient purity for further processing or analysis. The present invention relates particularly to methods for separating or isolating biological target materials, using particles that respond magnetically and are capable of reversibly binding the material. The present invention relates more specifically to methods for separating or isolating biological target materials using at least one magnetically responsive particle comprising silica or a silica derivative, such as a silica gel, which reversibly binds the biological target material thereof.

BACKGROUND OF THE INVENTION Many molecular biological techniques such as reverse transcription, cloning, restriction and sequence analysis involve the processing or analysis of biological materials. These techniques usually require that these materials are essentially free of contaminants capable of interfering with this processing or analysis procedures. These contaminants usually include substances that block or inhibit chemical reactions, (eg, nucleic acid or protein h-ibridizations, enzymatically catalyzed reactions and other types of reactions used in molecular biological techniques), substances that catalyze the degradation or depolymerization of the nucleic acid or other material of biological interest, or substances that provide "background" indicative of the presence in a sample of a quantity of the biological target material of interest, when the nucleic acid is not in fact present in the sample. The contaminants also include macromolecular substances of the medium in vivo or in vitro from which a material of interest is isolated of nucleic acid, macromolecular substances such as enzymes, other types of proteins, polysaccharides or polynucleotides, as well as lower molecular weight substances such as lipids, low molecular weight enzyme inhibitors, or oligonucleotides. Contaminants can also be introduced into a biological target material of chemical substances or other materials used to isolate material from other substances. Common contaminants of the latter type include trace metals, dyes and organic solvents. Obtaining the DNA or RNA sufficiently - free of contaminants for molecular biological applications is complicated by the complex systems where the -DNA or RNA are typically found. These systems, e.g. tissue cells, body fluid cells such as blood, lymph, milk, urine, faeces, semen or the like, cells in culture, agarose or polyacrylamide gels, or solutions where the amplification of the target nucleic acid, typically include significant amounts of contaminants from which the DNA or RNA of interest must be isolated, before being used in the molecular biological process. Conventional protocols for obtaining -ADN or AKN of cells are described in the literature. See, e.g.

Chapter 2 (DNA) and Chapter 4 (DNA) of the article by F. Ausubel et al., editors, Current Protocols in Molecular Biology. iley-Interscience, New York (1993). Conventional DNA isolation protocols usually involve suspending cells in a solution and using enzymes and / or chemicals, gently paralyze the cells, thereby releasing the DNA contained within the cells to the resulting lysate solution. For RNA isolation, conventional lysis and solubilization procedures include measures for the inhibition of ribonucleases and contaminants that are to be separated from RNA including DNA. Many conventional protocols currently in use usually involve the use of phenol or a mixture of organic solvents containing phenol and chloroform to extract additional cellular material such as proteins and lipids from a conventional lysate solution produced as described in the foregoing. The phenol / chloroform extraction step is usually followed - by precipitation of the nucleic acid material remaining in the extracted aqueous phase, adding ethanol to that aqueous phase. The precipitated material typically removed from the solution by centrifugation, and the resulting granule of the precipitated material is allowed to dry before being resuspended in water or a stabilizing solution for further processing or analysis. Conventional nucleic acid isolation procedures have significant drawbacks. Among these inconveniences are the time required for the multiple processing steps necessary in the extractions and hazards of using phenol and chloroform. Phenol causes serious burns during contact- Chloroform is highly volatile, toxic and flammable. Those characteristics require that the phenol be handled and the phenol / chloroform extractions carried out in a smoke hood. Another undesirable characteristic of the phenol / chloroform extractions is that the phenol oxidation products can damage the nucleic acids. Only freshly redistilled phenol can be used effectively, and nucleic acids can not be left in the presence of phenol. Generally also, multi-step procedures are required to isolate the RNA after extraction of the phenol / chloroform. The precipitation of ethanol (or isopropanol) should be used to precipitate the -DNA from an aqueous solution extracted with phenol / chloroform DNA in order to remove residual phenol and chloroform from the DNA. In addition, the precipitation of ethanol (or isopropanol) is required to remove certain amount of the nucleoside triphosphate and select (ie, less than about 30 bases or base pairs) of single or double stranded oligonucleotide contaminants of the DNA. Furthermore, under the best circumstances, these methods produce relatively low yields of the isolated nucleic acid material, and / or isolated nucleic acid material contaminated with impurities. There is a recognized need in the art for methods that are simpler safer and more effective than traditional phenol / chloroform / ethanol precipitation methods for isolating DNA and / or RNA sufficiently for manipulation, using molecular biological methods. Fractionation of DN recovered from cells according to size is required for many molecular biological procedures. This fractionation is typically achieved by agarose electrophoresis or polyacrylamide gel. For analysis or treatment by a molecular biological procedure after fractionation, the DNA in the fraction (s) of interest must be separated from contaminants, such as agarose, other polysaccharides, polyacrylamide, acrylamide or acrylic acid, in the gel used in This electrophoresis. Therefore, there is also a need in the art for methods to achieve these separations.

Methods for amplifying nucleic acids or segments thereof, such as the well-known polymerase chain reaction (PCR) process (see, eg, US Patent Number 4,683,202), yield solutions of complex mixtures of enzymes, nucleoside triphosphates , oligonucleotides, or other nucleic acids. Typically, the methods are carried out to obtain a highly increased amount of a single nucleic acid segment ("target segment"). Frequently it is necessary to separate this target segment from the other components in the solution, after the amplification process has been carried out. In addition, there is an additional need in the art for simple methods to achieve these separations. Silica materials, including glass particles, such as glass powder, silica particles and glass microfibers prepared by grinding glass fiber filter papers and including diatomaceous earth, have been employed in combination with aqueous solutions of chaotropic salts to separate the DNA of the other substances and make the appropriate A-DN for use in molecular biological procedures. See U.S. Patent Number 5,075,430, and the references cited therein, including the article by Marko et al., A-nal. Biochem. 121, 382-387 (1982) and Vogelstein et al., Proc. Nati Acad. Sci. (USA) 76, 615-619 (1979). Also see de Boom et al., J. Clin. Microbiol. 28, 495-503 (1990). With reference to intact fiberglass filters used in combination with aqueous solutions of a chaotropic agent to separate DNA from other substances, see Chen and Thomas, Anal. Biochem. 101, 339-341 (1980). Vogelstein et al., Supra, suggests that silica gel is not suitable for use in DNA separations. With respect to the separation of RNA using silica materials and chaotropic agents, see Gillespie et al., U.S. Patent No. 5,155,018. The glass particles, the silica particles, the silica gel and mixtures of the above have been configured in different ways to produce matrices capable of reversibly binding the nucleic acid materials when placed in contact with a medium containing these materials. , in the presence of chaotropic agents. These matrices are designed to remain bound to the nucleic acid material while the matrix is exposed to an external force such as centrifugation or vacuum filtration, in order to separate the matrix and the nucleic acid material bound thereto from the components of the remaining medium. The nucleic acid material is then eluted from the matrix by exposing the matrix to an elution solution such as water or a elution stabilizer. Numerous commercial sources offer silica-based matrices designed for use in centrifugation and / or filtration isolation systems. See, e.g. the -ADN izard ™ purification system line of products from Promega Corporation (Madison, Wisconsin, United States of America); or the QiaPrep ™ line of -ADN isolation systems from Qiagen Corp. (Chatsworth, California, E.U.A.). The magnetically responsive particles (hereinafter referred to as "magnetic particles") have been conventionally used to isolate and purify polypeptide molecules such as proteins or antibodies. In recent years, however, magnetic particles and methods for using magnetic particles have been developed for the isolation of nucleic acid materials. Several different types of magnetic particles designed for use in nucleic acid isolation are described in the literature, and many of those types of particles are available from commercial sources. "These magnetic particles usually fall into any of two categories, those designed to reversibly bind nucleic acid materials directly and those designed to do so through at least one intermediate substance. The intermediate substance is referred to herein as an "irradiation". Magnetic particles designed to indirectly bind nucleic acid materials are generally used to isolate a specific nucleic acid material such as mRNA, according to the following basic isolation procedure. First, a medium containing a nucleic acid material is placed in contact with an irradiation substance capable of binding to the nucleic acid material of interest. For example, one of these commonly used irradiation substances, biotinylated oligonucleotide deoxythymidine (oligo-dT), forms hydrogen bonds with the poly-adenosine tails of the mRNA molecules in a medium. Each irradiation substance used in this manner is designed to be ligated with a magnetically responsive particle when placed in contact with the particle under the appropriate binding conditions. For example, the biotin terminus of a biotinylated oligo-dT / mRNA complex is capable of binding to streptavidin residues on the surface of a magnetically responsive particle coated with streptavidin. Different commercial sources are available for streptavidin magnetic particles and reagents designed for use in the isolation of mRNA using biotinylated oiigo-dT as described above.

See, the ANm Isolation System e.g., PolyATtract® of the 9600 ™ Series from Promega Corporation; or the ProActive ™ line of the streptavidin-coated microsphere particles from Bangs Laboratories (of Carmel, Indiana, E.U.A.). Magnetic particles and irradiation systems have also been developed that are capable of indirectly binding and isolating other types of nucleic acids such as double-stranded or single-stranded PCR templates. See, e.g., BioMag ™ superparamagnetic particles from Advanced Magnetics, Inc. (Cambridge, Mass., E.U.A.). Indirectly binding magnetic separation systems for the isolation or separation of the nucleic acid all require at least three components, ie, magnetic particles, a substance of irradiation and a medium containing the material of nucleic acid interest. The binding reaction of the irradiating substance / nucleic acid and the binding reaction of the irradiation substance / particle frequently require different reaction conditions of solution and / or temperature from one another. Each additional component or solution that is used in the nucleic acid isolation procedure is added to the risk of contamination of the isolated end product by nucleases, metals or other harmful substances.

A few types of magnetic particles have also been developed for use in direct bonding and isolation of biological materials, particularly nucleic acid. One of these types of particles is a glass bead that responds magnetically preferably of a controlled pore size, see, e.g. Porous Glass Magnetic Particles, (MPG) from CPG, Inc. (of Lincoln Park, New Jersey, E.U.A.); or the porous magnetic glass particles described in - U.S. Patent Nos. 4,395,271; 4,233,169; or 4,297,337. The nucleic acid material tends to be so tightly bound to the glass, however, it can be difficult to remove once it is bound to it. Therefore, the elution efficiencies of the magnetic glass particles tend to be low compared to the elution efficiencies of particles containing minor amounts of a nucleic acid binding material such as silica. A second type of magnetically responsive particles designed to be used in the direct binding and isolation of biological materials, particularly nucleic acid, are particles comprising agarose embedded with smaller ferromagnetic particles and coated with glass. See, e.g. U.S. Patent Number 5,395,498. A third type of A particle that responds magnetically, a particle capable of directly linking enzymes, proteins, hormones or antibodies, is produced by incorporating magnetic materials in the matrix of polymeric silicon dioxide compounds. See, eg, German Patent Number DE 43 07 262 Al. The last two types of magnetic particles, the agarose particle and the polymeric silicon dioxide matrix, tend to leach iron in a medium under the conditions required to directly bind the biological materials in each of these magnetic particles. It is also difficult to produce these particles with sufficiently uniform and concentrated magnetic capacity to ensure fast and efficient isolation of the nucleic acid materials bound thereto. What is needed is a method for isolating biological entities, particularly nucleic acids, by using a magnetically responsive particle capable of rapidly and efficiently isolating these entities sufficiently free of contaminants for use in molecular biology procedures.

COMPENDIUM OF THE INVENTION Abbreviating, in one aspect, the present invention comprises a method of isolating the biological target material from other materials in a medium by: providing a medium that includes the biological target material; providing silica magnetic particles; forming a complex of the magnetic particles of silica and the biological target material by combining the magnetic particles of silica and the medium; removing the complex from the medium by applying an external magnetic field, and separating the biological target material from the complex by eluting the biological target material whereby the isolated biological target material is obtained. In a further aspect, the present invention is a method of isolating a material of interest from biological blank from other materials in a medium using magnetic particles of silica capable of reversibly binding at least 2 micrograms of the biological target material per milligram of magnetic particles. of silica, and of releasing at least 60 percent of the biological target material bound to them. In the preferred practice of the present method, at least about 4 micrograms of the biological blank material per milligram of the silica magnetic particle it is ligated to it and at least about 75 percent of the biological target material adhered to the magnetic silica particles is subsequently eluted. The biological target material isolated according to the method of this invention is preferably the nucleic acid. A preferred practice of the method of the present invention comprises the following steps. First, a mixture comprising the medium and the magnetic particles of silica is formed. Second, the biological target material adheres to the magnetic particles of silica in the mixture. Third, the magnetic particles of silica are removed from the mixture using an external force, more preferably using a magnetic force, and Fourth, at least 60 percent of the biological target material adhered to the magnetic particle of silica is eluted, by contacting the particle with an elution solution. In another aspect, the present invention is a method of isolating the plasmid-DNA from other materials in a medium using a preferred form of the silica magnetic particle, i.e., a magnetic particle coated with silicon oxide, wherein the preferred particles are capable of binding to at least 2 micrograms of the plasmid DNA material per milligram of particle, and release at least 60 percent of the plasmid DNA material bound thereto. A preferred practice of the methods of this aspect of the invention comprises the following steps. First a mixture is formed comprising a medium including plasmid DNA, the magnetic particle coated with silicon oxide and a chaotropic salt. Second, the plasmid DNA is adhered to the magnetic particle coated with silicon oxide in the mixture. Third, the magnetic particle coated with silicon oxide is removed from the mixture using an external force of greater preference using a magnetic field. Fourth, at least 60 percent of the plasmid DNA adhered to the magnetic particle coated with silicon oxide is eluted, by contacting the particle, with an elution solution. In a further aspect, the present invention is a case for isolating a biological target material from a medium containing it, the case comprising an aliquot of the magnetic particles coated with the silicon oxide suspended in an aqueous solution in a first container, wherein the particles have the ability to reversibly bind at least 2 micrograms of the biological target material per milligram of particle. Optionally, the case may include other components necessary to isolate a biological target material of a medium containing the same, according to the methods of the present invention. As used herein, the term "magnetic particles" refers to materials that do not have a magnetic field but that form a magnetic dipole when exposed to a magnetic field, i.e., materials capable of being magnetized in the presence of a magnetic field but that by themselves are not magnetic in the absence of this field. The term "magnetic" as used in this context includes materials that are paramagnetic or superparamagnetic materials. The term "magnetic" as used herein also encompasses temporarily magnetic materials such as ferromagnetic or ferrimagnetic materials at low temperatures, as long as these magnetic materials are temporarily paramagnetic within the temperature scale at which the magnetic particles are used. of silica containing these materials, according to the methods present to isolate the biological materials. The term "silica magnetic particle" refers to a magnetic particle comprising silica in the form of silica gel, silicon oxide, solid silica, such as glass or diatomaceous earth, or a mixture of two or more of the above. cited. The term "silica gel" as used herein refers to gel chromatography grade silica, a substance that can be obtained commercially from a number of different sources. Silica gel is most commonly prepared by acidifying a silicate-containing solution, e.g., sodium silicate to a pH of less than 10 or 11 and then allowing the acidified solution to gel. See, e.g. the discussion of silica preparation in Kurt-Othmer Encyclopedia of Chemical Technology, Volume 6, Fourth Edition, Mary Howe-Grant, editors John Wiley & Sons, 1993, page 773-775. The term "silica magnetic particle" as used herein preferably refers to particles with the characteristics described above, which have the ability to bind at least 2 micrograms of biological target material per milligram of magnetic particles. of silica and, independently, the ability to release at least 60 percent of the biological blank material bound thereto in an elution step of the present method. The magnetic particles of silica used in the present invention preferably further comprise a ferromagnetic material incorporated in a silica gel matrix. The elution step in the isolation methods of this invention is preferably accomplished in considerable contamination of the nucleic acid material by metal or metal compounds, e.g., iron or iron compounds) or another objectionable species that originates from the magnetic particles of silica. The term "glass particles" as used herein means particles of crystalline silicas (eg alpha-quartz, vitreous silica), even though crystalline silicas are not formally "glasses" because they are not amorphous, or glass particles made mainly of silica. The term "magnetic particle coated with silicon acid" or "SOCM" particle is used herein to refer to the especially preferred form of the magnetic particle of silica used in the same and kits of the present invention. The SOCM particle comprises a silicon oxide coating, a core of at least one particle of superparamagnetic or paramagnetic material. The SOCM particle used in the present method and the kits also has an adsorption surface of the aqueous silicon oxide, a surface characterized in that it has silanol groups therein. The target nucleic acid material, such as DNA or RNA, adhere to the adsorptive surface of the particle while the other material, particularly harmful contaminants such as exonucleases, do not adhere to or co-elute from the particle with the nucleic acid materials. The physical characteristics of the SOCM particle and methods for producing these particles are disclosed in the North American Patent Application filed simultaneously in Serial Number, entitled "Silica Adsorbent in Magnetic Substrate", the disclosure of which is incorporated herein by reference. The present invention provides a convenient and efficient means for isolating the material of interest from biological blank from a variety of different media. A preferred aspect of the present method briefly described above where the magnetic force is used to remove the particles from the medium, offers significant advantages over conventional isolation methods where one biological target material is reversibly linked to the other material of silica - Specifically, the magnetic removal step of the method - replaces the vacuum filtration or centrifugation steps required in conventional silica bonding and elution isolation methods. Therefore, it is particularly subject to automation. Small laboratories or individual researchers often need to purchase specialized and expensive equipment to carry out these methods, such as vacuum and vacuum manifolds for use in vacuum filtration - or a microcentrifuge for centrifugation method.

In contrast, the magnetic separation of the present invention only requires a concentrated magnetic field such as that which is generated from an intense and easily obtainable magnet. Economical devices for all specifically for use in the context of molecular biology research are also commercially available such as the MagneSphere® Technology Magnetic Separation Stand or Poly-Sat® Multi-Magnet Series 96-00 ™ (both available from Promega Corporation, Madison, Wisconsin, USA). The biological target material isolated using the isolation method of the present invention is sufficiently free of contaminating material for further processing or analysis, using standard molecular biology techniques. The applications of the present methods for isolating different biological target materials from a variety of different means will become apparent from the detailed description of the invention presented below.

BRIEF DESCRIPTION OF THE FIGURES The Figure 1 is a trace of a number of micrograms of the plasmid-DNA linked by microgram of plasmid DNA added either the glass particles of Magnetic controlled pore (CPG) or magnetic particles of silica. Figure 2 is a trace of a number of micrograms of the plasmid DNA eluted from either the magnetic or magnetic particles of CPG versus the amount of plasmid DNA added to the particles before elution. Figure 3 is a trace of the binding data shown in Figure 1 and the elution data shown in Figure 2 which is obtained from magnetic particles of magnetic GCS of silica. Figure 4 is a fluorimage of the agarose gel stained with a fluorescent dye, after fractionation of the DNA fragments on the gel using gel electrophoresis, where the DNA fragments were produced by digesting lambda DNA with Hind III and ligand and eluting the fragments of the silica magnetic particles. Figure 5 is a fluorimage of an agarose gel stained with a fluorescent dye, after fractionation of the DNA fragments on the gel using gel electrophoresis where the DNA fragments were produced by digesting FX174 DNA with Hae III and ligand and eluting the fragments of the silica magnetic particles.

Figure 6 is a histogram trace of a number of counts per million (CPM) of RNA irradiated with 32P- that is applied to, bound to, and released from magnetic particles of silica.

DETAILED DESCRIPTION OF THE INVENTION The biological blank material isolated using the methods of the present invention is prably a nucleic acid or a protein, more prably, a nucleic acid material such as RNA, DNA or RNA / DNA hybrid. When the biological blank material isolated using the present methods is a nucleic acid, prably DNA or RNA including but not limited to plasmid DNA, the DNA fragments produced from the restriction enzyme digestion, the amplified DNA produced by amplification reaction such as polymerase chain reaction (PCR), single-stranded DNA, mRNA or total RNA. The nucleic acid material isolated according to the methods of the present invention most prably is a plasmid DNA or a total RNA. Since the nucleic acids are the especially prred biological blank material isolated using the methods of the present invention, most of the Detailed description of the invention presented below describes this prred aspect of the present invention. However, the detailed description of this specific aspect of the present invention is not intended to limit the scope of the invention. The present disclosure provides sufficient guidance to enable a person skilled in the art of the present invention to use the methods of the present invention to isolate biological target materials other than nucleic acid materials., e.g. proteins or antibodies. The present methods for isolating the biological target material can be carried out using any magnetic particle of silica, but the preferred methods are carried out using the SOCM form of the silica magnetic particles. The present methods are also preferably carried out using magnetic particles of silica with the following physical characteristics. The magnetic particles of silica used in the methods of this invention can be any of a number of different sizes. The smaller magnetic silica particles provide greater surface area (one per unit basis in weight) for adsorption, but the smaller particles are limited in the amount of magnetic material that can be incorporated in these particles compared to the larger particles. The median particle size and magnetic particles of silica used in the present invention are preferably from about 1 to 15 microns, more preferably from 3 to 10 microns, and especially preferably from about 4 to 7 microns. The particle size distribution can also be varied. However, a relatively critical monodal particle size distribution is preferred. The monodal particle size distribution is preferably such that about 80 weight percent of the particles fall within the 10 micron scale, about the median particle size, most preferably within a 8 micron scale , and especially preferably within a 6 micrometer scale. The magnetic silica particle preferably used in the present invention has pores that are accessible from the outside of the particle. The pores are preferably of a controlled size scale large enough to admit a biological target material, e.g. the nucleic acid inside the particle and to bind to the silica gel material on the inner surface of most of these pores. The pores of the especially preferred forms of the particles Silica magnetic materials are designed to provide a large surface area of the silica gel material capable of being bound to a biological target material, particularly the nucleic material. The total pore volume of the silica magnetic particle as measured by the nitrogen BET method is preferably at least about 0.2 milliliter per gram of the mass of particles. From the total pore volume measured by BET of nitrogen, preferably at least about 50 volume percent of the pore that is contained in pores having a diameter of 600 angstrom units or greater. The magnetic particles of silica may contain substances, such as transition metals or volatile organic materials that could detrimentally affect the usefulness of the isolated biological blank material contaminated considerably with these substances. Specifically, these contaminants could affect downstream processing, analysis and / or use of these materials, for example, by inhibiting enzyme activity or by degrading the target material itself. Any of these substances present in the magnetic particles of silica used in the present invention are preferably present in the form that is not easily leached from the particle and towards the isolated biological blank material produced according to the methods of the present invention. Iron is an undesirable contaminant particularly when the biological target material is a nucleic acid. Iron, in the form of magnetite, is present in the core of a particularly preferred form of the silica magnetic particles of the present invention, ie, SOCM particles. Iron has a broad absorption peak between 260 and 270 nanometers (nm). The nucleic acids have a maximum absorption of approximately 260 nanometers, so that contamination of the iron in a nucleic acid sample can detrimentally affect the accuracy of the results of the quantitative spectrophotometric analysis of these samples. Any of the iron-containing magnetic silica particles used to isolate the nucleic acids using the present invention preferably do not produce an isolated nucleic acid material sufficiently contaminated with iron so that the iron interferes with the spectrophotometric analysis of the material or about 260 nanometers Especially preferred silica magnetic particles used in the methods of the present invention, the SOCM particles are leached at no more than 50 parts per million, most preferably no more than 10 parts per million and especially preferably not more than 5 parts per million of the transition metals when tested in the following manner. Specifically, 0.33 gram of particles (oven dried at 110 ° C) in 20 milliliters of an aqueous solution of HCl of IN concentration (using deionized water). The resulting mixture is then stirred only to disperse the particles. After approximately 15 minutes of total contact time, a portion of the liquid in the mixture is analyzed to determine the content of the metals. Any conventional elemental analysis technique can be used to quantify the amount of the transition metal in the resulting liquid, but inductively coupled plasma (ICP) spectroscopy is preferred. The number of the simultaneously filed patent application, entitled "Adsorbent Silica in the Magnetic Substrate" which is incorporated by reference herein, discloses methods for producing the appropriate SOCM particles for use in the methods and kits of the present invention. invention. The especially preferred method for producing SOCM particles for use in the present invention comprises the general steps of: (1) preparing the magnetite core particles by aqueous precipitation of a mixture of FeCl 2 and FeCl 3, (2) depositing an oxide coating of silicon on magnetite core particles by exposing a slurry of the particles to a mixture of SiO2 and Na20 for at least about 45 minutes at a temperature of at least 60 ° C and then adding an acid solution to the mixture until the pH it is decreased at a pH less than 9, (3) allowing the resulting slurry to be aged for at least about 15 minutes preferably, while continuing to stir the slurry, (4) washing the particles. The deposition and firing steps of the preferred particle production method described above can be repeated to produce multiple layers of silicon oxide coating above the magnetite core, thus providing additional security against leaching of the metals from the core to the surrounding environment. The SOCM particles produced by the above described method are preferably treated by undergoing a mild oxidation step to further inhibit the leaching of the core. The biological target material isolated using the method of the present invention can be obtained from eukaryotic or prokaryotic cells in culture or from cells that are taken or obtained from tissues, multicellular organisms, including animals and plants; body fluids such as blood, lymph, urine, fecal matter or semen; embryos or fetus; foodstuffs; cosmetics; or other sources of cells. Some biological target materials such as certain DNA or A-RN species are isolated according to the present method of DNA or A-RN from organelles, viruses, bacteriophages, plasmids, viroids or the like that infect the cells. The cells will be subjected to lysate and the lysate will usually continue in various ways known to those skilled in the art in order to obtain an aqueous solution of DNA or -RNA, to which the methods of separation or isolation of the invention are applied. The DNA or RNA, in this solution, will typically encounter other components, such as proteins, RNA (in the case of DNA separation), DNA (in the case of RNA separation, or other types of components). Regardless of the nature of the source of this material, the biological target material to be isolated in the present methods is provided in a medium comprising the biological target material and other species. The medium in a form that is available to adhere to the silica magnetic particles in the first step of the method When the nucleic acid material is contained within a cell , the walls of the cell or the membrane of the cell can cause the material is not available for adhesion to the particles. Even when these cells are lysed or ruptured sufficiently to cause the nucleic acid material contained therein to be released into the surrounding solution, the cellular debris in the solution could interfere with the adhesion of the nucleic acid material in the cells. magnetic particles of silica. Therefore, in cases where the nucleic acid material to be isolated using the methods of the present invention is contained within a cell, the cell is preferably first processed by lysate or by breaking the cell to produce a lysate, and more preferably it is further processed by clearing the lysing of the cellular debris (eg by vacuum filtration or centrifugation) which has the potential to interfere with the adhesion of the nucleic acid material to the silica magnetic particles when provided as the medium in the methods of the present invention. Any of a number of different known methods for lysing or breaking cells in order to release the nucleic acid materials contained therein are suitable for use in producing a cell medium for use in the present invention. The method selected to release the nucleic acid material from a cell will depend on the nature of the cell that contains the material For example, in order to cause a cell with a relatively hard cell wall with a fungus cell or a plant cell to release the nucleic acid material contained therein, one needs to use hard treatments such as potent proteases and Mechanical cutting with a homogenizer or break with sound waves using a sonicizer. In contrast, the nucleic acid material can be easily released from cells with lipid bi-layer membranes such as E. coli bacteria or animal blood cells by suspending only these cells in an aqueous solution and adding a detergent to the solution . Once the nucleic acid material is released from the cells subjected to Used or broken as described above, the cellular debris will tend to interfere with the adhesion of the nucleic acid material to the magnetic particles of silica can be removed using a number of different known techniques or the combination of techniques. The solution of the used or broken cells is preferably centrifuged to remove the debris from the cell into particles. The supernatant liquid is then preferably further processed by adding a second solution to the supernatant liquid which causes a material precipitated from another additional material to form and then removing the material precipitate of the resulting solution by centrifugation. In a particularly preferred aspect of the present method, the nucleic acid material of interest isolated according to the method of the present invention is the plasmid DNA initially contained in the cell of the E. coli bacterium. The nucleic acid material is especially preferably released from the bacterium cell by the addition of an alkaline solution such as a sodium hydroxide solution to form a lysate. The lysate is then preferably further treated by centrifugation to remove the waste from the cell. A neutralization solution such as an acidic stabilizer is preferably added to the resulting supernatant liquid to form an additional material that potentially interferes. The precipitated material formed in this way is preferably removed by centrifugation. A neutralization solution such as an acidic stabilizer is preferably added to the resulting supernatant liquid to form a precipitated material of additional potentially interfering material. The precipitated material formed in this way is preferably removed by centrifugation. The remaining supernatant fluid from the cleared lysate is the medium that is provides in the first step of this particularly preferred aspect of the present method. The medium that is provided in the first step of the method of this invention does not need to contain the nucleic acid material released directly from the cells. The nucleic acid material can be the product of an amplification reaction such as the amplified -DNA produced using the polymerase chain reaction (PCR). The nucleic acid material may also be capable of forming DNA fragments produced by digesting DNA "with a restriction enzyme." The medium may also be in the form of a mixture of enzymatically digested or fused electrophoresis gel and a nucleic acid material.

The magnetic particles of silica that are provided in the second step of the methods of the present invention, preferably have the ability to complex with the nucleic acid material in the medium, reversibly binding to at least two micrograms of the material of nucleic acid per milligram of the particle. The particles that are provided for use in the present invention of greater preference have the ability to reversibly bind at least 4 micrograms and more preferably at least 8 micrograms of the nucleic acid material per milligram of the particle. The magnetic particles of silica Preferably they must have a capacity to release at least 60 percent of the nucleic acid material adhered thereto. The most preferred particles have the ability to release at least 70 percent and more preferably at least 90 percent of the nucleic acid material adhered thereto. The silica magnetic particles that are provided in the first step of the methods of the present invention, more preferably, are SOCM particles. A complex of the magnetic particles of silica and the biological target material is formed in the third step by preferably exposing the particles to the medium containing the target material under conditions designed to promote complex formation. The most preferred form complex in a mixture of the silica magnetic particle, the medium and a chaotropic salt. Chaotropic salts are salts of chaotropic ions. These salts are highly soluble in aqueous solutions. The chaotropic ions that are provided by these salts and the sufficiently high concentration in the aqueous solutions of proteins or nucleic acids, cause the proteins to unfold, the nucleic acids lose the secondary structure or in the case of nucleic acids from double chain, they are founded (that is, they are separated by the chain). It is believed that chaotropic ions have these effects because they break the hydrogen bonding networks that exist in liquid water and thus render denatured proteins and thermodynamically more stable nucleic acids than their doubled or correctly structured duplicates. Chaotropic ions include guanidinium, iodide, perchlorate and trichloroacetate. The guanidinium ion is preferred in the present invention. Chaotropic salts include guanidine hydrochloride, guanidine thiocyanate (which is sometimes referred to as guanidine isothiocyanate), sodium iodide, sodium perchlorate and sodium trichloroacetate. Guanidinium salts are preferred, and guanidine hydrochloride is particularly preferred. The concentration of the chaotropic ions in the mixture formed in this practice of the present method is preferably between about 0.1 M and 7 M, but more preferably between about 0.5 M and 5 M. The concentration of the chaotropic ions in the mixture should be high enough to cause "the biological target material to adhere to the magnetic particles of silica in the mixture, but not so high as to denature essentially to degrade or to cause the white material to precipitate out of the mixture. Proteins and large molecules of double-stranded DNA such as chromosomal DNA are stable at chaotropic salt concentrations of between 0.5 and 2 molar, but are known to precipitate out of solution at chaotropic salt concentrations greater than approximately 2 molar. See, e.g., U.S. Patent Number 5,346,994 issued in favor of Piotr Chomczynski, column 2, lines 56-63. In contrast, the -RNA and the smaller DNA molecules such as plasmid DNA, the restriction or PCR fragments of chromosomal DNA, or the single-stranded DNA remain undegraded and in solution at concentrations of the chaotropic salt between 2 and 5 molar. With any chaotropic salt used in the invention, it is desirable that the concentration of the salt, in any of the solutions where the salt is employed to carry out the invention, remain at less than the solubility of the salt in the solution under all the conditions to which the solution is subjected to carry out the invention. One practice of the present methods, the mixture formed as described above is incubated until at least a certain amount of the nucleic acid material is adhered to the magnetic particle of silica for form a complex. This incubation step is carried out at a temperature of at least 0 ° C, preferably at least at 4 ° C, and more preferably at least at 20 ° C, as long as the incubation temperature is no greater of 67 ° C. The incubation step should be carried out at a temperature lower than the temperature at which the silica magnetic particles begin to lose their ability to bind reversibly to the nucleic acid material. The most preferred incubation step is carried out at about room temperature (ie, at about 25 ° C). The complex is removed from the mixture using a magnetic field. Other forms of external force in addition to the magnetic field can also be used to isolate the biological target substance in accordance with the methods of the present invention after the initial removal step. Additional appropriate forms of external force include, but are not limited to, filtration by gravity, vacuum filtration and centrifugation. The external magnetic field used to remove the complex from the medium can be generated appropriately in the medium using any of a number of different known means. For example, a magnet may be placed on the outer surface of a container of a solution containing the particles, causing the particles migrate through the solution and collect on the inner surface of the container adjacent to the magnet. The magnet can then be held in position on the outer surface of the package such that the particles are retained in the package by the magnetic field generated by the magnet, while the solution is decanted out of the package and discarded. A second solution can then be added to the package and the magnet can be removed so that the particles migrate towards the second solution. Alternatively, a magnetizable test probe could be inserted into the solution and the probe could be magnetized in such a way that the particles are deposited at the end of the test probe submerged in the solution. The test probe could then be removed from the solution while remaining magnetized, immersed in a second solution and the magnetic field is discontinued allowing the particles to go towards the second solution. There are commercial sources for magnets designed to be used in both types of magnetic transfer and removal techniques described in general terms in the foregoing. See, e.g., MagneSphere® Technology Magnetic Separation Stand or PolyATract® 9600 ™ Multi-Magnet Series, both obtainable from Promega Corporation; Magnetight Separation Stand (de Novagen, Madison, Wl); or Dynal Magnetic Particle Concentrator (Dynal, from Oslo, Norway).

In a preferred aspect of the methods of the present invention, the complex removed from the medium in the third step is washed at least once by rinsing in a washing solution. The wash solution used in this preferred additional step of the method preferably comprises a solution capable of removing contaminants from the magnetic particle of silica. The washing solution preferably comprises a salt and a solvent, preferably an alcohol. The concentration of alcohol in this preferred last form of the wash solution is preferably at least 30 volume percent, more preferably at least 40 volume percent, and especially at least 50 percent by volume in volume. The alcohol used in this manner is preferably ethanol or isopropanol, more preferably ethanol. The salt is preferably in the form of a stabilizer and more preferably in the form of an acetate stabilizer. The concentration of the salt in the wash solution is high enough to ensure that the nucleic acid material does not elute from the magnetic particles of silica during the wash step (s). The complex preferably is washed after being removed from the medium by resuspending the complex in the washing solution. The complex of preference is removed of the wash solution after the first wash and washed at least once more and more preferably three times more using a fresh washing solution for each wash step. Fourth and finally, the nucleic acid material is eluted from the magnetic silica particle by exposing the complex to an elution solution. The elution solution is preferably an aqueous solution of low ionic strength and more preferably water or a stabilizer of low ionic strength at about a pH at which the nucleic acid material is stable and essentially intact. Any aqueous solution with an ionic strength of or less than that of the TE stabilizer (ie, 10 mM Tris-HCl, lmM ethylenediamine tetraacetic acid (EDTA), pH 8.0) is suitable for use in the elution steps of the present methods, but the elution solution is preferably stabilized to a pH between about 6.5 and 8.5, and more preferably a pH between about 7.0 and 8.0 is stabilized. The TE Stabilizer and the distilled or deionized water are particularly preferred elution solutions for use in the present invention. The low ionic strength of the preferred forms of the elution solution described above ensures that the nucleic acid material is released from the particle. Other elution solutions suitable for use in the methods of this invention will be readily apparent to a person skilled in the art. The nucleic acid material eluted from the complex in the elution step of the method of preference is separated from the magnetic particles of silica and complexed with the rest of the elution mixture by an external force such as centrifugation or a magnetic field, but more preferably using centrifugation. Centrifugation is preferred because it can result in the removal of particles or fragments of particles and they are also small or not magnetically responsible enough to be removed using a magnetic field. The nucleic acid material eluted using the method of the present invention is suitable, without additional isolation, for further analysis or processing by molecular biological methods. The eluted nucleic acid can be analyzed by, for example, sequencing, restriction analysis or hybridization of nucleic acid probe. In this way the methods of the invention can be applied as part of methods based on the analysis of DNA or RNA, for among other things, diagnosis of diseases; identification pathogens; test foods, cosmetics, blood or blood products, or other products for contamination by pathogens; forensic test; Paternity test; and sex identification of fetus or embryos. The eluted DNA or RNA that is provided by the method of the invention can be processed by any of several exonucleases and endonucleases that catalyze the reactions with DNA or RNA, respectively, and in the case of DNA can be digested with restriction enzymes that are cut into the restriction sites present in the DNA. The restriction fragments of the eluted DNA can be ligated and transformed into appropriate hosts for cloning or expression. The eluted DNA or RNA segments can be amplified by any of the methods known in the art to amplify the target nucleic acid segments. If the eluted DNA is a plasmid and another type of autonomously duplicating DNA can be transformed into an appropriate host. for cloning for expression of genes in the -DNA that are capable of expression in the transformed host. Plasmid DNAs isolated by methods of the present invention have been found to be transfected more efficiently in eukaryotic cells than those isolated by the method of the prior art, wherein soil is used. diate instead of silica gel in the methods of the invention of this application. The following non-limiting examples disclose various embodiments of the invention. In the examples and elsewhere in the specification and claims, the volumes and concentrations are at room temperature unless otherwise specified. Only the especially preferred form of the magnetic silica particles was used in each of the examples that follow, that is, the SOCM particles. However, a person skilled in the art of the present invention will be able to use the teachings of the present disclosure to select and use the forms of the magnetic silica particles other than the SOCM particles whose use is illustrated in the aspects of the methods of the present invention that are demonstrated in the Examples presented below.

The same batch of SOCM particles was used to produce the test results presented in Examples 1 and 6 below, while a second batch of SOCM particles was used to generate the results presented in Examples 2 to 4 and 7. However, both batches of SOCM particles were found to produce acceptable results when tested as will be described below. The first batch of particles of SOCM, ie, the particles used in Examples 1 and 6, were found to have the following physical characteristics: surface area of 55 square meters per gram, pore volume of 0.181 milliliter per gram for particles and a diameter of < 600 angstrom units, pore volume of 0.163 milliliter per gram for diameter particles of > 600 angstrom units, median particle size of 5.3 microns, and iron leaching of 2.8 parts per million when tested as described herein using ICP. The other batch of SOCM particles used in the Examples presented below were found to have the following characteristics: surface area of 49 square meters per gram, pore volume of 0.160 milliliter per gram (diameter of < 600 angstrom units) pore volume of 0.153 milliliter per gram (diameter of> 600 angstrom units), median particle size of 5.5 micrometers and iron leaching of 2.0 parts per million.

EXAMPLE 1 - TESTING OF LIGAZON CAPACITY AND EFFICIENCY OF ELLUTION OF THE MAGNETIC PARTICLES OF SILICA FOR PLASMID DNA The bonding capacity of the SOCM form of the magnetic particles of silica and of the particles of Magically controlled pore glass (CPG) was determined by evaluating increased amounts of the plasmid against a constant amount of particles in a total volume of 600 microliters of 4.16M guanidine hydrochloride (GHC1). The magnetic CPG particles used were 5 micron magnetic glass particles with an average pore size of 500 angstrom units obtained from CPG Inc., from Lincoln Park, N.J. E.U.A. Part Number MCPG0510). In the present example, an amount of 140 milligrams of the magnetic silica was suspended in 10 milliliters of deionized water (DI H2O) and then washed 3 times with 10 milliliter of 5M GHC1 before being suspended at a final concentration of 14 milligrams per milliliter. in the same solution. A ligation mixture was formed by adding increased volumes of pGEM® 3zf (+) -DNA of plasmid from Promega Corporation (Catalog Number P2271) in DI H2O at a concentration of 1.0 microgram (μg) per microliter corresponding to 5 μg, 10 μg, 20 μg, 40 μg, 60 μg and 80 μg of -ADN up to 500 μl of the particles and was brought to a final volume of 600 μl by the addition of DI H2O. The plasmid / particle binding mixture was then incubated for 2 to 3 minutes at room temperature. The amount of the plasmid bound to the magnetic silica was determined by subtracting the amount of -DNA from plasmid remaining in the solution from the total amount of the plasmid that was added to the particles in each sample, from the following -many. The liquid fraction of the test mixture was separated from the magnetic silica by centrifugation at 14,000 x grams for 20 seconds. The amount of plasmid DNA remaining in the supernatant liquid was determined by monitoring the absorbance of the solution at 260 nanometers. An absorbance unit at 260 nanometers is equivalent to a plasmid DNA concentration of 50 μg per milliliter. The magnetic silica particles remaining in the ligation mixture were then separated from the mixture and washed in the following manner. A magnet was placed on the outside of the container that retains the ligation mixture but near one side of the container, causing the magnetic particles of silica in the mixture to settle on the side of the container closest to the magnet. The magnet was then held in this position on the side of the container while the mixture was decanted out of the container leaving essentially all of the magnetic particles of silica in the container. The remaining silica magnetic particles were then washed four times with 1 milliliter of a wash solution of 80mM KOAc and lOμM EDTA containing 55 percent EtOH, stirring the magnet on the side of the container during each washing step and placing the magnet, again, on one side of the container to ensure that the particles remain in the container while the washing solution is decanted after each washing step. The remaining particles in the container after the first wash step were then air dried for 3 to 5 minutes. Finally, the plasmid DNA was eluted from the magnetic silica particles by adding 1 milliliter of deionized water at room temperature. The particles were removed from the resulting isolated plasmid DNA solution by centrifugation. The amount of the eluted plasmid DNA was then determined by measuring the absorbance of the solution at 260 nanometers. The total efficiency of the plasmid isolation process was determined as the percentage of DNA recovered in the final elution compared to the amount of DNA incubated with the particle. Bonding capacity was determined at the point where the total efficiency decreased to 90 percent. The results of the binding assay described above are presented in Figure 1, and together with the elution results in Figure 3. The results of DNA binding capacity obtained with the magnetic silica particles ( ?) and magnetic CPG (+) are shown separately in Figure 1. The results show that as they are added Increased amounts of the plasmid DNA to the magnetic silica particles, the particles continued to ligate increased amounts of DNA, ligating as much as 90 μg per 130 μg of the added plasmid. In contrast, the magnetic CPG particles stopped binding to more than 40 μg of the plasmid DNA even when 130 μg of the plasmid DNA was added. The total binding capacity of the magnetic particles of silica was 8 μg of plasmid per milligram of particle. This is significantly higher than the bonding capacity of the magnetic CPG particles and at least 4 times higher than the binding capacity of 10 μM of a silica count used in the Wizard ™ DNA Purification Systems More DNA Plasmid of Promega Corporation. The results of the elution assay described above are presented in Figure 2, and together with the elution results in Figure 3. The results show that more than 90 percent of the plasmid DNA bound to the magnetic particles of The silica in this example was eluted from the particles while less than 60 percent of the plasmid DNA bound to the CPG particles was eluted therefrom. The results presented in Figures 1 to 3 clearly demonstrate that the magnetic particles of The silica tested here exhibits excellent binding and elution characteristics.

EXAMPLE 2 - TEST OF CAPACITY OF LIGAZON AND EFFICIENCY OF ELUTIONS OF MAGNETIC PARTICLES OF SILICA FOR FRAGMENTS OF DNA Purified native lambda DNA from Promega Corporation (Catalog Number D150) was digested with the restriction enzyme Hind III, an enzyme that cuts native lambda DNA into 8 fragments ranging in size from 23,000 bp to 125 bp. This digested lambda DNA Hind III is referred to below as a compendium of "digestive? Hind III". The magnetic silica was prepared as described above and resuspended in 5M GHC1 at a concentration of 14 milligrams per milliliter. One milliliter of the solution of resuspended particles was incubated with 80 μlitres of digestive collection? Hind III (0.44 μg per μl) for 2 to 3 minutes at room temperature. The amount of DNA bound to the magnetic silica was determined by subtracting the DNA remaining in the solution from the total amount of DNA added to the particles after separation of the liquid and solid phases by centrifugation at 14,000 x g for 20 seconds. DNA concentrations were determined by measurement of absorbance at 260 nanometers. An absorbance unit at 260 nanometers is equivalent to a concentration of -ADN of 50 μg per milliliter. The remaining silica magnetic particles in the ligation mixture were then separated from the mixture and washed in the following manner. A magnet was placed on the outside of the container that retains the ligation mixture but close to one side of the container causing the magnetic particles of silica in the mixture to be deposited on the side of the container closest to the magnet. The magnet was then held in its position on the side of the container while the mixture was decanted out of the container leaving essentially all the magnetic particles of silica in the container. The remaining magnetic silica particles were then washed 4 times with 1 milliliter of a wash solution of 80 mM KOAc and 10 μM EDTA containing 55 percent EtOH, stirring the magnet on the side of the container during each washing pair and placing the magnet, again, on one side of the container to ensure that the particles remained in the container while the washing solution was decanted after each washing step. The remaining particles in the container after the last wash step were air-dried for 3 to 5 minutes. Finally a digestive was eluted? Hind III adding 200 μl of deionized water at room temperature ambient. The particles were removed from the digestive solution? isolated resulting by centrifugation. The amount of digestive DNA? Hind III was eluted and then determined by measuring the absorbance of the solution at 260 nanometers. Similar silica magnetic particle binding and elution assays were performed using FX174 DNA digested with the restriction enzyme Hae III, a digestion reaction that produces 10 DNA fragments ranging from 1353bp to 72bp in size. The data for these experiments are summarized in Table 1, which is presented below.

TABLE 1 Type of -AUN -AUN A-DN DNA - > Added Attached Eluido * Recovery * Digestive? HindlII 35 μg 33.6μg 28.4μg Digestive X174 HaelII 40 μg 39.4μg 33.7μg * A second elution step wherein 200 μl of deionized water was added to the magnetic particles of silica after the first elution step which resulted in a recovery of > 97 percent of the -DNA bound ** Based on the amount of input DNA EXAMPLE 3 - ELECTROPHORESIS OF DNA FRAGMENTS AFTER THE ELLENTION OF THE SILICONE PARTICLES OF SILICA In order to determine whether the silica magnetic particles bound or released from the DNA fragments of different molecular weights at different weights, the DNA fragments bound to and eluted from the magnetic particles of silica in Example 2 were tested using electrophoresis as follows. The digestive samples? Hind III eluted from two different samples of silica magnetic particles were loaded and fractionated on an agarose gel together with a control sample from the untreated DNA digestive. The samples of bound and eluted digestive FX174 Hae III were also fractionated on an agarose gel together with a control sample from the untreated -DNA digest. The gels resulting from the fractionated DNA were then stained with a fluorescent dye able to stain DNA and stained gels were analyzed using a Molecular Dynamics Fluoroimager Apparatus. The fluorescent intensity of the DNA fragments eluted from each of the restriction enzyme digestives was compared with the control digestives before capture and elution in magnetic silica. Figures 4 and 5 show the visual image generated by the fluorine meter of the fluorescent dye agarose gel of the captured DNA fragments and fractionated elutes produced as described above. Figure 4 shows 2 μg of the digestive? HindI I I subjected to electrophoresis in 1 percent agarose gel. Figure 5 shows 5 μg of the digest of FX174 -Hae III subjected to electrophoresis in 3 percent agarose gel. In both panels, sample 1 is the control of the untreated DNA digestive, while samples 2 and 3 are samples of the DNA digestive bound to and eluted from two different samples of the magnetic particles of silica. No considerable difference in the relative band intensity or the background between the control and the samples of any of the sets of digestive samples analyzed herein was observed, indicating that the silica magnetic particles tested herein do not they bind or selectively release DNA fragments according to molecular weight.

EXAMPLE 4 - ISOLATION OF PLASMID DNA FROM BACTERIAL CROPS USING MAGNETIC PARTICLES OF SILICA AND MAGNETIC FORCE Some of the resuspended silica magnetic particles prepared in Example 1 were used to isolate the plasmid DNA pGEM®-3zf (+) from a culture of DH5a E. coli bacteria were transformed with any form of the plasmid DNA. The following solutions were used in the isolation procedure: 1. Cell Resuspension Solution: 50 mM Tris-HCl, pH 7.5 10 M EDTA 100 μg per milliliter of DNase A-free ribonuclease (RNase A) 2. Column Wash Solution: Prepared by producing an aqueous stabilizer consisting of either 200mM NaCl, 20mM Tris-HCl, 5mM EDTA, pH 7.5, or 190mM KOAc, 20mM Tris-HCl, 0. ImM EDTA, pH 7.5, and diluting the aqueous stabilizer to 1: 1.4 with 95 percent ethanol (EtOH). 3. TE stabilizer: 10mM Tns-HCl, pH 7.5 lmM EDTA 4. Neutralization solution: 1.32 M KOAc (potassium acetate), pH 4.8 . Cell Lysis Solution: 0.2M NaOH 1% SDS (sodium dodecyl sulfate) The battery culture was treated to produce a clear lysate, following the steps briefly described below: 1. Cells of 1 to 3 milliliters of culture of bacteria were harvested by centrifuging the culture during 1 to 2 minutes at top speed in a microcentrifuge. The harvested cells were resuspended in 200 μl of the Cell Resuspension Solution and transferred to a microcentrifuge tube. The resulting solution of resuspended cells was cloudy. 2. 200 μL of the Cell Lysis Solution were then added to the solution of resuspended cells and mixed by inversion until the solution became relatively clear indicating that the resuspended cells had been lysed. 3. 200 μL of the Neutralization Solution was added to the lysate solution, and mixed by inversion. The lysate became cloudy after the Neutralization Solution was added. 4. The solution was then spun in a microcentrifuge at high speed for 5 minutes to rinse the lysate. 5. The supernatant liquid resulting from the clarified lysate was transferred to a new tube of the microcentrifuge. The plasmid DNA was isolated after the lysed rinse using the magnetic silica particles suspended in a guanidine hydrochloride solution which was prepared in Example 1 Essentially the same procedure was used to isolate the plasmid DNA using the particles and magnetic force as used in the plasmid-binding assay described in Example 2. However, the present isolation process was initiated by adding 1 milliliter of the suspended silica magnetic particles to the lysed lysate produced from step 5, immediately above, instead of starting the process by adding 500 μl of the suspended particles to 5 to 80 μg of purified plasmid DNA. The volumes of each solution added to the magnetic silica particles in each subsequent step of the present isolation procedure which were followed were adjusted in proportion to the amount of the largest starting volume. The resulting isolated plasmid DNA was qualitatively assayed using gel electrophoresis and quantitatively using a spectrophotometer. The results of the gel assay showed high percentage of intact plasmid DNA present in the sample. Optical density measurements accurately reflected the DNA performance as evidenced by the absorbance ratios (eg, 260/250 nm and 260/280 nm) on the expected DNA scale.

EXAMPLE 5 - ISOLATION OF PLASMID DNA FROM BACTERIAL CROPS USING MAGNETIC SILICONE PARTICLES AND VACUUM FILTRATION The same procedure was used to produce a clear lysate of a culture of E. coli bacteria transformed with the plasmid DNA, such as the clear lysate production procedure using in Example 4. The plasmid was then isolated from the resulting clear lysate using the suspension of silica magnetic particles of Example 1, but using vacuum filtration instead of magnetic force to separate the particles from the binding mixture once the plasmid DNA had adhered to the particles. Vacuum filtration is also used to remove the wash solution of the particles in the wash steps of the isolation process.

EXAMPLE 6 - ILLUSTRATING THE RNA STRIPE Using magnetic silica at 14 milligrams per milliliter in 4M Guanidine Thiocyanate, 700 μL of resuspended silica magnetic particles prepared as in Example 1 were added to 30 microliters of the Promega RNA Markers, Catalog Number 1550, irradiated with 32 (approximately 200,000 cpms), and 5 μlitres of a solution of 1 milligram per milliliter of cold Promega RNA markers (ie without irradiating) part Number # G3191 in a container. The resulting mixture was incubated for 5 minutes at room temperature after which the particles were captured using magnetic force to attract the particles to one side of the container while the supernatant liquid was decanted into a second container. The supernatant liquid collected in the second container was preserved and counted. The particles captured in the first container were then washed three times with the washing solution of column prepared as described in Example 1 above. The particles were captured after each washing step and the washing solution was decanted. Each decanted washing solution was preserved and counted. The sum of the supernatant liquid accounts and the wash gap accounts was used to determine the unbound CPMs. After the third wash step, the RNA was eluted from the captured and washed particles by resuspending the particles in 250 μl of Nanopura water heated at 37 ° C and then using magnetic force to retain the particles on the side of the package while the eluent was he decanted and picked himself up. Then 100 μl of the eluent was counted. The remaining particles were resuspended in 500 μl of Nanopura and then counted to determine the amount of remaining eluted CPMs. The aforementioned analysis was carried out in duplicate. The results shown-in Figure 6 reflect the averaged accounts for each set of duplicates and accounts collected in each experiment. Figure 6 shows that 200,000 CPMs of RNA were exposed to magnetic silica particles in this assay, an average of 125,000 CPMs were bound to the particles and approximately 100,000 CPMs bound to the particles were released and eluted from the particles in the step of final elution.

These binding and elution assay results from -RNA are comparable to the DNA binding and elution results in Example 2, cited above. The present assays show the potential application of the magnetic silica particles according to the methods of the present invention to isolate the RNA.

EXAMPLE 7 - IRON LEACHING ANALYSIS OF SILICONE MAGNETIC PARTICLES Magnetic silica particles such as those used in the above Examples were selected because of their tendency to leach iron or other materials with a tendency to interfere with the quantitative analysis of the nucleic acid materials, producing a maximum absorbency of about 260. nm when the solutions exposed to the particles were analyzed in a spectrophotometer. The silica magnetic particles were analyzed in the following manner. 140 milligrams of magnetic particles of silica were resuspended in 10 milliliters of deionized water and vortexed briefly. The particles were exposed to a magnetic field for 1 minute, placing a magnet against the outside of the container that retained the particle / water mixture. The particles in the The mixture was collected in the water of the container closest to the surface of the magnet and held against the side of the container by the magnet, while the supernatant liquid was decanted out of the container. The magnet was then removed and the remaining particles in the package were resuspended in another 10 milliliters of deionized water. The steps of collection, decanting and resuspension were repeated three times. After the third of these steps, the resuspended particles were sequentially washed twice with 10 milliliters each of 7M guanidine hydrochloride of pH 5.9, twice with 10 milliliters of deionized water and two twice with 10m. 50 mM EDTA (pH 8.0). The supernatants from each of these three washes were examined from 230 nm to 300 nm using a Hewlett Packard Diode-forming spectrometer covered with a blanket against each of the control solutions. No absorbance above the bottom at 260 nm was observed in any of the wash solutions obtained by testing the magnetic silica particles used in the above-cited Examples.

Claims (30)

CLAIMS:

1. A method for isolating a biological blank material from another material in a medium by: a. providing a medium that includes the biological target material; providing magnetic particles of silica capable of reversibly binding the biological target material; b. form a complex of the magnetic particles of silica and the biological target material by combining the magnetic particles of silica and the medium; c. removing the complex from the medium by applying an external magnetic field, and d. Separate the biological target material from the complex by eluting the biological target material whereby the isolated biological target material is obtained.

2. A method of isolating the biological target material according to claim 1, wherein the biological material isolated according to the method consists of a nucleic acid.

3. A method of isolating a biological target material according to claim 1, wherein the magnetic particles of silica provided in step (b) are capable of reversibly binding at least 2 micrograms of the biological target material per milligram of particle.

4. A method of isolating a biological target material according to claim 3, wherein the magnetic particles of silica provided in step (b) of the method are magnetic particles coated with silicon oxide.

5. A method of isolating a biological target material according to claim 1, wherein at least 60 percent of the biological target material in the complex is eluted from the particles in step (d).

6. A method of isolating a biological target material according to claim 1, wherein the biological target material eluted from the complex in step (d) does not contain more than 50 parts per million of the transition metal contaminants.

7. A method of isolating a biological blank material from other materials in a medium comprising the steps of: a) providing a medium containing the biological target material; b) providing a magnetic particle of silica with the ability to reversibly bind at least 2 micrograms of the biological target material per milligram of particle; c) forming a mixture comprising the medium and the magnetic particle of silica; d) adhering the biological blank material to the magnetic silica particle in the mixture; e) remove the magnetic particle of silica with the biological target material adhered thereto of the mixture by application of an external magnetic field; and f) eluting at least 60 percent of the biological blank material from the silica magnetic particle by exposing the particle to an elution solution.

8. A method of isolating the biological target material according to claim 7, wherein the biological material isolated according to the method consists of a nucleic acid material.

9. A method of isolating the biological target material according to claim 8, wherein the biological target material of nucleic acid isolated according to the method consists of a plasmid -DNA material.

10. A method of isolating a biological target material according to claim 8, wherein the isolated nucleic acid biological target material consists of a material of a DNA fragment.

11. A method of isolating a biological target material according to claim 7, wherein the magnetic silica particles that are provided in step (b) of the method are magnetic particles coated with silicon oxide.

12. A method of isolating the biological target material according to claim 7, wherein the mixture formed in step (c) comprises the medium, the magnetic particle of silica and a chaotropic salt, wherein the salt concentration chaotropic is high enough to cause the biological target material to adhere to the silica magnetic particle in step (d).

13. A method of isolating a biological target material according to claim 12, wherein the chaotropic salt in the mixture formed in step (c) consists of a chaotropic guanidinium salt consisting of guanidine hydrochloride or guanidine thiocyanate.

14. A method of isolating the biological target material according to claim 12, wherein The concentration of the chaotropic salt in the mixture formed in step (c) is at least 2 molar.

15. A method of isolating a biological target material according to claim 7, wherein the biological target material is adhered to the magnetic silica particle in step (d) by incubating the mixture.

16. A method of isolating a biological target material according to claim 15, wherein the biological target material is adhered to the magnetic silica particle in step (d) by incubating the mixture at room temperature for at least 30 minutes. seconds-

17. A method of isolating a biological target material according to claim 7, further comprising a step of washing the magnetic particle of silica after removal of the medium, before eluting the biological target material from the particle. .

18. A method of isolating a biological target material according to claim 17, wherein the step of washing is carried out using a washing solution comprising an alcohol and a salt.

19. A method of isolating a biological target material according to claim 18, wherein the step of washing is carried out using a solution of washing comprising at least 30 percent alcohol by volume and a stabilizer.

20. A method of isolating a biological target material according to claim 7, wherein the biological target material is eluted from the magnetic particle of silica in step (f) using water or an elution solution with low ionic strength. .

21. A method of isolating a biological target material according to claim 7, wherein the biological target material eluted from the magnetic silica particle in step (f) is essentially free of macromolecular or metal contaminants.

22. A method of isolating a plasmid DNA material from other materials in a medium comprising the steps of: a) providing a medium containing the plasmid DNA; b) providing a magnetic particle coated with silicon oxide with the ability to reversibly bind at least 2 micrograms of the biological target material per milligram of the particle. c) forming a mixture comprising the medium, the magnetic particle coated with silicon oxide and a chaotrophic salt, wherein the The concentration of the chaotropic salt in the mixture is high enough to cause the plasmid DNA to adhere to the particle; d) incubating the mixture more or less at room temperature until at least some amount of the biological blank material is adhered to the magnetic particle coated with silicon oxide; e) removing the magnetic particle coated with silicon oxide from a mixture using an external magnetic force; and f) eluting at least 60 percent of the plasmid DNA adhered to the magnetic particle coated with silicon oxide by exposing the particle to an elution solution.

23. A method of isolating a plasmid DNA material according to claim 22, wherein the chaotropic salt in the mixture formed in step (c) is a chaotropic guanidinium salt consisting of guanidine hydrochloride or guanidine thiocyanate. .

24. A method of isolating a plasmid DNA material according to claim 22 wherein the concentration of the chaotropic salt in the mixture formed in step (c) is between about 0.1 M and 7 M.

25. A method of isolating a plasmid DNA material according to claim 22, further comprising a step of washing the magnetic particle coated with silicon oxide after removal of the medium, and before eluting the plasmid DNA material from the particle.

26. A method of isolating a plasmid DNA material according to claim 25, wherein the step of washing is carried out using a washing solution comprising an alcohol and a salt.

27. A method of isolating a plasmid DNA material according to claim 25, wherein the step of washing is carried out using a washing solution comprising at least 30 percent alcohol by volume and a stabilizer.

28. A method of isolating a plasmid DNA material according to claim 22, wherein the plasmid DNA eluted from the magnetic silica particle in step (f) is essentially free of macromolecular or metal contaminants prone to interfere with further processing or analysis.

29. A case for isolating a biological blank material from a medium, the case comprising: an aliquot of the magnetic particles coated with silicon oxide suspended in a solution aqueous in a first container, wherein the particles have the ability to reversibly bind at least 2 micrograms of the biological target material per milligram of particle-

30. A case for isolating a biological target material according to claim 29, which further comprises: a chaotrophic salt in a second container; and a washing solution in a third container.