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  Cell concentration and lysate clearance using paramagnetic particlesUSPTO Application #: 20060240448Title: Cell concentration and lysate clearance using paramagnetic particles Abstract: Methods are disclosed for using paramagnetic particles to concentrate or harvest cells. Methods are also disclosed for clearing a solution of disrupted biological material, such as a lysate of cells or a homogenate of mammalian tissue. Methods are also disclosed for using paramagnetic particles to isolate target nucleic acids, such as RNA or DNA, from a solution cleared of disrupted biological material using the same type or a different type of paramagnetic particle. Kits are also disclosed for use with the various methods of the present invention. Nucleic acids isolated according to the present methods and using the present kits are suitable for immediate use in downstream processing, without further purification. (end of abstract) Agent: Michael Best & Friedrich, LLP - Madison, WI, US Inventors: Rex Bitner, Craig E. Smith, Douglas H. White, Braeden L. Butler, Jacqui Sankbeil USPTO Applicaton #: 20060240448 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060240448. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. application Ser. No. 09/475,958, filed Dec. 30, 1999, which claims the benefit of U.S. Provisional Application No. 60/134,156, filed May 14, 1999, and is a continuation-in-part of U.S. application Ser. No. 09/064,449, filed Apr. 22, 1998, now U.S. Pat. No. 6,194,562. This application claims priority to each of these applications and hereby fully incorporates the subject matter of each of these applications. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] Not applicable. TECHNICAL FIELD [0003] This invention relates generally to the use of magnetically responsive particles, such as magnetically responsive silica gel particles or magnetically responsive ion exchange particles, to harvest or to concentrate cells or biological tissue. This invention also relates to the use of such particles to clear lysates or homogenates of such cells or tissue. This invention relates, furthermore, to the use of such particles to isolate target nucleic acids, such as plasmid DNA, chromosomal DNA, DNA fragments, total RNA, mRNA, or RNA/DNA hybrids from non-target material in a cell lysate. BACKGROUND OF THE INVENTION [0004] Cells in a liquid culture must be concentrated or harvested before they can be preserved for later use, stained for direct analysis, or processed to isolate target specific materials therefrom. Most cell harvesting and concentration techniques involve centrifugation, filtration, or a combination of centrifugation and filtration. (See, e.g., Molecular Cloning, (1989) ed. by Sambrook et al., pp. 222 and filtration system reference). Unfortunately, neither filtration nor centrifugation is amenable to automation. Specifically, neither can be performed at basic pipettor-diluter robotics stations, such as the Biomec.RTM.. When it becomes necessary to isolate or analyze certain types of material in the interior of a cell, such as a target nucleic acid or a protein, the cell membrane must be disrupted and the contents of the cell released into the solution surrounding the cell. Such disruption can be accomplished by mechanical means (e.g., by sonication or by blending in a mixer), by enzymatic digestion (e.g. by digestion with proteases), or by chemical means (e.g., by alkaline lysis followed by addition of a neutralization solution). Whatever means is used to disrupt a cell, the end product, referred to herein as a lysate solution, consists of the target material and many contaminants, including cell debris. The lysate solution must be cleared of as many of the large contaminants as possible before the target material can be further isolated therefrom. Either or both of the same two means described above, i.e. centrifugation and filtration, have been used to clear lysate solutions prior to further processing. However, for reasons given above, neither means of clearing a lysate solution is amenable to automation. [0005] Many different systems of materials and methods have been developed for use in the isolation of nucleic acids from cleared lysate solutions. Many such systems are silica based, such as those which employ controlled pore glass, filters embedded with silica particles, silica gel particles, resins comprising silica in the form of diatomaceous earth, glass fibers or mixtures of the above. Each such silica-based solid phase separation system is configured to reversibly bind nucleic acid materials when placed in contact with a medium containing such materials in the presence of chaotropic agents. The silica-based solid phases are designed to remain bound to the nucleic acid material while the solid phase is exposed to an external force such as centrifugation or vacuum filtration to separate the matrix and nucleic acid material bound thereto from the remaining media components. The nucleic acid material is then eluted from the solid phase by exposing the solid phase to an elution solution, such as water or an elution buffer. Numerous commercial sources offer silica-based resins designed for use in centrifugation and/or filtration isolation systems, e.g. Wizard.RTM. DNA purification systems products from Promega Corporation (Madison, Wis., U.S.A.), or the QiaPrep.RTM. DNA isolation systems from Qiagen Corp. (Chatsworth, Calif., U.S.A.). Unfortunately, the type of silica-based solid phases described above all require one use centrifugation or filtration to perform the various isolation steps in each method, limiting the utility of such solid phases in automated systems. [0006] Magnetically responsive solid phases, such as paramagnetic or superparamagnetic particles, offer an advantage not offered by any of the silica-based solid phases described above. Such particles could be separated from a solution by turning on and off a magnetic force field, or by moving a container on to and off of a magnetic separator. Such activities would be readily adaptable to automation. [0007] Magnetically responsive particles have been developed for use in the isolation of nucleic acids. Such particles generally fall into either of two categories, those designed to reversibly bind nucleic acid materials directly, and those designed to reversibly bind nucleic acid materials through an intermediary. For an example of particles of the first type, see silica based porous particles designed to reversibly bind directly to DNA, such as MagneSil.TM. particles from Promega, or BioMag.RTM. magnetic particles from PerSeptive Biosystems. For examples of particles and systems of the second type designed to reversibly bind one particular type of nucleic acid (mRNA), see the PolyATract.RTM. Series 9600.TM. mRNA Isolation System from Promega Corporation (Madison, Wis., U.S.A.); or the streptavidin coated microsphere particles from Bangs Laboratories (Carmel, Ind., U.S.A.). Both of these systems employ magnetically responsive particles with streptavidin subunits covalently attached thereto, and biotin with an oligo(dT) moiety covalently attached thereto. The biotin-oligo(dT) molecules act as intermediaries, hybridizing to the poly(A) tail of mRNA molecules when placed into contact therewith, then binding to the streptavidin on the particles. The mRNA molecules are then released in water. [0008] Indirect binding magnetic separation systems for nucleic acid isolation or separation require at least three components, i.e. magnetic particles, an intermediary, and a medium containing the nucleic acid material of interest. The intermediary/nucleic acid hybridization reaction and intermediary/particle binding reaction often require different solution and/or temperature reaction conditions from one another. Each additional component or solution used in the nucleic acid isolation procedure adds to the risk of contamination of the isolated end product by nucleases, metals, and other deleterious substances. [0009] Various types of magnetically responsive silica based particles have been developed for use as solid phases in direct or indirect nucleic acid binding isolation methods. One such particle type is a magnetically responsive glass bead, preferably of a controlled pore size. See, e.g. Magnetic Porous Glass (MPG) particles from CPG, Inc. (Lincoln Park, N.J., U.S.A.); or porous magnetic glass particles described in U.S. Pat. Nos. 4,395,271; 4,233,169; or 4,297,337. Nucleic acid material tends to bind very tightly to glass, however, so that it can be difficult to remove once bound thereto. Therefore, elution efficiencies from magnetic glass particles tend to be low compared to elution efficiencies from particles containing lower amounts of a nucleic acid binding material such as silica. [0010] Another type of magnetically responsive particle designed for use as a solid phase in direct binding and isolation of nucleic acids, particularly DNA, is a particle comprised of agarose embedded with smaller ferromagnetic particles and coated with glass, e.g. U.S. Pat. No. 5,395,498. Yet another type of magnetically responsive particle designed for direct binding and isolation of nucleic acids is produced by incorporating magnetic materials into the matrix of polymeric silicon dioxide compounds, e.g. German Patent Application No. DE 43 07 262. The latter two types of magnetic particles, the agarose particle and the polymeric silicon dioxide matrix, tend to leach iron into a medium under the conditions required to bind nucleic acid materials directly to each such magnetic particle. It is also difficult to produce such particles with a sufficiently uniform and concentrated magnetic capacity to ensure rapid and efficient isolation of nucleic acid materials bound thereto. [0011] Magnetically responsive beads designed for use in the isolation of target polymers, such as nucleic acids, and methods for their use therein are described in U.S. Pat. No. 5,681,946 and in International Publication No. WO 91/12079. These last beads are designed to become nonspecifically associated with the target polymer, only after the target polymer is precipitated out of a solution comprising the target polymer and the beads. Magnetic force is used to isolate the beads and polymer associated therewith from the solution. The magnetically responsive beads recommended for use in this last system are "finely divided magnetizable material encapsulated in organic polymer." ('946 Patent, col. 2, line 53). [0012] A variety of solid phases have also been developed with ion exchange ligands capable of exchanging with nucleic acids. However, such systems are generally designed for use as a solid phase of a liquid chromatography system, for use in a filtration system, or for use with centrifugation to separate the solid phase from various solutions. Such systems range in complexity from a single species of ligand covalently attached to the surface of a filter, as in DEAE modified filters (e.g., CONCERT.RTM. isolation system, Life Technology Inc., Gaithersburg, Md., U.S.A.), to a column containing two different solid phases separated by a porous divider (e.g., U.S. Pat. No. 5,660,984), to a chromatography resin with pH dependent ionizable ligands covalently attached thereto (e.g., U.S. Pat. No. 5,652,348). [0013] Materials and methods are needed which enable one to automate as many steps as possible to quickly and efficiently isolate target nucleic acids from cells or mammalian tissue. Specifically, methods and materials are needed for the concentration or harvesting of cells, for the clearing of solutions of disrupted cells or tissue, and for the isolation of target nucleic acids from such cleared solutions, wherein labor-intensive steps such as filtration or centrifugation are not required. The present invention addresses each of these needs. Nucleic acids isolated according to the present method can be used in a variety of applications, including restriction digestion and sequencing. BRIEF SUMMARY OF THE INVENTION [0014] In the methods of the present invention, magnetic particles are used to process biological material. In one embodiment, the present invention is a method of concentrating or harvesting cells comprising the steps of: (a) combining a solution with cells contained therein, such as an overnight culture of bacteria in a growth medium or white cells in whole blood with magnetic particles under conditions wherein the cells form a complex with the magnetic particles; and (b) isolating the magnetic particle/cell complex from the solution by application of magnetic force, e.g., by means of a magnet. [0015] In another embodiment, the present invention is a method of clearing disrupted biological material, such as a cell lysate or a homogenate of mammalian tissue, comprising the steps of: (a) providing a solution comprising a disrupted biological material, such as a cell lysate or homogenized tissue; (b) combining the solution with magnetic particles under conditions wherein the disrupted biological material forms a complex with the magnetic particles; and (c) isolating the complex from the solution by application of magnetic force. [0016] In yet another embodiment, the present invention is a method of isolating a target nucleic acid from a solution of disrupted biological material, comprising the target nucleic acid, a first non-target material, and a second non-target material, comprising the steps of: (a) combining a solution of the disrupted biological material with first magnetic particles under conditions wherein the first non-target material forms a first complex with the first magnetic particles; (b) separating the first complex from the solution of disrupted biological material by application of magnetic force, forming a cleared solution comprising the target nucleic acid and the second non-target material; (c) combining the cleared solution with second magnetic particles under conditions wherein the target nucleic acid adsorbs to the second magnetic particles, forming a second complex; (d) isolating the second complex from the cleared solution; (e) washing the second complex by combining the second complex with a wash solution and separating the second complex from the wash solution by magnetic force; and (f) combining the washed second complex with an elution solution, under conditions wherein the target material is desorbed from the second magnetic particles. [0017] In yet another embodiment, the present invention also consists of kits with at least one type of magnetic particle and at least one solution needed to practice one or more of the methods of the invention, described above. In one such embodiment, the present invention is a kit comprising: (a) a first container of first magnetic particles with the capacity to form a first complex with first non-target material in a first solution of disrupted biological material comprising the first non-target material and the target nucleic acid; and (b) a second container of second magnetic particles with the capacity to form a second complex with the target nucleic acid, under solution conditions designed to promote the specific adsorption of the target nucleic acid to the second magnetic particles. [0018] The methods and materials of the present invention can be used to isolate target nucleic acids including, but not limited to plasmid DNA, total RNA, mRNA, RNA/DNA hybrids, amplified nucleic acids, and genomic DNA from a variety of contaminants, including but not limited to agarose and components of a bacteria, animal tissue, blood cells, and non-target nucleic acids. Applications of the methods and compositions of the present invention to isolate nucleic acids from a variety of different media will become apparent from the detailed description of the invention below. Those skilled in the art of this invention will appreciate that the detailed description of the invention is meant to be exemplary only and should not be viewed as limiting the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Continue reading... 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