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KITS FOR CELL CONCENTRATION AND
LYSATE CLEARANCE USING
CROSS-REFERENCE TO RELATED 5
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. This application is also 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.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 15
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
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 35 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 2.22 and filtration system reference). Unfortunately, neither filtration nor centrifuga- 40 tion is amenable to automation. Specifically, neither can be performed at basic pipettor-diluter robotics stations, such as the Biomec®. 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 45 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 50 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 55 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 go means of clearing a lysate solution is amenable to automation.
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, 65 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 fiom 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® DNA purification systems products from Promega Corporation (Madison, Wis., U.S.A.), or the QiaPrep® 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.
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.
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 MagneSilTM particles from Promega, or BioMag® 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® Series 9600TM 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.
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.
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. 5 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.
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 15 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. Ger- 20 man 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 25 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.
Magnetically responsive beads designed for use in the isolation of target polymers, such as nucleic acids, and 30 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 35 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. 40 2, line 53).
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 45 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® isolation system, Life 50 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). 55
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 60 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 65 method can be used in a variety of applications, including restriction digestion and sequencing.
BRIEF SUMMARY OF THE INVENTION
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.
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.
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.
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.
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. 5
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a photograph of samples of plasmid DNA isolated with MagneSilTM particles (Promega) or varying amounts of MaglE-glycidyl-histidine particles, fractionated 10 by gel electrophoresis and visualized by staining with ethidium bromide, as described in Example 6.
FIG. 2 is a photograph of samples of plasmid DNA isolated from varying amounts of a culture of transformants oiE.coli DH5a cells using centrifugation ("Spin") on MagneSilTM particles (Promega Corp.) ("Mag"), followed by fractionation by gel electrophoresis on a short run gel, and visualization by staining with ethidium bromide, as described in Example 7.
FIG. 3 is a photograph of the same gel shown in FIG. 2, shot after electrophoresis was continued for a longer period of time.
FIG. 4 is a photograph of samples of DNA and RNA isolated from a mouse liver homogenate, using MaglE- 25 glycidyl-histidine particles, as described in Example 9, after fractionation by gel electrophoresis and visualization by staining with ethidium bromide.
FIG. 5 is a photograph of samples of DNA and RNA isolated from mouse spleen (lanes 2-5) and kidney (lanes 30 7-9), using MaglE-glycidyl-histidine particles, as described in Example 9, after the samples were fractionated by gel electrophoresis and visualized by staining with ethidium bromide, as described in Example 9.
FIG. 6 is a photograph of mouse liver RNA and DNA, 35 after digestion with DNase, fractionation by gel electrophoresis, and visualization by staining with ethidium bromide.
DETAILED DESCRIPTION OF THE 40
The present invention will now be described in detail, in part, by reference to the following definitions.
The term "solid phase" is used herein in a standard „
chromatographic sense, to refer to an insoluble, usually rigid, matrix or stationary phase which interacts with a solute, in this case a tissue or cell or target nucleic acid, in a solute mixture. In the methods and kits of the present invention magnetic particles function as a solid phase when 5Q added to various solute mixtures.
The term "surface", as used herein, refers to the portion of the support material of a solid phase which comes into direct contact with a solution when the solid phase is combined therewith. 55
The term "silica gel" as used herein refers to chromatography grade silica gel, a substance which is commercially available from a number of different sources. Silica gel is most commonly prepared by acidifying a solution containing silicate, e.g. by acidifying sodium silicate to a pH of less go than 11, and then allowing the acidified solution to gel. See, e.g. silica preparation discussion in Kurt-Othmer Encyclopedia of Chemical Technology, Vol. 21, 4th ed., Mary Howe-Grant, ed., John Wiley & Sons, pub., 1997, p. 1021.
As used herein, the term "silica magnetic particles" refers 65 to silica based solid phases which are further comprised of materials which have no magnetic field but which 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 which are not themselves magnetic in the absence of such a field.
The term "magnetic", as used herein refers to temporarily magnetic materials, such as fenimagnetic or ferromagnetic materials. The term encompasses paramagnetic and superparamagnetic materials.
The term "magnetic particle" refers to a matrix comprising a core of paramagnetic or superparamagnetc materials and a solid phase capable of forming a complex with a solute of interest.
The term "silica magnetic particles", as used herein refers to paramagnetic particles comprising a superparamagnetic core coated with siliceous oxide, having a hydrous siliceous oxide adsorptive surface (i.e. a surface characterized by the presence of silanol groups).
The term "magnetic ion exchange particles", as used herein, refers to paramagnetic particles with ion exchange ligands covalently attached thereto.
The term "pH dependent ion exchange magnetic particles", as used herein, refers to magnetic particles with a plurality of ion exchange ligands covalently attached thereto, which can act as cation exchangers at one pH and as anion exchangers at another pH. Such magnetic particles are particularly well suited for use in the methods and kits of the present invention, as their binding capacity to different substrates can be adjusted merely by varying the pH or salt conditions in a solution.
The term "pH dependent ion exchange silica magnetic particles", as used herein, refers to silica magnetic particles with a plurality of ion exchange ligands covalently attached thereto, which can act as cation exchangers at one pH and as anion exchangers at another pH. Such magnetic particles are particularly well suited for use in the methods and kits of the present invention, because substrates can selectively adsorb to the hydrous siliceous oxide adsorptive surface of the particle through hydrophobic interactions, to the ion exchange ligands through ion exchange, or to both the surface and ion exchange ligands, depending upon solution conditions.
The term "nucleic acid" as used herein refers to any DNA or RNA molecule or a DNA/RNA hybrid molecule. The term includes plasmid DNA, amplified DNA or RNA fragments, total RNA, mRNA, genomic DNA, and chromosomal DNA.
The term "target nucleic acid" as used herein refers to any particular species of nucleic acid to be isolated using magnetic particles according to a method of the present invention. The target nucleic acid is preferably at least 20 nucleotides long, more preferably at least 100 nucleotides long, and most preferably at least 1,000 nucleotides long.
The methods and kits of the present invention can be used to harvest or concentrate cells, to clear a solution of disrupted biological material, and/or to isolate a target nucleic acid from a solution, preferably from a solution of cleared disrupted biological material. In at least one step of each such method, a complex is formed in a solution between a solute and magnetic particles. The resulting complex is then isolated from or removed from the solution by the application of magnetic force. Magnetic particles suitable for use in any given step of the methods and kits of the present invention have the capacity to form a complex with the solute of interest in that particular step of the method.
The solute is the type of material to be isolated from or removed from a solution, using magnetic particles, accord8
ing to a method of the present invention. Cells to be concentrated or harvested are the solute in the harvesting method of the present invention. Disrupted biological material is the solute in the lysate or homogenate clearing method of the invention. A target nucleic acid is the solute when 5 magnetic particles are used to isolate the target nucleic acid from any solution comprising the target nucleic acid and other material, such as a cleared lysate or homogenate solution.
In one aspect of the methods of the present invention, 1° cells are harvested or concentrated using magnetic particles which can form a complex with the cells, under solution conditions designed to promote the formation of the complex. Silica magnetic particles and pH dependent ion exchange magnetic particles are both suitable for use in :5 harvesting or concentrating cells according to the method of the present invention. However, one of ordinary skill in the art could readily select other suitable magnetic particles for use in this particular embodiment of the invention.
Conditions which promote the formation of a magnetic 20 particle/solute complex vary, depending upon the nature of the solute and on the characteristics of the solid phase component of the magnetic particle. For example, when the magnetic particles are ion exchange magnetic particles or pH dependent ion exchange particles, the complex is pref- 25 erably formed as a result of ion exchange between the solute and ion exchange ligands at the surface of the particles. In order to promote such ion exchange interaction, there must be at least some salt present in the solution to promote ion exchange with the solute, and the pH of the solution must be within the range wherein the ion exchange ligand has a charge appropriate to exchange with the solute. When the magnetic particles are silica magnetic particles, the complex is preferably formed as a result of hydrophobic interactions between the solute and particles. When the magnetic particles are pH dependent ion exchange silica magnetic particles, the complex can be formed as a result of hydrophobic interactions between the solute and the siliceous oxide surface of the particles, as a result of ion exchange between the solute and the ion exchange ligands, or as a result of a combination of the two types of interactions. Preferred salt, pH, and other solution conditions to be used to promote formation of a complex with any given preferred substrate isolated according to the present methods or using the present kits are described below.
When the solute is intact cells, the complex is preferably formed in the presence of a low molecular weight alcohol, such as ethanol or isopropanol.
When the solute is disrupted biological material, such as 50 one finds in a cell lysate or tissue homogenate, and the magnetic particles are silica-based particles, the magnetic particle/solute complex is preferably formed in a solution which does not contain any more than trace amounts of alcohol or of chaotropic salts. Both alcohol and chaotropic 55 salts, such as guanidine thiocyanate or guanidine isothiocyanate, promote adsorption of nucleic acid materials to such particles. It is contemplated, however, that one could practice the present method of cell lysate clearance in the presence of alcohol or chaotropic salts if the concentration 60 of magnetic particles in a homogenate or lysate solution were low enough to clear the solution, but not high enough to adhere to a significant amount of the target nucleic acid in the solution.
When the solute is a target nucleic acid, formation of the 65 complex is preferably done in the presence of at least one agent known to promote reversible adsorption of the target
nucleic acid to the magnetic particles. The reversible adsorption reaction is preferably done through specific adsorption between the target nucleic acid and magnetic particles, leaving non-target material in solution. For example, when the target nucleic acid is plasmid DNA being isolated from a cleared lysate solution, the plasmid DNA is combined with magnetic particles under conditions wherein the plasmid DNA fonns a complex therewith while non-target materials, such as proteins, lipids, and chromosomal DNA remain in solution. When the magnetic particle is an ion exchange magnetic particle, the complex is formed in the presence of a counterion and in a solution with a pH at which the ion exchange ligands have the capacity to exchange with the target nucleic acid. When the magnetic particles are silica magnetic particles, formation of the complex is preferably done in the presence of an agent selected from the group consisting of a low molecular weight alcohol, a high concentration of a non-chaotropic salt, and a chaotropic salt, or a combination of any of the above. For methods of adsorption and desorption of target nucleic acids to silica magnetic particles, which are suitable for use in the present invention, see international patent application number PCT/US98/ 01149 for METHODS OF ISOLATING BIOLOGICAL TARGET MATERIALS USING SILICA MAGNETIC PARTICLES, published as WO 98/31840, incorporated by reference herein.
The solid phase of the magnetic particles used in the present methods can be made of any common support material, including soft gel supports such as agarose, polyacrylamide, or cellulose, or hard support material such as polystyrene, latex, methacrylate, or silica.
When the solid phase support material is silica, it is preferably in the form of silica gel, siliceous oxide, solid silica such as glass or diatomaceous earth, or a mixture of two or more of the above. Silica based solid phases suitable for use in the pH dependent ion exchange matrixes of the present invention include the mixture of silica gel and glass described in U.S. Pat. No. 5,658,548, the silica magnetic particles described in PCT Publication Number WO 98/31840, and solid phases sold by Promega Corporation for use in plasmid DNA isolation, i.e. Wizard® Minipreps DNA Purification Resin. Silica gel particles are particularly preferred for use as the solid phase in the pH dependent ion exchange matrix and methods of the present invention. Silica gel particles are stable at much higher pressures than solid phases made from soft gel support material, making the silica gel solid phases suitable for HPLC as well as LC and batch separation applications.
Silica magnetic particles can be used to concentrate cells, clear lysates, or isolate target nucleic acids according to the methods the present invention. When silica magnetic particles are employed, the silica-based surface material of the particle specifically interacts with the various solutes isolated or removed therewith.
When the silica magnetic particles have ion exchange ligands covalently attached thereto, the silica-based surface material acts primarily as a solid support for the ion exchange ligands, which enable the particles to form complexes with the various solutes to be isolated or removed from any given solution. When used to isolate a target nucleic acid, the ion exchange ligands are preferably capable of forming a complex with the target nucleic acid by exchanging therewith at one pH, and of releasing the target nucleic acid at another pH. The most preferred ion exchange ligands are ones which complex with the target nucleic acid at a pH which is lower than a neutral pH, and which release the target nucleic acid at about a neutral pH and in low salt