Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
  • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
  • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Definitions

• This invention relates to an apparatus and method that can be used for separating, isolating, and purifying polynucleotides, including single-stranded and double-stranded DNA and RNA. In some embodiments, this invention relates to methods and devices for separating target polynucleotides having a predetermined size or range of sizes. BACKGROUND OF THE INVENTION
• RNA RNA
• separation and purification of polynucleotides such as DNA (both double and single stranded) and RNA is of critical importance in molecular biology, and improved methods are a focus of current interest.
• a variety of methodologies have been developed for achieving these separations.
• separation techniques have often relied on gel electrophoresis.
• a polynucleotide of interest can be purified from a sample by gel electrophoresis of the sample followed by physical excision of the band corresponding to the polynucleotide (e.g., cutting out the band and recovery from low melting temperature agarose, electroelution, electrophoresis onto NA-45 paper (Schleicher and Schuell)).
• Disadvantages of gel based techniques include the time and effort required for sample preparation, gel preparation, electrophoresis, band detection, band excision/recovery, and post-excision clean-up. These disadvantages can be particularly burdensome where the high-throughput processing of multiple samples is desired. Furthermore, polynucleotides can become covalently modified by the chemicals used during the fractionation process (e.g., formaldehyde or acrylamide), and these techniques often involve the used of hazardous chemicals (e.g., acrylamide, ethidium bromide, methylmercuric hydroxide).
• the chemicals used during the fractionation process e.g., formaldehyde or acrylamide
• hazardous chemicals e.g., acrylamide, ethidium bromide, methylmercuric hydroxide
• a further disadvantage of methods that rely on binding of anionic DNA is the required use of high concentrations of nonvolatile salts in the mobile phase; this interferes with subsequent isolation and measurement (e.g. mass spectrometry analysis) on the separated fragments.
• spin columns containing beads coated with poly-T oligomers are often used (e.g., Poly(A)PureTM mRNA Purification Kit, Ambion, Inc., Austin, TX; OligotexTM mRNA Purification System, Qiagen, Inc., Valencia, CA).
• the disadvantages of this technique include a requirement for high amounts of total RNA sample due to low recovery of mRNA, contamination of the product (e.g.
• the instant invention provides a non-HPLC chromatographic method for purifying a target polynucleotide or polynucleoitdes or separating a target polynucleotide or plurality of polynucleotides.
• the invention provides a non-HPLC chromatographic method of purifying a polynucleotide comprising the steps of applying the target polynucleotide to a separation medium having a non-polar separation surface in the presence of a counterion agent, whereby the polynucleotide is bound to the separation medium;eluting the target polynucleotide from the separation medium by passing through the separation medium an elution solution containing a concentration of organic solvent sufficient to elute the target polynucleotide from the separation medium; and collecting the eluted target polynucleotide.
• the target polynucleotide is applied to the separation medium as a component of a loading solution containing a non-target molecule.
• the non-target molecule is not bound to the separation medium in the presence of the loading solution, and is thereby eluted from the separation medium and separated from the target polynucleotide by passing the loading solution through the separation medium.
• the non-target molecule is bound to the separation medium in the presence of the loading solution, and including an additional step between steps (a) and (b) of eluting the non-target molecule from the separation medium by passing through the separation medium a wash solution containing a counterion agent and a concentration of organic solvent sufficient to elute the non-target molecule, but insufficient to elute the target polynucleotide from the separation medium, whereby the non-target molecule is separated from the target polynucleotide.
• the non-target molecule remains bound to the separation medium in the presence of the elution solution, and is thereby separated from the target polynucleotide during the elution step.
• the separation medium has a nonpolar separation surface that is substantially free of multivalent cations that are capable of interfering with polynucleotide separations, and/or the solutions used are substantially free of multivalent cations capable of interfering with polynucleotide separations.
• the non-target molecule is a polynucleotide.
• the polynucleotide is double-stranded DNA, RNA, or single-stranded DNA.
• the DNA can be an oligonucleotide.
• a mixture of polynucleotide fragments of varying nucleotide length is applied to the separation medium, and the elution solution contains a concentration of organic solvent that has been predetermined to elute polynucleotide fragments falling within a defined range of nucleotide lengths, whereby polynucleotide fragments falling within the defined range of nucleotide lengths are eluted from the separation medium and thereby separated from other polynucleotides of the mixture.
• the polynucleotide fragments are double-stranded DNA fragments, single-stranded DNA fragments, or RNA fragments.
• the separation medium that is supported in a spin column.
• the separation medium is preferably in communication with an upper solution input chamber and a lower eluant receiving chamber, wherein the loading solution containing the polynucleotide and a counterion agent is applied to the separation medium by introducing the solution into the upper solution input chamber and centrifuging the spin column under conditions where the polynucleotide substantially binds to the separation medium, wherein the elution solution is passed through the separation medium by centrifugation of the spin column, and wherein the eluted polynucleotide is collected in the lower eluant receiving chamber.
• the polynucleotide is eluted from separation medium that is supported in a vacuum tray separation device.
• the separation medium comprises particles selected from the group consisting of silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon, insoluble polysaccharide, and diatomaceous earth, the particles having separation surfaces which are coated with a hydrocarbon or non-polar hydrocarbon substituted polymer, or have substantially all polar groups reacted with a non-polar hydrocarbon or substituted hydrocarbon group, wherein the surfaces are non-polar.
• the separation medium comprises polymer beads having an average diameter of 0.5 to 100 microns, the beads being unsubstituted polymer beads or polymer beads substituted with a moiety selected from the group consisting of hydrocarbon having from one to 1 ,000,000 carbons.
• the separation medium comprises a monolith.
• the separation medium comprises capillary channels.
• the separation medium has been subjected to acid wash treatment to remove any residual surface metal contaminants and/or has been subjected to treatment with a multivalent cation binding agent.
• the organic solvent employed is selected from the group consisting of alcohol, nitrile, dimethylformamide, tetrahydrofuran, ester, ether, and mixtures of one or more thereof.
• a particularly preferred organic solvent comprises acetonitrile.
• the counterion agent is selected from the group consisting of lower alkyl primary amine, lower alkyl secondary amine, lower alkyl tertiary amine, lower trialkylammonium salt, quaternary ammonium salt, and mixtures of one or more thereof.
• Particularly preferred counterion agents include octylammonium acetate, octadimethylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium
• the target polynucleotide is applied to the separation medium under denaturing conditions.
• the method is used to separate a sample containing RNA and genomic into a RNA-containing fraction and a genomic DNA-containing fraction.
• Another aspect of the invention is a device for purifying a target polynucleotide comprising a tube having: an upper solution input chamber; a lower eluant receiving chamber; and a fixed unit of separation medium supported therebetween, wherein the separation medium has a nonpolar separation surface that is substantially free of multivalent cations that are capable of interfering with polynucleotide separations.
• Preferred embodiments of this aspect of the invention employ a separation medium selected from the group consisting of beads, capillary channels and monolith structure.
• the fixed unit of separation medium comprise a fixed bed of separation medium particles, especially particles selected from the group consisting of organic polymer and inorganic particles having a nonpolar surface.
• the device for purifying a target polynucleotide has a closed lower chamber and/or the the lower chamber has an open bottom portion.
• the device can include an eluant container shaped to receive said lower chamber.
• the eluant chamber is a centrifuge vial.
• the afore-mentionid cylinder is a member of an array of cylinders and the eluant container is a member of an array of eluant containers, and the array of cylinders and array of containers have matching configurations.
• the invention provides a separation system comprising a multicavity separation plate having outer sealing edges, a multiwell collection plate and a vacuum system having a separation plate sealing means forming a sealed engagement with the outer sealing edges of the multicavity separation plate and a vacuum cavity receiving the multiwell collection plate;
• the multicavity separation plate including an array of tubes, each tube having an upper solution input chamber, a lower eluant receiving chamber with an bottom opening therein, and a fixed unit of separation medium supported therein, the separation medium having nonpolar separation surfaces that are free from multivalent cations that are capable of interfering with polynucleotide separations;
• the multiwell collection plate having collection wells which are positioned to receive liquid from the bottom opening of the lower eluant receiving chamber.
• Preferred embodiments of this aspect of the invention employ a separation medium selected from the group consisting of beads, capillary channels and monolith structure.
• the fixed unit of separation medium comprise a fixed bed of separation medium particles, especially particles selected from the group consisting of organic polymer and inorganic particles having a nonpolar surface.
• FIG. 1 is a cross-sectional representation of a spin vial system for low pressure separations according to this invention.
• FIG. 2 is a multiwell plate separation system of this invention in combination with a vacuum attachment.
• FIG. 3 is the top view of a multiwell plate of FIG. 2.
• FIG. 4 is a cross-sectional view of the separation tray of FIG. 2 taken along
• FIG. 5 is an enlarged view of a single separation cell of the multiwell plate of
• FIG. 6 is a chromatogram from a MIPC analysis of RNA size markers. Peaks are labeled with the number of nucleotides of the eluted molecules.
• FIG. 7 is a chromatogram from a MIPC analysis of RNA size markers.
• FIG. 8 is a chromatogram from a MIPC analysis of total RNA from a plant extract.
• FIG. 9 is a chromatogram from a MIPC analysis of RNA from a plant extract after a first affinity purification.
• FIG. 10 is a chromatogram from a MIPC analysis of RNA from a plant extract after a second affinity purification.
• FIG. 11 is a chromatogram from a MIPC analysis of mouse brain mRNA.
• FIG. 12 is a chromatogram from a MIPC analysis of human brain mRNA.
• FIG. 13 is a chromatogram from a MIPC analysis of human brain mRNA.
• FIG. 14 shows the release of eight DNA fragments from polymer beads in single equilibria bulk separations (under conditions as described in TABLE 1 ) showing the dependence on the acetonitrile concentration.
• FIG. 15 is a separation of pUC18-DNA Haelll digest on two discs containing binding media placed in series and containing nonporous poly(styrene- divinylbenzene) polymer beads. The dimensions of each disc was 0.7 mm x 4.6 mm i.d.
• Fig. 16 is a is a chromatogram of a pUC 18 Msp I standard mixture of dsDNA fragments used in Example 14.
• Fig. 17 is a chromatogram of the low molecular weight and small base-pair length fraction eluant obtained in Example 14.
• Fig. 18 is a chromatogram of the high base-pair length fraction eluant obtained in Example 14, demonstrating the efficacy of the spin column device for purifying high base-pair length components of a mixture of DNA fragments.
• Fig. 19 is a chromatogram of a pBR322 standard mixture of dsDNA fragments used in Example 15.
• Fig. 20 is a chromatogram of the low molecular weight and small base-pair length fraction eluant obtained in Example 15.
• Fig. 21 is a chromatogram of the high base-pair length fraction eluant obtained in Example 15.
• Fig. 22 is a chromatogram obtained in the procedure of Example 16.
• Fig. 23 is a chromatogram obtained in the procedure of Example 16.
• FIG. 24 shows a chromatogram obtained for unpurified product of polynucleotide kinase reaction.
• FIG. 25 shows a chromatogram obtained for product of polynucleotide kinase reaction subsequent to spin column purification.
• FIG. 26a is a chromatogram representing the IP-RP-HPLC separation of the
• FIG. 26b is a chromatogram representing the IP-RP-HPLC separation of the
• FIG. 26c is a chromatogram representing the IP-RP-HPLC separation of the
• FIG. 27a is a chromatogram representing the IP-RP-HPLC separation of the
• FIG. 27b is a chromatogram representing the IP-RP-HPLC separation of the
• IP-RP-HPLC Ion pairing reverse phase HPLC
• a reverse phase i.e., hydrophobic stationary phase
• a mobile phase that includes an alkylated cation (e.g., triethylammonium) that is believed to form a bridging interaction between the negatively charged polynucleotide and non-polar stationary phase.
• alkylated cation e.g., triethylammonium
• the alkylated cation-mediated interaction of polynucleotide and stationary phase can be modulated by the polarity of the mobile phase, conveniently adjusted by means of a solvent that is less polar than water, e.g., acetonitrile.
• Performance is enhanced by the use of a non-porous separation medium, as described in U.S. Patent Application No. 5,585,236, incorporated by reference herein in its entirety. It has been shown, for example, that under non-denaturing conditions the retention time of a double-stranded DNA fragment is dictated by the size of the fragment; the base composition or sequence of the fragment does not appreciably affect the separation, see U.S. Patent Application No. 5,772,889.
• IP-RP-HPLC Matched Ion Polynucleotide Chromatography
• MIPC is characterized by the use of solvents and chromatographic surfaces that are substantially free of multivalent cation contamination that can interfere with polynucleotide separation.
• IP-RP-HPLC is able to rapidly achieve good polynucleotide separations, the columns and other components of the system are relatively expensive. This can limit the application of the techniques for the use in the routine processing a large number of samples. It would be desirable to have available less expensive purification methods and apparatus that at least to some extent achieve the superior performance of IP-RP-HPLC, but in a more affordable format suited to the economical and rapid preparation of multiple polynucleotide.
• the instant invention achieves this aim, thus providing a valuable contribution to related fields of endeavour such as molecular biology and medicine.
• polynucleotide e.g., double-stranded DNA, RNA
• the instant invention pertains to the processing of polynucleotides in general, and is not intended to be limited to any particular species.
• the present invention provides novel methods and apparatus for separating and purifying polynucleotides. This process exploits the ability of polynucleotides, in the presence of certain counterions, to bind non-specifically and reversibly to a solid phase separation medium having a hydrophobic surface, e.g., chromatography beads.
• the polynucleotide can be present in solution with water or in a reaction buffer.
• a solution can also contain other components, such as other biomolecules, inorganic compounds and organic compounds as long as such other components do not interfere significantly with the binding process of the invention.
• the solution can be a preparation of total RNA and/or genomic DNA from a cell type or organism.
• the process can be applied with any system which can retain the separation medium and provides means to rapidly pass liquids through the separation medium.
• IP-RP-HPLC has been shown to be effective for separating polynucleotides.
• the instant invention pertains to the use of non-HPLC chromatographic methods for separating and/or purifying polynucleotides.
• non-HPLC chromatographic method is intended to encompass any chromatographic method which does not involve the use of a pump to generate high pressure to force eluant through a chromatography column.
• HPLC separations are typically achieved at pressures greater than 1000 psi, often reaching 2000 to 3000 psi, and in some instances reaching 6000 psi or higher.
• Non-HPLC chromatographic methods are characterized by the use of alternate means for driving the eluant through the column.
• alternate means include gravity, low or medium pressure pumps (e.g., peristaltic pumps), centrifugal force, high pressure gas and vacuum pressure.
• non-HPLC chromatography methods are more economical than HPLC, which represents a significant advantage of the instant invention.
• the method is able to isolate a target polynucleotide, or target polynucleotides sharing predetermined physical characteristics (e.g., size ranges, hydrophobicity, poly-A tails, etc.), from a larger pool of non-target molecules (e.g., biomolecules, non-target polynucleotides).
• predetermined physical characteristics e.g., size ranges, hydrophobicity, poly-A tails, etc.
• Exemplary applications of the invention include purification of plasmid DNA (e.g., from a miniprep), purification of the product of PCR amplification, purification of chemically synthesized oligonucleotide, and recovery of polynucleotide after enzymatic modification (e.g., phosphorylation by polynucleotide kinase).
• the invention can be used to isolate a pool of RNAs enriched for a particular class of RNA molecules (e.g., mRNAs, rRNAs, tRNAs).
• the method can also be used to separate selected RNA molecules from other macromolecules (e.g., genomic DNA, proteins, carbohydrates) or small molecule contaminants.
• the method can be used to stabilize RNA molecules by separating the RNA from species capable of promoting RNA degradation, particularly RNases and other nucleases.
• the method is used to separated polynucleotides (double- or single-stranded) of a length greater than 100 nucleotides, more preferably greater than 500 nucleotides, still more preferably longer than 1000 nucleotides, even more preferably longer than 1500 nucleotides, and most preferably greater than 2500 nucleotides.
• the invention is particularly useful for the separation of tagged polynucleotides.
• Non-limiting examples of polynucleotides tags suitable for use with the instant invention include fluorescent groups, hydrophobic or hydrophillic groupls, biotin, digoxigenin, etc..
• fluorescent groups suitable for use with the instant invention include 5-carboxyfluorescein (5-FAM), 6- carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), N,N,N'-N-tetramethyl-6-carboxy rhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4 ) 7,2 , ,4 , ,5 ⁇ 7'-hexachloro-6-carboxy-fluorescein (HEX-1 ), 4,7,2',4 , ) 5 , I T- hexachloro-5-carboxy-fluorescein (HEX-2), 2',4',5',7 ,
• Fluorescent labels can be attached to DNA using standard procedures, e.g. for a review see Haugland, "Covalent Fluorescent Probes," in Excited States of Biopolymers, Steiner, Ed. (Plenum Press, New York, 1983), incorporated by reference herein in its entirety.
• a fluorescent group can be covalently attached to a desired primer by reaction with a 5'-amino-modified oligonucleotide in the presence of sodium bicarbonate and dimethylformamide, as described in U.S. Patent Application No. 09/169,440.
• the reactive amine can be attached by means of the linking agents disclosed in U.S. patent No. 4,757,141.
• covalently tagged primers can be obtained commercially (e.g., from Midland Certified Reagent, Co.). Fluorescent dyes are available form Molecular Probes, Inc. (Eugene, OR), Operon Technologies, Inc., (Alameda, CA) and Amersham Pharmacia Biotech (Piscataway, NJ), or can be synthesized using standard techniques. Fluorescent labeling is described in U.S. Patent No. 4,855,225.
• Polynucleotides for use in the disclosed method can be part of a crude cellular or nuclear extract, partially purified, or extensively purified.
• DNA molecules can be the product of in vivo or in vitro amplification (e.g., PCR) or chemical synthesis (oligonucleotides).
• RNA molecules can also be made by in vitro transcription or by direct synthesis.
• the method can be used, for example, to purify an individual polynucleotide or a plurality of polynucleotides (e.g., a synthetic oligonucleotide or PCR amplification product), to separate polynucleotides from other biomolecules, to separate one species of polyucleotide from another (e.g., genomic DNA from RNA or plasmid DNA, mRNA from other RNA species).
• Polynucleotides can be prepared using known methods for preparing cellular extracts and for purifying polynucleotides. Methods for preparing extracts containing DNA and/or RNA molecules are described in, for example, Sambrook et al., and Ausubel et al.
• DNA molecules can also be produced recombinantly using known techniques, by in vitro transcription, and by direct synthesis.
• DNA encoding RNA molecules can be obtained from known clones, by synthesizing a DNA molecule encoding an RNA molecule, or by cloning the gene encoding the RNA molecules.
• Techniques for in vitro transcription of RNA molecules and methods for cloning genes encoding known RNA molecules are described by, for example, Sambrook et al. Polynucleotides can be prepared, for example, on an Applied Biosystems (Foster City, CA) 392 DNA/RNA synthesizer using standard phosporamidite chemistry.
• the method can be applied to an RNA preparation that has been enriched for RNA containing poly-A tails (associated with most mature mRNA molecules) from total RNA.
• RNA preparation that has been enriched for RNA containing poly-A tails (associated with most mature mRNA molecules) from total RNA.
• These can be prepared by affinity chromatography using beads coated with poly-T oligomers, as described, for example, in Sambrook and Ausubel. Separation columns containing such beads are commercially available from a number of sources (e.g., Poly(A)PureTM mRNA Purification Kit, Ambion, Inc., Austin, TX; OligotexTM mRNA Purification System, Qiagen, Inc., Valencia, CA).
• a first solution containing a polynucleotide, or a collection of polynucleotides is applied to a separation medium having a nonpolar, preferably nonporous surface, the first solution containing counterion and a polynucleotide- binding concentration of organic solvent, whereby a target polynucleotide, or plurality of polynucleotides, is non-specifically and reversibly bound to the medium.
• the target polynucleotide or polynucleotides are then removed from the medium by contacting the medium with a second solution containing counterion and a concentration of organic solvent suffficient to elute the target polynucleotide or polynucleotides from the separation medium into a distinct segment of eluant.
• the concentration of organic solvent sufficient to elute the target polynucleotides is predetermined based on the length and/or physical characteristics of the target.
• the separation can be conducted as a batch process in a container.
• the volume of the container can vary widely depending on the amount of mixture to be separated.
• the container can be, for example, a low-pressure (e.g., ambient pressure) column, a spin column, a web, a pad, a flask, a well, or a tank.
• the size of such a container can be as small as a well on a multi-well microtiter plate or as large as a multi-liter vat, for example.
• the separation medium takes the form of chromatographic beads. Beads useful in the batch process can be a variety of shapes, which can be regular or irregular; preferably the shape maximizes the surface area of the beads.
• the beads should be of such a size that their separation from solution, for example by filtration or centrifugation, is not difficult.
• polynucleotide is defined as a polymer containing an indefinite number of nucleotides, linked from one ribose (or deoxyribose) to another via phosphodiester bonds.
• the present invention can be used in the separation of RNA or of double- or single-stranded DNA or of synthetic nucleic acid analogs.
• the polynucleotide can be a linear molecule or a closed circle and can be modified, e.g. labeled with biotin or fluorescent molecules.
• oligonucleotides For purposes of simplifying the description of the invention, and not by way of limitation, the separation of a particular species of polynucleotide (e.g., dsDNA, RNA, ssDNA) will be described in the examples herein, it being understood that all polynucleotides are intended to be included within the scope of this invention. Short, typically single-stranded polynucleotides are referred to as oligonucleotides, and are often used in molecular biology as primers and probes.
• counterion agent is defined herein as a compound used to form a ionic pair with a polynucleotide that is capable of separation by the methods described herein.
• Preferred counterion agents comprise a cationic species having a hydrophobic character (e.g., an alkylated cation such as triethylammonium), believed to be capable of forming a bridging interaction between negatively charged polynucleotides and the hydrophobic surface of a separation medium of the invention.
• Non-specific binding refers to the binding of a plurality of polynucleotides in a mixture despite differences in the sequence or size of the different polynucleotides.
• Separatation medium refers to a solid phase having a hydrophobic surface suitable for binding polynucleotides in the presence of an aqueous phase containing a suitable counterion agent. Examples include beads, particles and monoliths.
• Elution solution refers to an aqueous solution containing a concentration of organic solvent sufficient to cause the elution of a polynucleotide, especially a target polynucleotide, from the hydrophobic surface of the separation medium.
• concentration of organic solvent need not be sufficient to result in the elution of all polynucleotide species, e.g., non-target polynucleotides.
• organic solvent refers to a solvent of sufficient non-polar character to cause elution of a polynucleotide from a separation medium when used as a component of an elution solution. Preparation of elution solution is facilitated by the used of an organic solvent that is suitably water-soluble.
• loading solution refers to a solution containing a target polynucleotide that is applied to a separation medium for purification according to the present invention.
• the loading solution is aqueous and includes a counterion agent.
• purify is used in the present invention to describe the separation of a target polynucleotide from some other molecular constituent of the loading solution, i.e., a non-target molecule, such as a different polynucleotide or other biomolecule.
• a target polynucleotide e.g., a target polynucleotide from some other molecular constituent of the loading solution, i.e., a non-target molecule, such as a different polynucleotide or other biomolecule.
• purify does not necessarily imply a total separation from all other polynucleotides or molecular species.
• a family of related polynucleotides e.g., mRNAs
• another class of polynucleotide e.g., genomic DNA
• wash solution refers to a solution used to wash non-target molecule from the separation medium with no substantial release of target polynucleotide.
• a wash solution will generally contain a concentration of organic solvent sufficient to elute non-target molecule, but insufficient to elute target polynucleotide.
• the wash solution contains a counterion agent.
• the apparatus of this invention provides a novel and unique method for separating and purifying single-stranded oligonucleotides and single-stranded DNA fragments, RNA, double-stranded DNA fragments, plasmids and the like. This process exploits the binding characteristics of separation media with nonpolar surfaces in the presence of counterion and materials to be separated.
• the separation medium is a unique aspect of this invention.
• the separation medium should have a surface that is either intrinsically non-polar or bonded with a material that forms a surface having sufficient non-polarity to interact with a counterion agent.
• the medium takes the form of chromatographic beads.
• the media surfaces can be porous or nonporous.
• porous media examples include porous media, particularly multivalent metal ions.
• nonporous media surfaces are preferred, i.e., beads having a pore size that essentially excludes the polynucleotides being separated from entering the bead, although porous beads can also be used.
• nonporous is defined to denote a bead that has surface pores having a diameter that is sufficiently small so as to effectively exclude the smallest RNA fragment in the separation in the solvent medium used therein. Included in this definition are polymer beads having these specified maximum size restrictions in their natural state or which have been treated to reduce their pore size to meet the maximum effective pore size required.
• the surface conformations of nonporous beads of the present invention can include depressions and shallow pit-like structures that do not interfere with the separation process.
• Non-porous polymeric beads useful in the practice of the present invention can be prepared by a two-step process in which small seed beads are initially produced by emulsion polymerization of suitable polymerizable monomers.
• the emulsion polymerization procedure is a modification of the procedure of Goodwin, et al. (Colloid & Polymer Sci., 252:464-471 (1974)).
• Monomers which can be used in the emulsion polymerization process to produce the seed beads include styrene, alkyl substituted styrenes, alpha-methyl styrene, and alkyl substituted alpha-methyl styrene.
• the seed beads are then enlarged and, optionally, modified by substitution with various groups to produce the nonporous polymeric beads of the present invention.
• the seed beads produced by emulsion polymerization can be enlarged by any known process for increasing the size of the polymer beads.
• polymer beads can be enlarged by the activated swelling process disclosed in U.S. Patent No. 4,563,510.
• the enlarged or swollen polymer beads are further swollen with a crosslinking polymerizable monomer and a polymerization initiator.
• Polymerization increases the crosslinking density of the enlarged polymeric bead and reduces the surface porosity of the bead.
• Suitable crosslinking monomers contain at least two carbon-carbon double bonds capable of polymerization in the presence of an initiator.
• Preferred crosslinking monomers are divinyl monomers, preferably alkyl and aryl (phenyl, naphthyl, etc.) divinyl monomers and include divinyl benzene, butadiene, etc.
• Activated swelling of the polymeric seed beads is useful to produce polymer beads having an average diameter ranging from 1 up to about 100 microns.
• the polymer seed beads can be enlarged simply by heating the seed latex resulting from emulsion polymerization.
• This alternative eliminates the need for activated swelling of the seed beads with an activating solvent.
• the seed latex is mixed with the crosslinking monomer and polymerization initiator described above, together with or without a water-miscible solvent for the crosslinking monomer. Suitable solvents include acetone, tetrahydrofuran (THF), methanol, and dioxane.
• THF tetrahydrofuran
• methanol methanol
• dioxane dioxane
• the temperature of the mixture can be increased by 10 - 20% and the mixture heated for an additional 1 to 4 hours.
• the ratio of monomer to polymerization initiator is at least 100:1 , preferably in the range of about 100:1 to about 500:1 , more preferably about 200:1 in order to ensure a degree of polymerization of at least 200.
• Beads having this degree of polymerization are sufficiently pressure-stable to be used in HPLC applications.
• This thermal swelling process allows one to increase the size of the bead by about 110 - 160% to obtain polymer beads having an average diameter up to about 5 microns, preferably about 2 - 3 microns.
• the thermal swelling procedure can, therefore, be used to produce smaller particle sizes previously accessible only by the activated swelling procedure.
• polymerization can be conducted, for example, by heating of the enlarged particles to the activation temperature of the polymerization initiator and continuing polymerization until the desired degree of polymerization has been achieved. Continued heating and polymerization allows one to obtain beads having a degree of polymerization greater than 500.
• packing material disclosed by U.S. Patent No. 4,563,510 can be modified through substitution of the polymeric beads with alkyl groups or can be used in its unmodified state.
• the polymer beads can be alkylated with 1 or 2 carbon atoms by contacting the beads with an alkylating agent, such as methyl iodide or ethyl iodide.
• Alkylation can be achieved by mixing the polymer beads with the alkyl halide in the presence of a Friedel-Crafts catalyst to effect electrophilic aromatic substitution on the aromatic rings at the surface of the polymer blend.
• Suitable Friedel-Crafts catalysts are well-known in the art and include Lewis acids such as aluminum chloride, boron trifluoride, tin tetrachloride, etc.
• the beads can be hydrocarbon substituted by substituting the corresponding hydrocarbon halide for methyl iodide in the above procedure, for example.
• alkyl as used herein in reference to the beads useful in the practice of the present invention is defined to include alkyl and alkyl substituted aryl groups, having from 1 to 1 ,000,000 carbons, the alkyl groups including straight chained, branch chained, cyclic, saturated, unsaturated nonionic functional groups of various types including aldehyde, ketone, ester, ether, alkyl groups, and the like, and the aryl groups including as monocyclic, bicyclic, and tricyclic aromatic hydrocarbon groups including phenyl, naphthyl, and the like.
• Methods for alkyl substitution are conventional and well-known in the art and are not an aspect of this invention.
• the substitution can also contain hydroxy, cyano, nitro groups, or the like which are considered to be non-polar, reverse phase functional groups.
• Non-limiting examples of base polymers suitable for use in producing such polymer beads include mono- and di-vinyl substituted aromatics such as styrene, substituted styrenes, alpha-substituted styrenes and divinylbenzene; acrylates and methacrylates; polyolefins such as polypropylene and polyethylene; polyesters; polyurethanes; polyamides; polycarbonates; and substituted polymers including fluorosubstituted ethylenes commonly known under the trademark TEFLON.
• mono- and di-vinyl substituted aromatics such as styrene, substituted styrenes, alpha-substituted styrenes and divinylbenzene
• acrylates and methacrylates polyolefins such as polypropylene and polyethylene
• polyesters polyurethanes
• polyamides polyamides
• polycarbonates and substituted polymers including fluorosubstituted
• the base polymer can also be mixtures of polymers, non-limiting examples of which include poly(styrene-divinylbenzene) and poly(ethylvinylbenzene-divinylbenzene).
• Methods for making beads from these polymers are conventional and well known in the art (for example, see U.S. Patent No. 4,906,378).
• the physical properties of the surface and near-surface areas of the beads are the primary determinant of chromatographic efficiency.
• the polymer, whether derivatized or not, should provide a nonporous, non-reactive, and non-polar surface for the IP-RP-HPLC separation.
• the separation medium consists of octadecyl modified, nonporous alkylated poly(styrene-divinylbenzene) beads. Separation columns employing these particularly preferred beads, referred to as
• DNASep® columns are commercially available from Transgenomic, Inc.
• a separation bead used in the invention can comprise a nonporous particle which has non-polar molecules or a non-polar polymer attached to or coated on its surface.
• such beads comprise nonporous particles which have been coated with a polymer or which have substantially all surface substrate groups reacted with a non-polar hydrocarbon or substituted hydrocarbon group, and any remaining surface substrate groups endcapped with a tri(lower alkyl)chlorosilane or tetra(lower alkyl)dichlorodisilazane as described in U.S Patent No. 6,056,877.
• the nonporous particle is preferably an inorganic particle, but can be a nonporous organic particle.
• the nonporous particle can be, for example, silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon, insoluble polysaccharides such as cellulose, or diatomaceous earth, or any of these materials which have been modified to be nonporous.
• carbon particles include diamond and graphite which have been treated to remove any interfering contaminants.
• the preferred particles are essentially non-deformable and can withstand high pressures.
• the nonporous particle is prepared by known procedures.
• the preferred particle size is about 0.5 -100 microns; preferably, 1 - 10 microns; more preferably, 1 - 5 microns. Beads having an average diameter of 1.0 - 3.0 microns are most preferred.
• An inorganic particle must have a hydrophobic surface to function as a separation medium in the instant invention.
• the hydrophobic surface can be an organic polymer supported on the inorganic particle.
• the hydrophobic surface includes long chain hydrocarbons having from 1-24 carbons, and preferably 8-24 cabons, bonded to the inorganic oxide particle.
• An example is a silica particle having substantially all surface substrate groups reacted with a hydrocarbon group and then endcapped with a non-polar hydrocarbon or substituted hydrocarbon group, preferably a tri(iower alkyl)chlorosilane or tetra(lower alkyl)dichlorodisilazane.
• the particle can be end-capped with trimethylsilyl chloride or hexamethyldisilazane.
• the nonporous beads of the invention are characterized by having minimum exposed silanol groups after reaction with the coating or silating reagents.
• Minimum silanol groups are needed to reduce the interaction of the RNA with the substrate and also to improve the stability of the material in a high pH and aqueous environment.
• Silanol groups can be harmful because they can repel the negative charge of the RNA molecule, preventing or limiting the interaction of the RNA with the stationary phase of the column.
• Another possible mechanism of interaction is that the silanol can act as ion exchange sites, taking up metals such as iron (III) or chromium (III). Iron (III) or other metals which are trapped on the column can distort the RNA peaks or even prevent RNA from being eluted from the column.
• Silanol groups can be hydrolyzed by the aqueous-based mobile phase. Hydrolysis will increase the polarity and reactivity of the stationary phase by exposing more silanol sites, or by exposing metals that can be present in the silica core. Hydrolysis will be more prevalent with increased underivatized silanol groups.
• the effect of silanol groups on the RNA separation depends on which mechanism of interference is most prevalent. For example, iron (III) can become attached to the exposed silanol sites, depending on whether the iron (III) is present in the eluent, instrument or sample.
• metals can occur if metals are present within the system or reagents. Metals present within the system or reagents can get trapped by ion exchange sites on the silica. However, if no metals are present within the system or reagents, then the silanol groups themselves can cause interference with RNA separations. Hydrolysis of the exposed silanol sites by the aqueous environment can expose metals that might be present in the silica core.
• Fully hydrolyzed silica contains a concentration of about 8 ⁇ moles of silanol groups per square meter of surface. At best, because of steric considerations, a maximum of about 4.5 ⁇ moles of silanol groups per square meter can be reacted, the remainder of the silanol being sterically shielded by the reacted groups. Minimum silanol groups is defined as reaching the theoretical limit of or having sufficient shield to prevent silanol groups from interfering with the separation.
• Nonporous silica core particles Numerous methods exist for forming nonporous silica core particles. For example, sodium silicate solution poured into methanol will produce a suspension of finely divided spherical particles of sodium silicate. These particles are neutralized by reaction with acid. In this way, globular particles of silica gel are obtained having a diameter of about 1 - 2 microns.
• Silica can be precipitated from organic liquids or from a vapor. At high temperature (about 2000°C), silica is vaporized, and the vapors can be condensed to form finely divided silica either by a reduction in temperature or by using an oxidizing gas. The synthesis and properties of silica are described by R. K. Her in The Chemistry of Silica, Solubility, Polymerization, Colloid and Surface Properties, and Biochemistry, John Wiley & Sons:New York (1979).
• the nonporous particle can be coated with a polymer or reacted and endcapped so that substantially all surface substrate groups of the nonporous particle are blocked with a non-polar hydrocarbon or substituted hydrocarbon group. This can be accomplished by any of several methods described in U.S. Patent No. 6,056,877. Care should be taken during the preparation of the beads to ensure that the surface of the beads has minimum silanol or metal oxide exposure and that the surface remains nonporous.
• Nonporous silica core beads can be obtained from Micra Scientific (Northbrook, IL) and from Chemie Uetikkon (Lausanne, Switzerland).
• Beads useful in the present process can be a variety of shapes, which can be regular or irregular; preferably the shape maximizes the surface area of the beads.
• the beads should be of a size such that their separation from solution, for example by filtration or centrifugation, is not difficult.
• the separation medium can be in the form of a polymeric monolith, e.g., a rod-like monolithic column.
• a monolith is a polymer separation medium, formed inside a column, having a unitary structure with through pores or interstitial spaces that allow eluting solvent and analyte to pass through and which provide the non-polar separation surface, as described in U.S. Patent No. 6,066,258 and U.S. Patent Application No. 09/562,069.
• the interstitial separation surfaces can be porous, but are preferably nonporous.
• the rod is substantially free of contamination capable of reacting with polynucleotides and interfering with its separation, e.g., multivalent cations.
• a molded polymeric monolith rod that can be used in practicing the present invention can be prepared, for example, by bulk free radical polymerization within the confines of a chromatographic column.
• the base polymer of the rod can be produced from a variety of polymerizable monomers.
• the monolithic rod can be made from polymers, including mono- and di-vinyl substituted aromatic compounds such as styrene, substituted styrenes, alpha-substituted styrenes and divinylbenzene; acrylates and methacrylates; polyolefins such as polypropylene and polyethylene; polyesters; polyurethanes; polyamides; polycarbonates; and substituted polymers including fluorosubstituted ethylenes commonly known under the trademark TEFLON.
• polymers including mono- and di-vinyl substituted aromatic compounds such as styrene, substituted styrenes, alpha-substituted styrenes and divinylbenzene; acrylates and methacrylates; polyolefins such as polypropylene and polyethylene; polyesters; polyurethanes; polyamides; polycarbonates; and substituted polymers including fluorosubstituted ethylenes commonly known under the
• the base polymer can also be mixtures of polymers, non- limiting examples of which include poly(glycidyl methacrylate-co-ethylene dimethacrylate), poly(styrene-divinylbenzene) and poly(ethylvinylbenzene- divinylbenzene.
• the rod can be unsubsituted or substituted with a substituent such as a hydrocarbon alkyl or an aryl group.
• the alkyl group optionally has 1 to ⁇ 1 ,000,000 carbons inclusive in a straight or branched chain, and includes straight chained, branch chained, cyclic, saturated, unsaturated nonionic functional groups of various types including aldehyde, ketone, ester, ether, alkyl groups, and the like, and the aryl groups includes as monocyclic, bicyclic, and tricyclic aromatic hydrocarbon groups including phenyl, naphthyl, and the like.
• the alkyl group has 1-24 carbons.
• the alkyl group has 1-8 carbons.
• the substitution can also contain hydroxy, cyano, nitro groups, or the like which are considered to be non-polar, reverse phase functional groups.
• the separation medium can take the form of a continuous monolithic silica gel.
• a molded monolith can be prepared by polymerization within the confines of a column (e.g., to form a rod) or other containment system.
• a monolith is preferably obtained by the hydrolysis and polycondensation of alkoxysilanes.
• a preferred monolith is derivatized in order to produce non-polar interstitial surfaces. Chemical modification of silica monoliths with ocatdecyl, methyl or other ligands can be carried out.
• An example of a preferred derivatized monolith is one which is polyfunctionally derivatized with octadecylsilyl groups.
• the present invention preferably employs a separation medium having low amounts of metal contaminants or other contaminants that can bind RNA.
• preferred beads have been produced under conditions where precautions have been taken to substantially eliminate any multivalent cation contaminants (e.g. Fe(lll), Cr(lll), or colloidal metal contaminants), including a decontamination treatment, e.g., an acid wash treatment. Only very pure, non-metal containing materials should be used in the production of the beads in order to minimize the metal content of the resulting beads.
• all process solutions and materials contacting the medium are preferably substantially free of multivalent cation contaminants (e.g. Fe(lll), Cr(lll), and colloidal metal contaminants). For example, all surfaces contacting the separation medium are preferably substantially free of multivalent cation contaminants (e.g. Fe(lll), Cr(lll), and colloidal metal contaminants). For example, all surfaces contacting the separation medium are preferably substantially free of multivalent cation contaminants (e.g. Fe(lll), Cr(lll), and colloidal metal contaminants
• 5 separation medium or process solution are preferably made of material which does not release multivalent cations, as described (in the context of HPLC) in U.S. Patent Nos. 5,772,889, 5,997,742 and 6,017,457.
• Preferred materials include titanium, coated stainless steel, passivated stainless steel, and organic polymer. Metals found in stainless steel, for example, do not harm the separation, unless they are in
• I0 an oxidized or colloidal partially oxidized state.
• multivalent cations in mobile phase solutions and sample solutions can be removed by contacting these solutions with a multivalent cation capture resin.
• the multivalent capture resin is preferably cation exchange resin and/or chelating resin.
• An example of a suitable chelating resin is available
• a multivalent cation-binding agent can be added to solutions used in the invention.
• the multivalent cation-binding agent can be a coordination compound. Examples of preferred coordination compounds include
• Non-limiting examples of multivalent cation-binding agents which can be used in the present invention include acetylacetone, alizarin, aluminon, chloranilic acid, kojic acid, morin, rhodizonic acid,
• furildioxime cupferron, ⁇ -nitroso- ⁇ -naphthol, nitroso-R-salt, diphenylthiocarbazone,
• a preferred multivalent cation- binding agent is EDTA.
• the present invention requires a counterion agent for forming a hydrophobic salt with anionic RNA to enable the hydrophobic interaction of the RNA-counterion with the separation medium.
• trialkylammonium acetate and trialkylammonium carbonate are preferred for use in the process of the invention, with triethylammonium acetate (TEAA) and triethylammonium hexafluoroisopropyl alcohol being most preferred.
• Trialkylammonium phosphate can also be used.
• the counterion agent can be added to the RNA preparation first, or the RNA preparation can be injected into a polar
• counterion agents are those which are easily removed after the separation process.
• volatile salts are desired because they are easily removed from the purified product by evaporation.
• the ability to use non-volatile salts is a significant advantage of the instant invention.
• the counterion agent is preferably selected from the group consisting of lower alkyl primary amine, lower alkyl secondary amine, lower alkyl tertiary amine, lower trialkyammonium salt, quaternary ammonium salt, and mixtures of one or more thereof.
• Non-limiting examples of counterion agents include octylammonium acetate, octadimethylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylam
• anion in the above examples is acetate
• other anions may also be used, including carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride, and bromide, or any combination of cation and anion.
• the pH of solutions used in the present invention is preferably within the range of about pH 5 to about pH 9, and optimally within the range of about pH 6 to about pH 7.5.
• denaturing conditions refers to conditions where polynucleotides of interest (normally mRNA molecules on the context of the instant invention) are denatured, resulting in substantial loss of secondary structure and/or tertiary structure, which can improve the separation.
• separation of single-stranded polynucleotides under denaturing conditions can result in enhanced size-dependency of the separation.
• Denaturing conditions can be achieved, for example, by conducting chromatography at high temperature (usually at about 50°C or greater, preferably at about 50°C or greater, and most preferably at about 75°C or greater), at a pH sufficient to cause denaturation, in the presence of a chemical denaturant, or a combination thereof. Normally, extreme pH is not a preferred means of achieving denaturation owing to the instability of RNA under both acid and base conditions.
• High temperature can be achieved by heating the separation medium during separation. For example, a spin column can be centrifuged in a heated environment.
• a multicavity separation system as described below can be heated by means of a heat block or similar structure.
• the present invention involves polynucleotide elution by means of an elution solution containing an appropriate concentration of organic solvent.
• organic solvents are able to release the polynucleotide-counterion complex from the separation medium surface while maintaining the complex in solution. Preferred organic solvents do not interfere with the isolation or recovery of the fragments and are easily removed after the separation.
• suitable organic solvents include alcohol, nitrile, dimethylformamide, tetrahydrofuran, ester, ether, and mixtures of one or more thereof, e.g., methanol, ethanol, 2-propanol, 1-propanol, tetrahydrofuran, ethyl acetate, acetonitrile.
• the most preferred organic solvent is acetonitrile.
• concentrations of acetonitrile sufficient to elute a polynucleotide according to the instant invention have been shown not to inhibit various enzymes used in molecular biology protocols, such as polymerases and restriction enzymes. Thus, in many cases a polynucleotide purified according to the instant invention can be used directly in downstream applications (e.g. RT-PCR, sequencing), without removal of the acetonitrile.
• the release of the fragments from the surface can be modulated by exposing the surface of the separation medium to variations in parameters such as temperature and pH.
• the release of fragments can also be modulated by chemical interactions, such as the use of an additive (e.g. a second, more polar counterion agent in the stripping solvent capable of competing with the first counterion to form a complex with the RNA molecules, thereby promoting the release of the molecules from the surface of the medium).
• an additive e.g. a second, more polar counterion agent in the stripping solvent capable of competing with the first counterion to form a complex with the RNA molecules, thereby promoting the release of the molecules from the surface of the medium.
• the temperature at which the separation is performed affects the choice of organic solvents used in the separation.
• the solvents affect the temperature at which a polynucleotide becomes denatured, losing secondary and tertiary structure, which can affect affinity for the separation medium. Some solvents can stabilize such structure better than other solvents.
• a solvent is important is because it affects the distribution of the polynucleotide between the mobile phase and the stationary phase. Acetonitrile and 1-propanol are preferred solvents in these cases.
• the toxicity (and cost) of the solvent can be important. In this case, methanol is preferred over acetonitrile and 1-propanol is preferred over methanol.
• the process of the invention preferably includes precautions to prevent contamination with multivalent cations such as Fe(lll), Cr(lll), or colloidal metal contaminants.
• Multivalent cations can cause non-specific binding of the DNA to the surfaces of conduits and containers which can lead to low recovery.
• the inner surfaces, which contact liquids within the system preferably are treated to remove multivalent contaminants, e.g. treating with an acid such as nitric acid.
• the efficiency of the separation process may be enhanced by the optional addition of a chelating agent such as EDTA, e.g. at a concentration of 0 to 0.1 M.
• Suitable precautions are described in U.S. Patent No. 5,772,889. Precautions can also be taken during the manufacture of the separation medium to prevent contamination with multivalent cations. Examples of suitable precautions in the manufacture of beads, for example, are described in U.S. Patent No. 6,056,877.
• Polynucleotides in solution can be detected by any suitable method, e.g., by
• UV absorbance UV absorbance, fluorescence or radioactivity.
• the general process of separating and/or purifying polynucleotides includes the following steps:
• the eluted polynucleotides can be collected for further processing or analysis (optional). 5.
• the eluted polynucleotides can be detected (optional).
• Steps 3-5 can optionally be repeated one or more additional times, resulting in fractionation of target polynucleotides into multiple fractions.
• the geometry, volume and configuration of the container supporting the separation medium can be varied without loss of the ability to predictably separate polynucleotides on the basis of the physical characteristics, including size and base composition.
• the container can be, for example, a low-pressure column, a spin column, a web, a pad, a flask, a well, or a tank.
• separation can be achieved in a batch process.
• a relatively polar sample solution containing polynucleotide, including a counterion agent are mixed in bulk with separation beads in a container, whereby polynucleotides of interest bind to the beads.
• all of the polynucleotide-counterion aggregates will bind nonspecifically to the beads under the initial loading conditions.
• the beads are brought into contact with an elution solution with a sufficient concentration of organic solvent to effect elution of the desired polynucleotides.
• Elution conditions for specific polynucleotides, or classes of polynucleoitdes can often be predetermined, e.g. by determining the elution profile of a standard polynucleotide mixture at various concentrations of organic solvent.
• This calibration procedure can be conducted on a small scale and applied to a large-scale process.
• An example of the high resolution which can be obtained in a single equlibria bulk process, in the context of DNA, is exemplified by referring to FIG. 14 and EXAMPLE 9, where isolation of a 102 base pair fragment was achieved by incrementally increasing the ACN concentration from 14.6% to 15.9%.
• a wash solution is applied in a first release step in which the organic solvent is applied at a concentration which will release non-target polynucleotides.
• the beads are then separated from the solvent, e.g. by centrifugation or by filtration.
• An elution solution is then applied to the beads in a second release step in which the elution solution contains an incrementally elevated concentration of organic solvent, which selectively releases the target polynucleotide, plurality of polynucleotides, or target class of polynucleotides (e.g., mRNA molecules).
• the process can be repeated with the application of elution solutions containing increasing concentrations of organic solvent in order to successively release polynucleotide fractions characterized by increasing affinity for the separation medium.
• Each fraction can be recovered, e.g. by collecting the elution solution at each concentration of organic solvent. It is possible to have multiple wash steps at a single concentration of organic solvent to ensure complete removal of target molecules.
• the separation is performed using a column, e.g. an open column under gravity flow conditions or a low pressure column equipped with a peristaltic pump.
• the separation medium comprises beads having a diameter large enough to permit flow of stripping solvent without requiring high pressure pumps.
• Preferred beads have a diameter of about 20 to 1000 microns and can be made from various materials as described hereinabove.
• the dimensions of the column can range from about 10 cm to 1 m in length, and 1 to 100 cm in diameter, for example.
• the column is first conditioned using a polar solvent.
• an RNA-counterion mixture is applied to the column in a convenient volume such as from 1 to 50 mL.
• the sample can be applied continuously, or in stages, to "load" the column.
• all of the RNA-counterion aggregate will bind to the separation medium under the initial conditions in which the loading solution has low concentration of organic solvent.
• the beads are brought into contact with an elution solution having a sufficient concentration of organic solvent. Elution conditions for specific RNA molecules, or classes of RNA molecules, can be pre-determined, e.g.
• RNA contaminating species e.g.
• an elution solution is then applied in a second release step in which the organic solvent is present at an elevated concentration, e.g. an incrementally elevated concentration, which selectively releases the target RNA molecule.
• organic solvents can be applied in a gradient of increasing concentration, e.g. a step-gradient or continuous gradient, in order to progressively release RNA molecules having increasing affinity for the separation medium.
• Each fraction is recovered, for example, by collecting the elution solution at each concentration of organic solvent.
• the separation process can be repeated, if necessary, e.g. by application to another column.
• the separation medium can be retained in a web or pad.
• An example is a web of inert fiber matrix with hydrophobic separation medium, such as the beads as described hereinabove, enmeshed in the matrix.
• the web of the present invention is a composite article comprising separation medium which has been incorporated into a fabric or membrane.
• incorporated into a fabric membrane means that the separation medium is encapsulated by or trapped within a fabric or membrane, is stabilized within a fabric or membrane or is covalently attached to a fabric or membrane such that the separation medium does not exist as free flowable particulate bulk material and is not separable from the web under liquid chromatography conditions.
• the separation medium is incorporated into a web, which may be woven or non-woven.
• the spaces between fibers of the web should be small enough to prevent separation medium material from passing through the web.
• the density of non-woven fibers and the density of warp and weft fibers of the web can be routinely adjusted to provide the desired density and porosity.
• the web fibers can be made of any suitable material so long as the material is porous. Suitable materials are described in U.S. Patent No. 5,338,448. Generally, the fibers will be made of a porous synthetic or natural polymeric material, e.g. polytetrafluoroethylene, cellulose, polyvinyl chloride, nylon, etc.
• the RNA in the sample preferably binds only to the separation medium and the binding is not detrimentally affected by the fiber matrix material.
• the separation medium consists of polymeric beads
• the ratio of beads to fiber matrix material can be in the range of 19:1 to 4:1 by weight, for example.
• the web is mounted on a support and the sample is applied and eluted in a manner analogous to the open column process as described hereinabove.
• the web material can be packed into a column.
• An advantage of using a web material is that it provides flexibility in how thin a column bed can be made, e.g. the web can be formed as a disk. Also, several uniform beds can be made at once. Multiple webs can be supported in a row or adapted to a matrix well format, e.g. a multi-well plate.
• the web can be used in analogy to the bulk equilibria process or column as described hereinabove with a binding step followed by release steps.
• a suitable fibril matrix is polytetrafluoroethylene (PTFE) as described in U.S. Pat. No. 4906378 to Hagen.
• PTFE polytetrafluoroethylene
• the ratio of beads to PTFE fibril matrix can be in the range of 19:1 to 4:1 by weight, for example.
• FIG. 1 is a cross-sectional view of a spin column separation device suitable for such a use.
• a standard laboratory centrifuge is used to rapidly pass liquids through the separation medium.
• the system uses a standard cylindrical centrifuge vial or eluant container 142 into which a separator tube or cylinder 144 is inserted.
• the separator cylinder can have a cylindrical body 146, open at top end 148 and bottom end 150, and sized to fit within the vial 142.
• the upper end 148 has an outwardly extending upper flange 152 which is sized to rest on the upper rim 154 of the cylindrical vial 142.
• the lower end 150 has an inwardly extending lower flange 156 which is sized to support the separation unit 158.
• the separation unit comprises a porous support disk 160 which rests on flange 156, an optional outer cylinder 162 within which the separation medium 164 is positioned.
• the separation unit can also comprise an optional upper porous disk 166 to prevent disruption of the separation medium and an optional ring 168.
• the optional ring 168 preferably has a slightly elastic or yielding composition and an outer diameter which is sized to establish a frictional engagement with the inner wall of cylinder 146. The ring 168, when pressed against the disk 166, holds the disk in place during use of the column.
• a solution containing the polynucleotide (or polynucleotides) of interest is diluted in a loading solution containing an appropriate counterion agent and no organic solvent, or a concentration of organic solvent below that which is required to cause elution of the polynucleotide of interest. I0 2.
• the diluted mixture is placed into chamber 170 and the spin column is placed in a standard laboratory centrifuge and spun until all of the free liquid has passed into the chamber 172.
• the inner cylinder 144 is removed from the vial, and the contents of chamber 172 are discarded (or saved if so desired).
• the polynucleotides to be separated bind to the 15 separation medium in this step.
• a wash solution containing counterion and an organic solvent is added to chamber 170.
• the organic solvent concentration is calculated to be the amount which will remove non-target molecules that have less affinity for the separation medium than the target
• the appropriate concentration of organic solvent can be pre-determined as described supra.
• the separation device is spun in a centrifuge until all of the free wash solution has passed into the chamber 172.
• the inner cylinder 144 is removed from the vial, and the contents of chamber 172
• This step removes from the separation medium contaminants and other non-target molecules that have less affinity for the separation medium than the target polynucleotide.
• An elution solution containing counterion and a higher concentration of organic solvent is prepared and placed in the chamber 170.
• concentration of organic solvent is calculated to be the amount which will remove target polynucleotides from the separation medium. In some instances, it will be desirable to use a concentration of organic solvent that is low enough to cause non-target molecules with greater affinity for separation medium than the target polynucleotide to remain bound to the
• the separation device is centrifuged until all of the free elution solution has passed into the chamber 172.
• the inner cylinder 144 is removed from the vial, and the contents of chamber 172, containing purified target
• RNA molecule or molecules is removed for further processing.
• vial 142 can be replaced between steps or cleaned between steps to prevent contamination of the product fraction or fractions.
• the concentration of organic solvent in the elution solution can be selected to remove a single polynucleotide species or a plurality of polynucleotides sharing .0 similar physical characteristics and hence affinity for the separation medium.
• Steps (5) and (6) can be repeated with successively higher concentrations of organic solvent to remove a series of polynucleotide-containing fractions.
• FIG. 2 is a cross-sectional view of a vacuum tray separation device suitable for use in this invention
• FIG. 3 is a top view of the separation tray of FIG. 3.
• the separator tray 200 is a single plate with rows and columns of tubular separation channels 202, preferably having regular, repeated spaces between the rows and columns for indexing the spaces.
• the dimensions of the tray 200 and separation channels can correspond and match the dimensions of standard multi-well plates, such as the 96 cavity microtiter plate.
• the multicavity separation plate 200 is supported on support flange and vacuum seals 204 formed in the internal cavity of an upper plate 206 of the vacuum assembly 207.
• the vacuum assembly 207 further comprises a vacuum cavity 208 defined by housing 210.
• the upper plate 206 positioned on the housing 210 by locating pins 212, and the upper plate 206 and the housing 210 have a sealed engagement with the seals 204.
• the housing 210 has an exhaust outlet channel 214 communicating with the vacuum chamber 208 and with a vacuum conduit 216 and vacuum valve 218.
• the vacuum conduit 216 and vacuum valve 218 communicate with a vacuum source (not shown).
• a multi-well collection plate 220 is supported in the vacuum chamber 208.
• the multi-well collection plate 220 is a single plate with rows and columns of separation channels 222, preferably having regular, repeated spaces between the rows and columns for indexing the spaces.
• the dimensions of the tray 220 and collection channels can correspond and match the dimensions of standard multi-well plates, such as the 96 cavity microtiter plate.
• the collection plate 220 is held in a position which aligns each of the collection wells 222 with a corresponding separating channel 202 of the separation plate 200 so each well 222 can collect liquid falling from the corresponding separation channel 202.
• FIG. 4 is a cross-sectional view of the separation tray of FIG. 3 taken along
• the separation channels 202 each have an evenly spaced upper
• FIG. 5 is an enlarged view of the separation components of the separation tray of FIG. 4.
• the bottom of the separation cavity 224 supports a porous disk 230, which in turn supports separation medium 232.
• An optional containment disk 234 rests on the separation medium 232, and the containment disk 234 can be optionally held in place by friction ring 236 or an equivalent device.
• the separation medium 232 can be the same nonpolar medium as described above.
• the separation of polynucleotides using the device of FIGS. 2-5 can be achieved by the following sequence of steps.
• a loading solution is prepared containing the polynucleotide (or polynucleotides) of interest, an appropriate counterion agent and no organic solvent, or a concentration of organic solvent below that which is required to cause elution of the polynucleotide of interest.
• the loading solution is placed in one of the chambers 202 of the fully assembled vacuum device.
• the other chambers 202 are filled with other polynucleotide containing loading solutions to be separated by the same procedure.
• Vacuum is applied to the vacuum chamber 208 by opening vacuum valve 218 until all of the liquid from the mixtures contained in each chamber has collected in chambers 222.
• the vacuum device is disassembled, and the contents of chambers 222 are discarded.
• the polynucleotides to be separated bind to the separation medium 232 in each chamber 202 in this step. 4)
• the vacuum apparatus and plates are reassembled, and a wash solution containing counterion and an organic solvent is added to the chambers 202.
• the organic solvent concentration is calculated to be the amount which will remove non-target molecules that have less affinity for the separation medium than the target polynucleotide.
• the appropriate concentration of organic solvent can be pre-determined as described supra.
• Vacuum is applied to the vacuum chamber 208 by opening vacuum valve 218 until all of the liquid from the mixtures contained in each chamber has collected in chambers 222.
• the vacuum device is disassembled, and the contents of chambers 222 are removed This step removes from the separation medium contaminants and other non-target molecules that have less affinity for the separation medium than the target polynucleotide.
• the vacuum apparatus and plates are reassembled, and an elution solution containing counterion and an organic solvent is placed in the chambers 202. .
• the concentration of organic solvent is calculated to be the amount which will remove target polynucleotides from the separation medium. In some instances, it will be desirable to use a concentration of organic solvent that is low enough to cause non-target molecules with greater affinity for separation medium than the target polynucleotide to remain bound to the column, thereby effectively separatingthese molecules from target polynucleotide.
• Vacuum is applied to the vacuum chamber 208 by opening vacuum valve 218 until the liquid from the mixtures contained in each chamber has 5 collected in chambers 222.
• the vacuum device is disassembled, and the contents of chambers 222, containing purified target polynucleotide or polynucleotides of interest, is removed for further processing.
• the plate 220 can be replaced between steps or cleaned between steps to prevent contamination of the product fraction or fractions.
• the concentration of organic solvent in the elution solution can be selected to remove a single polynucleotide or a genus of polynucleotides sharing similar physical characteristics and hence affinity for the separation medium.
• Steps (6) and (7) can be repeated with successively higher concentrations of organic solvent to remove a series of polynucleotide fractions. 15 It will be readily apparent to a person skilled in the art that other variations can be applied to remove a series of purified fractions in much the same manner as is shown above and illustrated in the Examples and Figures of this application.
• the spin column components 142 and 146 of FIG. 1 and the plates 200 and 220 in FIGS. 2-5 are made of a material which does not interfere with the separation 20 process such as polystyrene, polypropylene, or polycarbonate.
• the upper plate 206 and housing 210 can be made of any materials having the requisite strength such as a rigid organic polymer, aluminum, stainless steel or the like.
• the vacuum chamber walls are preferably coated with Teflon film.
• the vacuum conduit and valve can also be made of Teflon coated aluminum or the like.
• Sodium chloride (0.236 g) was added to 354 mL of deionized water in a reactor having a volume of 1.0 liter.
• the reactor was equipped with a mechanical stirrer, reflux condenser, and a gas introduction tube.
• the dissolution of the sodium chloride was carried out under inert atmosphere (argon), assisted by stirring (350 rpm), and at an elevated temperature (87°C).
• Freshly distilled styrene (33.7 g) and 0.2184 g of potassium peroxodisulfate (K 2 S 2 O 8 ) dissolved in 50 mL of deionized water were then added.
• the gas introduction tube was pulled out of the solution and positioned above the liquid surface.
• the reaction mixture was subsequently stirred for 6.5 hours at 87°C. After this, the contents of the reactor were cooled down to ambient temperature and diluted to a volume yielding a concentration of 54.6 g of polymerized styrene in 1000 mL volume of suspension resulting from the first step.
• the amount of polymerized styrene in 1000 mL was calculated to include the quantity of the polymer still sticking to the mechanical stirrer (approximately 5 - 10 g).
• the diameter of the spherical beads in the suspension was determined by light microscopy to be about 1.0 micron. Beads resulting from the first step are still generally too small and too soft (low pressure stability) for use as chromatographic packings. The softness of these beads is caused by an insufficient degree of crosslinking. In a second step, the beads are enlarged and the degree of crosslinking is increased.
• the protocol for the second step is based on the activated swelling method described by Ugelstad et al. (Adv. Colloid Interface Sci., 13:101-140 (1980)).
• the aqueous suspension of polystyrene seeds (200 ml) from the first step was mixed first with 60 mL of acetone and then with 60 mL of a 1-chlorododecane emulsion.
• the swollen beads were further grown by the addition of 310 g of a ethyldivinylbenzene and divinylbenzene (DVB) (1 :1.71 ) mixture also containing 2.5 g of dibenzoylperoxide as an initiator.
• DVD divinylbenzene
• reaction mixture was transferred into a separation funnel.
• the dried beads (10 g) from step two were washed four times with 100 mL of n-heptane, and then two times with each of the following: 100 mL of diethylether, 100 mL of dioxane, and 100 mL of methanol. Finally, the beads were dried.
• IP-RP-HPLC analysis of a 0.16-1.77 Kb RNA ladder (Catalog no. 15623010, Life Technologies) was performed using C-18 alkylated nonporous poly(styrene-
• RNAs having the nucleotide lengths as shown in FIG. 6.
• the column Prior to the injection, the column was equilibrated with 75% acetonitrile for 30- 45 min at a flow rate of 0.9 mL/min. The column was then equilibrated using 38%B for 30 min. Prior to the elution of RNA, two control gradient elutions (using the same
• Column Total RNA was extracted from the flower of tobacco plant (Nicotiana tabacum cv. Wisconsin 38) by an acid guanidinium thiocyanate phenol-chloroform extraction method, and precipitated with 4 M lithium chloride (Chomczynski, et al. (1987) Anal. Biochem. 162:156-159) as described in Bahrami, et al. (1999) Plant Molecular Biology 39:325-333.
• IP-RP-HPLC analysis of total RNA from the plant extract was performed using C-18 alkylated nonporous poly(styrene-divinylbenzene) beads packed in a 50 mm x
• OLIGOTEX mRNA Purification System from Qiagen and following the procedures supplied with the kit (catalog no. 70022). A portion of the extracted mRNA was analyzed by IP-RP-HPLC (FIG. 9) using the elution conditions described in Example 4. The product of the first OLIGOTEX extraction was re-extracted, and a portion of 0 the product was again analyzed by IP-RP-HPLC (FIG. 10).
• the experiments showed that the smaller fragments (80, 102, 174 bp) in this particular digest were released quantitatively from the resin surface by increasing the ACN concentration from 15 to 16% (in solution) and the larger fragments (257, 267, 298, 434, and 587 bp) by increasing the ACN concentration from 16 to 18.5%. Quantitative release for the 102 bp fragment was achieved by increasing the ACN concentration from 14.6% to 15.9%.
• FIG. 15 shows the separation of dsDNA fragments from a pUC18-DNA Haelll digest performed using 8 micron C-18 alkylated nonporous poly(styrene- divinylbenzene) polymer beads in two discs placed in series.
• the discs are available commercially under the trademark Guard DiscTM (Transgenomic, Inc., San Jose, CA) which contain beads enmeshed in a web of TEFLONTM fibril matrix at a weight ratio of 9:1 beads to fibril matrix.
• the DNA separation was run under the following conditions: Guard DiscTM
• EXAMPLE 11 Separation of PCR Reaction Products Using Discs
• the reaction products of a PCR preparation are separated under the conditions as described in EXAMPLE 10.
• Primer dinners elute in about 2-3 minutes and are well resolved from a 405 base pair PCR product which elutes in about 4-5 minutes.
• EXAMPLE 12 Spin Column Preparation A 50 mg portion of resin is added to each spin column (See schematic representation of column in FIG. 1) while pulling a vacuum on the columns. The sides of the columns were tapped to remove resin from the walls. A polyethylene filter was placed on the top of the resin in each vial, followed by a retaining ring, with gentle tapping with a hammer to position the retaining ring securely against the filter. The spin columns were washed with an aqueous solution containing 50% acetonitrile (ACN) and 0.1 M triethylammonium acetate (TEAA). The vials were then washed with an aqueous solution of 25% ACN and 0.1 M TEAA and then with an aqueous solution of 0.1 M TEAA.
• ACN acetonitrile
• TEAA triethylammonium acetate
• EXAMPLE 14 Separation ofPUC 18 Mspl with Spin Column A sample solution was prepared by diluting 35 ml stock pUC 18 Msp I to 1 ml with 0.1 M TEAA (1 ml total volume). A 400 ml aliquot (corresponding to 6.6 mg loaded on the column) was selected. Base pair length separation of the solution was performed using the WAVE separation system (Transgenomic, Inc., Omaha, NE) described in FIGS. 1-3, and the chromatograms obtained for two of the columns are shown in FIG. 16 for the pUC 18 Msp I standard.
• WAVE separation system Transgenomic, Inc., Omaha, NE
• FIG. 23 shows a purified PCR product recovery of 97.9% and a byproduct removal of greater than 99.2% This example demonstrates the ability of this procedure to elute PCR product with a high recovery and almost complete removal of PCR byproducts.
• EXAMPLE 17 Purification of oligonucleotide by spin column An 18-mer oligonucleotide (5'-CGCGCGTTCAGGCTCCGG-3'; SEQ ID NO.: 1) was phosphorylated by reaction with T4 polynucleotide kinase (PNK), using the following standard protocol. 5 ⁇ l (1.6 ⁇ g) of the single stranded 18-mer
• oligonucleotide was mixed with 2 ⁇ l 10X PNK buffer, 2 ⁇ l 10mM dATP and 10 ⁇ l T4
• the reaction was purified using a spin column as described above with the following protocol: (1 ) diluted PNK reaction 1 :5 with binding buffer (6%acetonitrile, 0.12M TEAA); (2) added to spin column and centrifuged @ 12000rpm for 1 minute;
• Tris-HCI Tris-HCI
• the eluant and unpurified reaction product were analyzed by IP-RP-HPLC using C-18 alkylated nonporous poly(styrene-divinylbenzene) beads packed in a 50
• Buffer A 0.1 M TEAA, pH 7.0
• buffer B 0.1 TEAA, 25% (v/v) acetonitrile, pH 7.0.
• the gradient conditions were as follows:
• the injection volume was 10 ⁇ L of the unpurified
• FIGS. 24 and 25 chromatograms are shown in FIGS. 24 and 25.
• the two peaks appearing at around 8 minutes are believed to represent the 18-mer oligonucleotide (main peak) and an N-1 mer that occurred during oligonucleotide synthesis and that elutes slightly ahead of the 18-mer.
• Quantification of the peaks revealed that about 42% of the N-1 mer and 82% of the 19mer were recovered following spin column purification. It is apparent from comparison of the chromatograms that the spin column removes substantial amounts of the by-products that elute at around 0.5-1 minutes, presumably salts and nucleotides from the PNK reaction.
• the spin column purification also results in high recovery of the oligonucleotide.
• oligonucleotides used in this example were synthesized on an Applied Biosystems 394 DNA synthesiser using cyanoethyl phosphoramidite chemistry. Following deprotection, the oligonucleotides were purified using denaturing PAGE, evaporated to dryness and desalted using a Pharmacia NAP 10 column according to the manufacturer's instructions. 5 pmol of labeled synthetic Holliday junction HJ50 was prepared by annealing and purifying the four 50-mer oligonucleotides HJ1 , HJ2, HJ3 and HJ4 (HJ1 5'
• HJ2 5'- ACGTCATAGACGATTACATTGCTACATGGAGCTGTCTAGAGGATCCGA (SEQ ID NO: 3);
• HJ3 5'-(6-FAM)-TGCCGAATTCTACCAGTGCCAGTGCCAGTGATGGACATCTT-
• HJ50 was added to a solution oflOOmM Ascorbate (Aldrich), followed by 5 ⁇ l
• RuvA a Holliday junction-binding protein. RuvA was purified as described in Sedelnikova et al. (1997) Acta. Cryst. D53:122-24.
• FIG. 26b shows that the protein protected strand HJ3 from cleavage in the right portion of the chromatogram.
• the labeled DNA was used to generate a
• reaction volume was then made up to 20 ⁇ l with MilliQ water and
• DNA was then precipitated for 15 mins at 15, 000 g, re- suspended in Milli Q water (20 ⁇ l) and purified by spin-column as described above.
• Example 19 Use of a spin column to separate genomic DNA from RNA
• RNA and genomic DNA from a single sample.
• the procedure involves binding a sample of RNA and gDNA to a column, a wash step to remove impurities, elution of RNA in an RNA isolation butter, and finally elution of gDNA with a gDNA isolation buffer.
• RNA and gDNA Prior to application of the sample the spin column is washed of multivalent cations and RNAse activity as described in Example 13.
• a sample containing RNA and gDNA is diluted 1 :5 in binding buffer (6.0% ACN/0.12 M TEAA), loaded on the spin column, and centrifuged at 7000 rpm for 1 min.
• 600 ⁇ L of Wash buffer (10.0% ACN/0.12M TEAA) is added to the spin column and the column is spun at 7000 rpm for 1 min.
• 100 ⁇ L of RNA Isolation buffer (18.0% ACN/0.1 M TEAA) is added to the spin column and the column is spun at 7000 rpm for 1 min, where the RNA comes out in the eluant.

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