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EP1525053A1 - Dispositif et procede de purification d'acides nucleiques - Google Patents

Dispositif et procede de purification d'acides nucleiques

Info

Publication number
EP1525053A1
EP1525053A1 EP03771854A EP03771854A EP1525053A1 EP 1525053 A1 EP1525053 A1 EP 1525053A1 EP 03771854 A EP03771854 A EP 03771854A EP 03771854 A EP03771854 A EP 03771854A EP 1525053 A1 EP1525053 A1 EP 1525053A1
Authority
EP
European Patent Office
Prior art keywords
ion
exchange
size
poly
particles
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03771854A
Other languages
German (de)
English (en)
Inventor
Kevin Hennessy
Aldrich Lau
Michael Harrold
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Applied Biosystems Inc
Original Assignee
Applera Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/413,797 external-priority patent/US20040016702A1/en
Priority claimed from US10/413,935 external-priority patent/US6833238B2/en
Application filed by Applera Corp filed Critical Applera Corp
Publication of EP1525053A1 publication Critical patent/EP1525053A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/14—Polymerisation; cross-linking
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J47/00—Ion-exchange processes in general; Apparatus therefor
    • B01J47/018—Granulation; Incorporation of ion-exchangers in a matrix; Mixing with inert materials
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4005—Concentrating samples by transferring a selected component through a membrane
    • G01N2001/4016—Concentrating samples by transferring a selected component through a membrane being a selective membrane, e.g. dialysis or osmosis

Definitions

  • the present teachings relate to apparatuses and methods for purifying a sample through ion-exchange.
  • PCR polymerase chain reaction
  • sequencing reaction can present a number of challenges for subsequent, downstream processing. Impurities can cause artifacts in subsequent processing steps. Numerous purification steps to eliminate artifacts can be cumbersome and inefficient. Further, purification, such as by size-exclusion chromatography or ion-exchange chromatography, requires a well-formed resin bed, without cracks, bubbles, or channels, as well as correct sample-loading techniques. The resin beds can be up to ten times the volume of the sample in size, requiring much space and increasing the cost of purification. A need exists for a purification method that addresses these and other problems associated with conventional techniques of purification.
  • an apparatus for filtration and or purification of a sample wherein the apparatus includes ion-exchange particles in contact with a substrate.
  • an apparatus for filtration and/or purification of a sample includes size-exclusion ion-exchange particles in contact with a substrate.
  • the size-exclusion ion-exchange (SEIE) particles can include an ion- exchange core micro-encapsulated by a shell.
  • the ion-exchange core can include a solid core material capable of ion-exchange.
  • the ion-exchange core can include a solid core material coated with an ion-exchange resin.
  • the ion-exchange resin can be formed in situ on the core material.
  • the shell can be capable of size-exclusion.
  • the shell can include a size-exclusion hydrogel, for example, the polymerization product of a water- soluble reactive monomer.
  • an apparatus for filtration and/or purification of a sample includes anionic ion-exchange particles embedded in a substrate, wherein the substrate is capable of cation exchange.
  • the anionic ion-exchange particles can be capable of size-exclusion.
  • an apparatus for filtration and/or purification of a sample wherein the apparatus includes ion-exchange particles in a substrate that includes a size-exclusion resin.
  • the apparatus can be attached or otherwise connected to a support, or placed in a sample well.
  • a method is provided to filter a sample solution.
  • the sample can contain, for example, primers, dye-labeled nucleotides, salts, oligonucleotides, and/or a mixture thereof.
  • the method can include placing an apparatus capable of ion-exchange and/or size-exclusion in contact with the sample for a period of time sufficient for the apparatus to adsorb unwanted materials from the sample, resulting in a purified sample solution.
  • the purification can occur in ten minutes or less, five minutes or less, or two minutes or less.
  • Fig. la is a schematic diagram of an interaction of an anion with a size-exclusion ion-exchange particle, according to various embodiments
  • Fig. lb is a cross-sectional view through different lines, of an SEIE particle used according to various embodiments.
  • FIGs. 2a-d are schematic diagrams illustrating the making of a coated stick from a substrate and size-exclusion ion-exchange particles
  • FIGs. 3a-d are schematic diagrams of a purification reaction using the coated stick of Fig. 2c;
  • FIGs. 4a-f are schematic diagrams illustrating the making of a purification dipstick from a substrate, ion-exchange particles, and a support;
  • Figs. 5 a-c are schematic diagrams of a purification reaction using the purification dipstick of Fig. 4f;
  • FIGs. 6a-e are schematic diagrams illustrating the making of a purification device from a substrate, ion-exchange particles, and a support;
  • FIGs. 7a-c are schematic diagrams of a purification reaction using the purification device of Fig. 6e;
  • size-exclusion ion-exchange (SEIE) particles having an ion-exchange core micro-encapsulated by a shell capable of size-exclusion are provided.
  • SEIE size-exclusion ion-exchange
  • micro-encapsulation refers to a process of encapsulation on the individual particle level.
  • a core of liquid, solid, and/or gas is micro-encapsulated with a shell to control access to the core.
  • micro-encapsulation can coat the entire exterior surface of the core (and optionally interior surfaces), or it can coat only a portion of the exterior surface of the core (and optionally interior surfaces).
  • micro-encapsulation of the core can be irreversible to permanently coat the core, or reversible to release the core upon dissolution of the coating.
  • micro-encapsulation can include encapsulation of an agglomerate of core material in a shell.
  • the agglomerate can be fused, sintered, pressed, compressed, or otherwise formed together core materials.
  • the core material can be a single particle and not an aggregate.
  • core or “core material” can refer to a single particle or an aggregate of particles.
  • shell refers to coating any portion of the core exterior surface and/or interior surface. The dimensions and formation of the shell are described below.
  • the term "material” refers to any substance on a molecular level or in bulk.
  • a material can be a liquid and/or solid, e.g. an emulsion or a resin.
  • a "mixture" can refer to more than one SEIE particle used together in a packed column, a mixed-bed, a homogenous bed, a fluidized bed, a static column with continuous flow, or a batch mixture, for example.
  • the mixture can include size-exclusion cation-exchange particles and size-exclusion anion-exchange particles, size-exclusion cation- exchange particles and anion-exchange particles, size-exclusion anion-exchange particles and cation-exchange particles, SEIE particles and inerts, and other materials that have affinity adsorption/absorption characteristic capable of removing organo- and bio-molecules from the analyte or a combination thereof.
  • the mixture can include any physical configuration known in the art of separations, and any chemical mixture known in the art of ion exchange.
  • Small molecules such as, for example, inorganic ions and nucleotides, can penetrate or permeate through the size-exclusion shell and can be retained by or ion- exchanged with the ion-exchange core.
  • the shell can prevent larger ions, such as, for example, DNA fragments, from penetrating or permeating through the shell and reacting with the ion-exchange core.
  • SEIE particles can have many uses such as, for example, in the purification of biomolecules. Applications can include, for example, purification of polymerase chain reaction (PCR) products, purification of DNA sequencing reaction mixtures, and purification of RNA. SELE particles can also be used for purification and/or separation of, for example, oligonucleotides, ligase chain reaction products, proteins, antibody binding reaction products, oligonucleotide ligation assay products, hybridization products, and antibodies. SEIE particles can also be used for desalting of biological products or reaction mixtures.
  • PCR polymerase chain reaction
  • LEC can differ from SEC, for example, in the order of elution of species from a column. According to various embodiments, LEC selectivity can be based on, for example, the charge of the analyte.
  • SELE particles can enable high quality separation of biomolecules by combining the effects of SEC and IEC.
  • SELE particles can use a size-exclusion shell to restrict the ability of large molecules to interact with an ion-exchange core.
  • SEIE particles can combine the high selectivity and binding ability of IEC resins with the size-exclusion benefits of SEC.
  • Small molecules that can penetrate the size-exclusion shell of the SELE particle can interact with the ion-exchange core and can be retained on the core.
  • Larger, highly charged species can be restricted from interacting with the ion-exchange core by the size-exclusion shell of the SEIE particle. Such larger, highly charged species can remain in solution rather than bind to the ion-exchange core.
  • a device for purification of a sample for example, a sample having nucleic acids
  • the device can be capable of size- exclusion, ion-exchange, or both.
  • the device can retain small molecules, such as, for example, inorganic ions and nucleotides, from a sample solution.
  • the device can selectively prevent adsorption of larger ions, such as, for example, DNA fragments, onto the device, leaving such larger ions in the sample solution.
  • the device can be used for purification of a sample of biological material at one or more of various steps in a processing sequence, for example, PCR, DNA sequencing reaction, and other processes described elsewhere herein.
  • the purification device can include a substrate capable of retaining particles, wherein the particles are capable of ion-exchange.
  • the particles can be embedded in the substrate in whole or in part.
  • the particles can be capable of size- exclusion.
  • the substrate can be capable of size-exclusion.
  • the device can be used to purify a sample solution by a combination of size-exclusion and ion- exchange, or SEIE.
  • FIG. la An interaction involving the SELE particle, is shown in Fig. la.
  • Figs, la and lb are not drawn to scale, and the relation between objects in the figures, such as the relation between core pore sizes and shell pore sizes, is not to scale, and can in fact be inverse, such that the core pore size is larger than the shell pore size.
  • ssDNA long single stranded DNA
  • dsDNA double stranded DNA
  • Small molecules such as deoxynucleotide triphosphates (dNTPs), dye-labeled deoxynucleotide triphosphates, dideoxynucelotide triphosphates (ddNTPs), dye-labeled dideoxynucelotide triphosphates, and small ions, such as chloride, can pass through the pores 125 of the size-exclusion shell 120 and can undergo ion-exchange with anion-exchange resin 113 at or near the interface 123 of the shell and ion-exchange core 111, or within the pores of ion-exchange core 111.
  • dNTPs deoxynucleotide triphosphates
  • ddNTPs dideoxynucelotide triphosphates
  • small ions such as chloride
  • the anion-exchange resin 113 for example, a cross-linked, macroporous copolymer of methyl methacrylate and 2-hydroxy-3- methacryloyloxypropyltrimethylammom ' um chloride, can be present on all internal and external surfaces of the solid core material 112. Together, the anion-exchange resin and solid core material or support 112 form the anion-exchange core 111.
  • Counter-anions released from the anion-exchange core 111 can react with a counter-cation, for example, hydronium, of a cation-exchanger 135 that can be provided in a mixture with the SEIE particle 130, to produce a neutral molecule such as water.
  • a counter-cation for example, hydronium
  • a cation-exchanger 135 that can be provided in a mixture with the SEIE particle 130
  • SEEB particles can be used in a mixture, a mixed bed, or a homogeneous bed of particles. Wherein a homogeneous bed of anionic- or cationic-SELE particles is used, the counter-ion can be released directly into a sample solution upon ion-exchange. In certain cases, the presence of the counter-ion in the sample solution does not affect further processing or reaction of the sample.
  • the selectivity of an SELE particle can be determined by the nature of the size-exclusion shell, the charge of the ion-exchange core, and the nature of the counter-ion.
  • the properties of the size-exclusion shell can be varied by, for example, choosing appropriate synthesis conditions that can affect the pore size of the resulting shell. Controlling an effective pore size " of the size-exclusion shell can allow the SEIE particle to be optimized for different size-exclusion applications.
  • a purification device can include SEIE particles embedded in a substrate.
  • the SEIE particles can be ion-exchange particles, as described elsewhere herein, micro-encapsulated by a size-exclusion resin, as described elsewhere herein.
  • anion-exchange resins or cation-exchange resins can be impregnated or retained on at least a portion of the internal surfaces, on at least a portion of the external surface, or on at least a portion of all surfaces of the solid core material of the ion-exchange particle.
  • a solid core material capable of ion-exchange can be micro-encapsulated by a size-exclusion resin to form an SEIE particle.
  • AnionicSEIE particles 2 and cationic SEIE particles 4 are placed in a receptacle 18, for example, a dish, sample well, plate, container, or other device capable of holding the particles.
  • a substrate having a support 10 with one or more protrusion 12 terminating in a ball-shaped distal end or portion 14 can be heated and/or chemically treated and pushed into the receptacle 18 containing the SEIE particles 2, 4.
  • the SEIE particles 2, 4 can be heated and/or chemically treated in addition to, or instead of, heating or chemically treating the substrate.
  • the substrate and/or SELE particles can be heated to a temperature of from the glass transition temperature Tg to the melting temperature T m of the substrate.
  • the polymeric substrate of a coated stick can be treated to function as a cation exchanger.
  • a polyslyrene-containing substrate can be treated with sulfonic acid to provide cation exchange groups on the substrate, for example, on the protrusion and/or terminal portion.
  • Anionic SEIE particles can be attached to and/or embedded in the terminal portion of the polymeric substrate.
  • Such a purification device can provide both cation- and anion-exchange functions for purification of a sample solution.
  • the polymeric substrate can be capable of cation-exchange and or anion-exchange. Exemplary suitable polymeric materials are set forth, for example, in U.S. Patent Nos.
  • the support and/or protrusions therefrom can be constructed of ion-exchange material and then at least partially coated with size-exclusion resin.
  • the support and protrusions is active, as opposed to being inert.
  • inert refers to a material that provides neither size- exclusion nor ion-exchange.
  • the purification device 5 can be inserted into the sample wells 30 such that support 10 contacts a top portion of sample wells 30, sealing the sample wells 30, preventing fluid loss from the sample wells 30, and/or preventing entrance of contaminants into the sample wells 30.
  • protrusions 12 extend from support 10 such that terminal ball-shaped portions 14 embedded with or otherwise in contact with SEIE particles are fitted snuggly into respective sample wells 30.
  • the terminal ball-shaped portion 14 having SEIE particles 2, 4 embedded thereon can be completely covered by sample 34 in each sample well 30.
  • the purification device 5 remains in sample wells 30 for a period of time sufficient for the purification device to remove substantially all impurities from sample 34.
  • Purification device 5 can be separated from the sample 34 after a sufficient time to remove substantially all impurities, for example, at least 70%, at least 80%, at least 90%, or at least 95% of impurities from sample 34, leaving purified sample 38 in sample wells 30, as shown in Fig. 3c.
  • the purified sample can be separated from the purification device, for example, by movement of the sample well array, or by opening a closable valve in the sample well to allow the purified sample to flow from the sample well.
  • a purification device can include a substrate capable of retaining ion-exchange particles, wherein the substrate is capable of size-exclusion.
  • the substrate can be on or in a support structure.
  • the substrate can be a size-exclusion resin as defined elsewhere herein.
  • the ion-exchange particles can be as defined elsewhere herein.
  • the support structure can include a polymeric material.
  • the support structure can include polystyrene or a copolymer of polystyrene.
  • the support structure can include a petroleum-based polymer, co-polymer or homopolymer.
  • the support structure can be glass or ceramic.
  • the support structure can be a planar structure capable of receiving, holding, or retaining the substrate.
  • the planar structure can be a cover having a recess for receiving the substrate.
  • the support structure can have one or more protrusion, wherein each protrusion has a terminal portion.
  • the terminal portion can be any suitable shape to provide a surface area for interaction with a sample solution.
  • the terminal portion can be ball-shaped, bell-shaped, flared, tubular, column-shaped, discshaped, ovoid, pin-shaped, or any other suitable shape.
  • the terminal portion of the support structure can be of a sufficient size to fit snuggly within a sample container while allowing a sample solution to flow between the interior walls of the sample container and the terminal portion of the support structure.
  • the monomer solution 26 can include one or more initiator, cross-linker, chain transferring agent, surfactant, catalyst, terminator, promoter, buffer, accelerator, or a combination thereof.
  • the monomer solution can be capable of polymerizing and/or cross-linking at about room temperature.
  • the monomer solution can be capable of polymerizing upon application of heat, application of radiation, addition of a catalyst, addition of an initiator, or a combination thereof.
  • the monomer solution can substantially cover the ion- exchange particles in the mold such that a portion or none of the ion-exchange particles protrudes above the monomer solution.
  • the dipstick is formed with a length and diameter complementary to, and slightly smaller than, a sample well with which the purification device is intended to be used.
  • the dipstick can be of a size and shape to fit snugly within the sample container.
  • the dipstick can be column-like in shape, having a blunt or rounded terminal end, and having a diameter less than that of the sample well, or less than about three millimeters, and a length sufficient to span the distance from just above the bottom of the sample well to the support when the support is fitted onto a surface of the sample well array.
  • the shape of the dipstick and sample container can be complimentary, and the dipstick can have a length exceeding its diameter, for example for use with a sample well array, or a diameter exceeding its length, for example, for use with a Petri dish.
  • One or more dipstick 60 can be attached to the support 10 permanently or removably by an adhesive, screw- fit, friction-fit, or any other retaining method known to one of ordinary skill in the art.
  • the purification device 65 can be inserted into a sample container, for example a sample well 30 having a sample solution 34 therein.
  • the purification device 65 can be inserted so that the support 10 contacts the sample well 30, sealing the sample well during reaction of the sample solution 34 and dipstick 60.
  • the purification device 65 is kept in contact with the sample solution 34 for a period of time sufficient to remove substantially all impurities, for example, at least 70%, at least 80%, at least 90%, or at least 95% of impurities from the sample solution 34.
  • the purification device 65 can be separated from the purified solution 38 in sample well 30.
  • the purified sample can be separated from the purification device, for example, by movement of the sample well, or by opening a closable valve in the sample well to allow the purified sample to flow from the sample well.
  • the used purification device can be discarded, washed and reused, or can be used to transfer the adsorbed material to another reaction chamber.
  • a purification device 75 in the form of a coated stick or popsicle stick can be fonned, as shown, for example, in Figs. 6 and 7.
  • One method of forming the popsicle stick is shown in Figs. 6a-e.
  • Support 10 having a protrusion 12 and a terminal end 14 can be inserted into the mixture of ion-exchange particles 22, 24 and monomer solution 26.
  • the monomer solution 26 is polymerized and/or cross-linked to form a substrate of size-exclusion resin 20 encapsulating the ion-exchange particles 22, 24, wherein the size-exclusion resin 20 is in the form of a gel plug surrounding and attached to the terminal end 14.
  • the gel plug can be attached to at least a portion of protrusion 12 of support 10.
  • the popsicle stick can be formed such that the gel plug is slightly smaller in diameter and height than the corresponding sample container.
  • the ion-exchange particles 22, 24 can protrude partially beyond the size-exclusion resin 20.
  • the purification device can include one popsicle stick or multiple popsicle sticks attached to the support 10. The number of popsicle sticks can be equal to, less than, or greater than the number of sample wells in, for example, a sample well array, allowing purification of multiple wells simultaneously.
  • the protrusion 12 of support 10 can be permanently or removably attached to the support 10 by an adhesive, screw-fit, friction- fit, or any other retaining method known to one of ordinary skill in the art, or can be made integral therewith.
  • a purification device can be formed from SEIE particles encapsulated by an inert porous resin that provides support for particles and access to the sample.
  • the purified sample can be separated from the purification device, for example, by movement of the sample well, or by opening a closable valve in the sample well to allow the purified sample to flow from the sample well.
  • the used purification device can be discarded, washed and reused.
  • the used purification device can be used to transfer the adsorbed material to another reaction chamber in a process also referred to herein as purification.
  • a purification device 70 in the form of a gel plug can be formed from a size-exclusion resin 20 and ion-exchange particles 22, 24.
  • the ion-exchange particles 22, 24 can be added to a receptacle, for example, a mold or a sample well 30. If the ion-exchange particles are added to a mold, the mold can be the same size as, or slightly smaller than, the sample well with which the gel plug can be used for purification of a sample solution.
  • a monomer solution 26 as described previously herein can be added to the ion-exchange particles 22, 24 in the sample well 30.
  • a cover 40 can be placed over the sample well and the monomer solution 26 can be polymerized and/or cross- linked to form a size-exclusion resin 20 in the form of a gel plug containing the ion-exchange particles 22, 24.
  • the ion-exchange particles can be below, flush with, or protrude slightly beyond the size-exclusion resin at a surface of the gel plug that contacts the sample solution.
  • the gel plug 70 can be used as a purification device in the receptacle in which it was formed, or in another receptacle.
  • a gel plug can be formed for use in a column, including a column of a microfluidic device, or a gel plug can be formed as a sheet for use as a filter material.
  • a gel plug can be formed from SEIE particles encapsulated by an inert porous resin that provides support for particles and access to the sample.
  • the gel plug 70 can be situated in the sample well in which it was formed, or placed in a sample well 30. According to various embodiments, gel plug 70 fits snugly into sample well 30. Gel plug 70 can be slightly smaller than sample well 30 such that a sample solution 34 can flow around ' gel plug 70 in sample well 30. For purification of a sample solution 34, the sample solution 34 is contacted with gel plug 70 in sample well 30. A cover 40 can be set over and in contact with sample well 30. Cover 40 can minimize loss of sample solution 34 due to evaporation, and/or prevent contaminants from entering sample solution 34.
  • a purification device can include both anionic ion-exchange particles, cationic ion-exchange particles, anionic SEIE particles, cationic SEIE particles, or mixtures of such. Wherein a mixture of anionic particles and cationic particles is used, whether the mixture is ion-exchange only, SEIE only, or a mixture of ion-exchange and SEIE, the particles can be present in stoichiometrically equal amounts, such that the ion- exchange capacity for anions and cations is approximately equivalent. According to various embodiments, the anionic particles and cationic particles can be present in amounts which are not stoichiometrically equal.
  • One or more of the purification device and sample container can be moved relative to one another to contact the purification device with a sample in the sample container, and to separate the purification device from the purified sample in the sample container.
  • the purification device can be inserted into and removed'from the sample container, or the sample container can be moved into a position surrounding the purification device and removed from the purification device after purification of the sample, or any combination thereof.
  • a sample container can include any arrangement suitable for containing or retaining the purification device and a sample.
  • the sample container can include a sample well of a sample well array, a test tube, a Petri dish, a column, a portion of a pathway of a microfluidic device, or any other suitable container known to those of ordinary skill in the art.
  • sample purification can occur in a bulk mode on a purification device as described herein.
  • the ion-exchange capacity of a purification device as described herein for a given ion is improved over prior art methods and apparatuses for ion-exchange.
  • purification of a sample using a purification device can occur in ten minutes or less, five minutes or less, or two minutes or less.
  • the selectivity of a purification device can be determined by the nature of the size-exclusion resin, the charge of the ion-exchange particle, or a combination thereof.
  • the properties of the size-exclusion resin can be varied by, for example, choice of synthesis conditions, which can affect the pore size of the resin. Controlling an effective pore size of the size-exclusion resin can enable optimization of the device for different applications.
  • a size-exclusion resin can be polymerized and/or a cross-linked monomer such as a hydrogel.
  • a cross-linked monomer such as a hydrogel.
  • the terms "polymer,” “polymerization,” “polymerize,” “cross-linked product,” “cross-linking,” “cross-link” and other like terms are meant to include both polymerization products and methods, and cross-linked products and methods wherein the resultant product is a three- dimensional structure, as opposed to, for example, a linear polymer.
  • the degree of cross- linking of the size-exclusion resin can be varied in order to vary the size of the pores of the size- exclusion resin.
  • the pore size of the size-exclusion resin can be large enough to allow relatively small ions, such as, ' for example, " chloride, nucleotides, or other small molecules, to permeate through the size-exclusion resin.
  • the pore size of the size-exclusion resin can be small enough to prevent any relatively large molecules, such as DNA, from permeating through the size-exclusion resin.
  • the size- exclusion resin can be hydrophilic to reduce passive adsorption or absorption of biomolecules such as, for example, ssDNA fragments.
  • a size-exclusion resin can be a cross-linked product of two or more reactive monomeric units.
  • the monomeric units can be water-soluble monomeric units.
  • water-soluble includes materials with any degree of water solubility from slightly water-soluble to highly water-soluble, and materials that are swellable in water.
  • the monomeric units can be nitrogen-containing, oxygen-containing, or both.
  • the size-exclusion resin can be a homopolymer or a copolymer.
  • the size-exclusion resin can be a reaction product of acrylamide and a cross-linker, for example, N,N'- methylenebisacrylamide, 2,2-bisacrylamidoacetic acid, N,N'-diacryloylpiperazine, 1ri(meth)acryloylperhydro-5-triazine, or a combination thereof.
  • a cross-linker for example, N,N'- methylenebisacrylamide, 2,2-bisacrylamidoacetic acid, N,N'-diacryloylpiperazine, 1ri(meth)acryloylperhydro-5-triazine, or a combination thereof.
  • exemplary water-soluble monomers suitable for preparing size-exclusion resin can include, but are not limited to, (meth)acrylamide, N-methyl(meth)acrylamide, N,N- dimethyl(meth)acrylamide, N-methyl-N-ethyl (meth)acrylamide, N-ethyl (meth)acrylamide, N,N-diethyl (meth)acrylamide, N-zso-propyl (meth)acrylamide, N-hydroxymethyl (meth)acrylamide, N-vinylformamide, N-vinylacetamide, N-methyl-N-vinylacetamide, precursor of vinyl alcohol, for example, vinyl acetate, 2-hydroxyethyl (meth)acrylate, 3- hydroxypropyl (meth)acrylate, vinypyrrolidone, vinyloxazolidone, vinylmethyloxazolidone, N-(meth)acrylylcinamide, poly(ethyleneglycol) mono(meth)acrylate, other suitable monomers
  • the size-exclusion resin can be neutral, anionic, for example, containing acrylic acid as a co-monomer, or cationic, for example, containing 2-acryloylethyl trimethyl ammonium chloride as a co-monomer.
  • the size-exclusion resin can be hydrophilic.
  • the size- exclusion resin can be a cross-linked polymer network of polymers capable of swelling in water, for example, hydrogels. Exemplary hydrogels are described, for example, in U.S. Patent No. 6,380,456 Bl, incorporated herein in its entirety by reference.
  • the size-exclusion resin, once formed, can be non-water soluble.
  • the size-exclusion resin can prevent adsorption of ssDNA fragments and/or double-stranded DNA (dsDNA) fragments.
  • the size-exclusion resin can be formed with pores of a pre-determined size. The pores can be of the same or varying size. The pores can function as the size-exclusion factor for preventing molecules larger than a certain size from passing through the size-exclusion resin to the ion-exchange core which is micro-encapsulated by the size-exclusion resin.
  • the size-exclusion resin can be formed by cross-linking one or more reactive monomer by addition of a cross-linker, for example, N,N'-methylenebisacrylamide, or a free-radical initiator.
  • a cross-linker for example, N,N'-methylenebisacrylamide
  • the cross-linker or initiator can be added in an amount of from 1.0 mol% to 100 mol%.
  • the cross-linker or initiator can be added in an amount of from 1.0 mol% to 80 mol%, from 2.0 mol% to 50 mol%, from 5 mol% to 30 mol%, or from 10 mol% to 20 mol%.
  • the amount of cross-linker or initiator used to form the size-exclusion resin is at least one factor in determining the size of the pores of the size-exclusion resin, and the size-exclusion ability of the size-exclusion resin.
  • the choice of cross-linker or initiator, and/or selection of the reaction conditions can control the amount of cross-linking of the size-exclusion resin.
  • various multifunctional cross-linkers for example, tri(meth)acryloylperhydro-5-triazine can be used that have varying amounts of functionality.
  • the appropriate amount of a cross-linker to use to form a desired size-exclusion resin pore size can be determined by those of ordinary skill in the art based on the functionality of the cross- linker chosen, the reaction conditions, and other factors as known to those of ordinary skill in the art.
  • the size-exclusion resin pore size can be equal to or smaller than a 10 nucleotides (“nt") ssDNA.
  • the size-exclusion resin pore size can be equal to or smaller than a 100 nt ssDNA.
  • the pores of the size-exclusion resin have a pore size capable of excluding a molecule equal to or larger than a 100 nt ssDNA, nucleotides, oligonucleoti.de primers less than 100 nt in size, and buffer salts can pass through the size-exclusion resin while 100 nt or larger molecules are deflected by the size- exclusion resin.
  • the size-exclusion resin can be used with ion-exchange particles for purification of biological samples, for example, PCR products, to separate larger DNA, for example dsDNA, from ssDNA, free nucleotides, and salts.
  • the size-exclusion resin can pass through the size-exclusion resin.
  • 50% or more of the pores of the size-exclusion resin have a pore size capable of excluding a molecule equal to or larger than 10 nt
  • the size-exclusion resin can be used with ion-exchange particles for purification of biological samples, for example, from a sequencing reaction.
  • Purification of a sequencing reaction sample can remove dye- labeled dideoxynucleotides and salts from the sequencing reaction sample by allowing such sample components to pass through the size-exclusion resin and react with the ion-exchange particles, leaving a purified sample containing ssDNA in an amount of 70% or more, 80% or more, 90% or more, or 95% or more of the eluted sample volume.
  • an ion-exchange particle can be an anionic or cationic material.
  • the ion-exchange particle can be a polymer, cross-linked polymer, or inorganic material, for example, silica.
  • the ion-exchange particle can be a solid core material capable of ion-exchange, or a solid core material treated with an ion-exchange resin.
  • the ion- exchange particle can be surface-activated.
  • the ion-exchange particle can be non-magnetic, paramagnetic, or magnetic.
  • Exemplary ion-exchange particle materials include Macro-Prep® ion-exchange resins from Bio-Rad, and Nucleosil® sihca-based ion-exchange resins from Macherey-Nagel.
  • the solid core material can be macroporous silica, controlled pore glass (CPG), a macroporous polymer microsphere with internal pores, other porous materials as known to one of ordinary skill in the art, or a combination thereof.
  • the solid core material can have various surface features, including, for example, pores, crevices, cracks, or depressions.
  • the solid core material can include sodium oxide, silicon dioxide, sodium borate, or a combination thereof.
  • the solid core material can be modified to be capable of ion-exchange, for example, cation-exchange or anion-exchange.
  • Modification of the solid core material can include treatment of the solid core material to form cationic or anionic substituent groups on the surfaces of the solid core material.
  • the term "surface" can include an external surface and internal surfaces, for example, the surfaces of voids or pores within the solid core material.
  • the solid core material can be modified to include tertiary amino groups, quaternarized ammonium groups, at least one carboxylic acid group, at least one sulfonic acid group, other cationic or anionic functional groups known to one of ordinary skill in the art, or a combination thereof on the surface of the solid core material.
  • the solid core material can be porous, microporous, or macroporous.
  • the solid core material can have an average pore size of less than or equal to 1000 Angstroms, from 100 Angstroms to 1000 Angstroms, or less than or equal to 100 Angstroms.
  • the average diameter of the solid core material can be from 0.1 ⁇ m to 100 ⁇ m, from 1 ⁇ m to 50 ⁇ m, or from 2 ⁇ m to 20 ⁇ m, according to various embodiments.
  • the average diameter of the solid core material can be 100 ⁇ m or less, 50 ⁇ m or less, or 20 ⁇ m or less.
  • a solid core material can adsorb an ion- exchange resin onto the external surface, internal surface, or both the external and the internal surface of the solid core material to form an ion-exchange particle.
  • the term "resin” can encompass a resin or a gel.
  • the ion-exchange resin can be a cation-exchange resin or an anion-exchange resin.
  • the ion-exchange resin can include tertiary amino groups, quaternarized ammonium groups, at least one carboxylic acid group, at least one sulfonic acid group, or a combination thereof. Suitable anion-exchange resins and cation-exchange resins are known to one of ordinary skill in the art.
  • the ion-exchange resin can be sequestered into the pores of the solid core material, for example, a macroporous silica particle. Filling at least a portion of the pores of the solid core material and/or coating the external surface of the solid core material with the ion-exchange resin can increase the ion-exchange capacity of the ion- exchange particle over traditional ion-exchange resins.
  • the ion-exchange capacity of the ion- exchange particle can be improved by increasing a mass of ion-exchange resin, such as quaternary ammonium resin, on the external surface and/or on the internal surfaces of the pores of the solid core material of the ion-exchange particle.
  • the ion-exchange capacity of the ion-exchange particle can be improved by selection of cationic or anionic functional groups on the external surface, internal surfaces of the pores, or both internal surfaces and external surface of the solid core material.
  • the ion-exchange resin can be formed in situ on the solid core material, as described, for example, in concurrently filed U.S. Patent Application No. 10/414,179, to Lau et al, entitled "SIZE-EXCLUSION ION-EXCHANGE PARTICLES,” Attorney Docket No. 4885, which is incorporated herein in its entirety by reference.
  • the ion-exchange resin can be the product of one or more monomer, one or more polymer, or a combination thereof, according to various embodiments.
  • a solid core material of S1O2 having an average pore size of about 1000 Angstroms, a void volume of about 0.95cc/g, and a diameter of about 5 ⁇ m, can be added to a solution of polyethyleneimine in methanol and incubated for a time sufficient to impregnate the polyethyleneimine on all internal and external surfaces of the solid core material.
  • polyethyleneimine can be adsorbed due to hydrogen bonding with silanol groups in the solid core material.
  • the adsorbed polyethyleneimine can be reacted with a second compound, such as, for example, 1,3-dibromopropane, in a solvent, for example, dioxane, followed by placement in water, to form a gel that functions as an anion-exchange resin on the solid core material, forming an ion-exchange particle.
  • a second compound such as, for example, 1,3-dibromopropane
  • a solvent for example, dioxane
  • the said anion-exchange resin can be quarternized by reacting the cross-linked network with an alkyl halide, for example, methyl bromide, resulting in an strong anion exchange resin.
  • anion-exchange resins or cation- exchange resins can be impregnated or retained on at least a portion of the internal surfaces, on at least a portion of the external surface, or on at least a portion of all surfaces of the solid core material of the ion-exchange particle.
  • purification of a sample can be accomplished by ion-exchange. Displacement of counter-ions from ion-exchange particles of a device as described herein during ion-exchange can release a large number of counter-ions into a sample solution.
  • anionic ion-exchange particles and cationic ion- exchange particles can both be present during purification of a sample such that counterions of the ion-exchange particles react to form a neutral molecule, for example, water.
  • the purification device can include an ion- exchange particle having a lower mobility counter-ion, for example, octane sulfonate.
  • the device can include an ion-exchange particle can contain a volatile counter-ion, for example, acetate, which can later be removed from a sample solution.
  • the counter-ion for an anionic ion- exchange particle can be, for example, a halide or hydroxide.
  • the counter-ion for a cationic ion-exchange particle can be, for example, hydrogen.
  • the sample for purification can be a PCR product solution containing, for example, buffer salts, metal ions, polymerase, nucleotides, oligonucleotide primers, and other components.
  • PCR products can be used in subsequent enzymatic reactions that can be sensitive to at least some of the artifacts found in a sample solution containing the PCR products. For example, free nucleotides and oligonucleotide primers can interfere with downstream enzymatic reactions.
  • a size-exclusion resin in a purification device can have a pore size capable of excluding a molecule equal to or larger than a 100 nt ssDNA, allowing nucleotides, oligonucleotide primers less than 100 nt in size, and buffer salts, to pass through the size-exclusion resin and make contact with the ion-exchange particle, becoming trapped therein. 100 nt or larger molecules can remain in the sample solution.
  • the surface of the size-exclusion resin is capable of excluding a molecule equal to or larger than a 100 nt ssDNA, and allowing nucleotides, oligonucleotide primers, and buffer salts less than 100 nt in size to pass through the size- exclusion and be trapped by the ion-exchange particles.
  • the resulting purified sample solution can contain purified PCR products in a desalted environment, and can be used in downstream reactions and analyses.
  • PCR purification can be directed toward purifying larger dsDNA separate from smaller ssDNA, free nucleotides, and salts.
  • PCR product purification using a purification device can isolate a 250-600 bp amphcon, can remove 44 nt primers and/or nucleotides, or can both isolate and remove.
  • a sample can be a DNA sequencing reaction solution containing, for example, buffer salts, metal ions, polymerase, nucleotides, oligonucleotide primers, and other components.
  • Purification of sequencing reaction solutions can have different requirements than purification of PCR reaction solutions.
  • finished sequencing reactions can contain residual dye- labeled dideoxynucleotides (terminators) that can be removed, according to various methods, prior to electrophoretic analysis and DNA sequencing or basecalling. Removing terminators can remove "blobs" that would otherwise be caused and lead to errors in DNA sequencing or basecalling.
  • capillary sequencers can use electrokinetic injection as a means to introduce DNA sequencing reaction samples.
  • the presence of salts in the samples can effect the introduction of the sample into the capillary.
  • DNA sequencing reaction samples can be highly desalted by purification with a purification device.
  • a sample solution purified with a purification device as described herein can have a salt connection less than or equal to 100 ⁇ M, or less than or equal to 50 ⁇ M.
  • a sample solution purified by a purification device according to various embodiments, suitable for electrokinetic capillary injection.
  • a sequencing reaction purification using the purification device can separate ssDNA, for example, ssDNA of from 10 nt to 800 nt in size, from, for example, dye-labeled nucleotides and salts.
  • a kit for forming a purification device, and/or conducting purification of a sample can include ion-exchange particles, a reactive monomer such as a polymerizable or cross-linkable solution capable of forming a size-exclusion resin, particles of an affinity adsorbent and a receptacle capable of receiving the particles, the solution, or both.
  • the kit can contain one or more of an initiator or cross- linker.
  • the kit can contain size-exclusion ion-exchange particles.
  • the kit can contain a substrate or support structure to which the ion-exchange particles in a size-exclusion resin, or size-exclusion ion-exchange particles, can be attached, adhered, embedded, or otherwise bound or contacted.
  • the kit can further include a chemical for softening a substrate or support structure, and/or a device for heating the substrate, support structure, or particles.
  • the kit can contain heparin.
  • the kit can contain a sample container.
  • the kit can be used to construct a purification device as described herein. The purification device can be used with the sample container of the kit, or any other sample container, to purify a sample solution as described herein.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Devices For Use In Laboratory Experiments (AREA)
  • Manufacturing Of Micro-Capsules (AREA)

Abstract

L'invention concerne un dispositif de purification d'un échantillon, comprenant des particules d'échange ionique en contact avec un substrat. Le dispositif peut comprendre un matériau d'exclusion de dimension et un matériau d'échange ionique.
EP03771854A 2002-07-26 2003-07-25 Dispositif et procede de purification d'acides nucleiques Withdrawn EP1525053A1 (fr)

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
US413935 1982-09-01
US39885202P 2002-07-26 2002-07-26
US398852P 2002-07-26
US10/413,797 US20040016702A1 (en) 2002-07-26 2003-04-14 Device and method for purification of nucleic acids
US10/414,179 US20040018559A1 (en) 2002-07-26 2003-04-14 Size-exclusion ion-exchange particles
US414179 2003-04-14
US10/413,935 US6833238B2 (en) 2002-01-04 2003-04-14 Petal-array support for use with microplates
US413797 2003-04-14
PCT/US2003/023338 WO2004011142A1 (fr) 2002-07-26 2003-07-25 Dispositif et procede de purification d'acides nucleiques

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EP1525053A1 true EP1525053A1 (fr) 2005-04-27

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EP03771853A Withdrawn EP1525052A1 (fr) 2002-07-26 2003-07-25 Particule d'echange ionique a exclusion de dimension
EP03771854A Withdrawn EP1525053A1 (fr) 2002-07-26 2003-07-25 Dispositif et procede de purification d'acides nucleiques
EP03771827A Withdrawn EP1525299A2 (fr) 2002-07-26 2003-07-25 Support de reseau de petales pour microplaques

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JP (1) JP2005533646A (fr)
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AU2003256787A8 (en) 2004-02-16
WO2004011142A1 (fr) 2004-02-05
WO2004011592A2 (fr) 2004-02-05
AU2003254175A1 (en) 2004-02-16
WO2004011592A3 (fr) 2004-04-22
EP1525052A1 (fr) 2005-04-27
WO2004011141A1 (fr) 2004-02-05
JP2005533646A (ja) 2005-11-10
EP1525299A2 (fr) 2005-04-27
AU2003254174A1 (en) 2004-02-16

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