Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
• • 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
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
  • B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
• • 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
  • B01J47/016—Modification or after-treatment of ion-exchangers
• • 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

Definitions

• the present teachings relate to apparatuses and methods for filtering and/or purifying
• reaction products obtained from, for example, a polymerase chain reaction (PCR) or a 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. It can be desirable to capture primers, unincorporated nucleotides, primer-dimers, small DNA fragments, and in some cases desalt PCR products. It can be desirable to capture primers, dye-labeled primers, nucleotides, dye-labeled nucleotides, dideoxynucleotides, and dye labeled dideoxynucleotides and desalt DNA sequencing reaction products. A need exists for separation that addresses these and other problems associated with conventional techniques of purification.
• the present teaching provide a particle including a core including ion-exchange material, and a coating including polyelectrolyte material, wherein the core and coating are adapted to separate PCR reaction products.
• the present teaching provide a method for purifying PCR reaction products, the method including providing a plurality of particles, wherein each particle includes a core for ion-exchange and a coating of polyelectrolyte, and contacting the PCR reaction products to separate dsDNA fragments.
• the present teaching provide a particle including a core including ion-exchange material, and a coating including polyelectrolyte material, wherein the core and coating are adapted to separate DNA sequencing reaction products.
• the present teaching provide a method for purifying DNA sequencing reaction products, the method including providing a plurality of particles, wherein each particle includes a core for ion-exchange and a coating of polyelectrolyte, and contacting the DNA sequencing reaction products to separate dye-labeled ssDNA fragments.
• the present teaching provide a method for forming a particle, the method including selecting core material and polyelectrolyte material adapted to separating at least one of PCR reaction products and DNA sequencing reaction products, providing the core including ion-exchange material, and coating the core with polyelectrolyte material.
• the present teaching provide a composition including polyelectrolyte material wherein the polyelectrolyte material is adapted to coating ion-exchange material and to providing separation of at least one of PCR reaction products or DNA sequencing reaction products.
• the present teaching provide a system for biological separation, the system including polyelectrolyte material wherein the polyelectrolyte material is adapted to coating ion- exchange material and to providing sieving for separation of at least one of PCR reaction products or DNA sequencing reaction products.
• FIG. 1 illustrates a cross-sectional view of a polyelectrolyte-coated particle, where the coating includes a biopolyrr ,
• Fig. 2 illustrates a cross-sectional view of a polyelectrolyte-coated particle, where the coating is a synthetic polymer
• Fig. 2a illustrates several synthetic polymers that can be included in the coating for the polyelectrolyte-coated particle.
• Figs. 3a-3d demonstrate separation of sequencing reaction products with polyelectrolyte-coated particles with biopolymer in comparison with standard separation techniques, where Figs. 3a-3c demonstrate separation with polyelectrolyte-coated particles, according to various embodiments, Fig. 3d demonstrates separation with an uncoated ion- exchange particle;
• Figs. 4a-4b demonstrate separation of PCR reaction products by polyelectrolyte- coated particles with biopolymer, where Fig. 4a illustrates unpurified PCR reaction products including a mixture of a dye-labeled amplicon and a dye-labeled primer, and Fig. 4b illustrates PCR reaction products separated with a polyelectrolyte-coated particle to remove the dye- labeled primer;
• Figs. 5a-5b is a set of graphs illustrating a detail of Figs. 4a-4b, respectively;
• Fig. 6 demonstrates separation of a sequencing reaction products with polyelectrolyte-coated particles with synthetic polymer;
• Figs. 7a-7b demonstrate separation of a sequencing reaction products with polyelectrolyte-coated particles synthetic polymer
• Fig. 8 demonstrates the size cutoff for separation by the polyelectrolyte-coated particles with synthetic polymer for separation using coating polymers with different molecular weights;
• Figs. 9a and 9b demonstrate the separation of sequencing reaction products by polyelectrolyte-coated particles with synthetic polymer;
• Fig. 10 demonstrates the size-based removal of small dsDNA fragments from larger dsDNA fragments using polyelectrolyte-coated particles with synthetic polymer
• Fig. 11 demonstrates the removal of an oligonucleotide primer from a PCR product using polyelectrolyte-coated particles by illustrating the result of separating components with gel electrophoresis using a 2% agarose gel;
• Fig. 12 demonstrates the DNA size discrimination using non-desalting polyelectrolyte-coated particles.
• the section headings used herein are for organizational purposes only, and are not to be construed as limiting the subject matter described. All documents cited in this application, including, but not limited to patents, patent applications, articles, books, and treatises, are expressly incorporated by reference in their entirety for any purpose.
• the term "particle” as used herein refers to an ion-exchange material of liquid, solid, and/or gas that can be coated.
• the coating can cover the entire exterior surface of the particle or substantial portions thereof.
• the coating can cover portions of the interior surfaces of the particle.
• the coating can be irreversible to permanently coat the particle, or reversible to release the particle upon dissolution of the coating.
• the particle can be a single material or an agglomerate of materials that can be prepared by, for example, fusion, sintering, pressing, compressing, phase separation, precipitation, aggregation and coalescence, or otherwise formed together.
• the particle can have any shape either regular or irregular such as spherical, elliptical, triangular, cylindrical, etc.
• material refers to any substance on a molecular level or in bulk and can be a liquid and/or solid, e.g. an emulsion or a resin.
• pore size refers to a mean measurement, providing a guideline that particles larger than the pore size are less likely to penetrate into the interior of the particle, while smaller particles are more likely to penetrate into the interior of the particle. It is to be understood that the particles admitted to or deflected from a pore are not necessarily exactly the "pore size" given. That is, admittance to or exclusion from the pore is based on many factors, including actual pore size (wherein each pore of a core can have a different size), steric hindrance factors, ionic attractions, polarizations, and the like. Additionally, some particles, such as microporous gel type ion exchange materials, do not have defined pores. The particles have a "pore size that is defined by the intermolecular spacing within the gel matrix to define the size exclusion limit.
• ion-exchange refers to the process wherein each charge equivalent that can be “coupled” or “captured” on the ion-exchange surface can release an equivalent charge into an appropriate solution.
• This displacement of counter-ions from the ion-exchange core can release a large number of counter-ions into a sample solution.
• the selectivity of the ion-exchange core can be greater for the ion to be removed from the sample solution than for the counter-ion of the ion-exchange core.
• Ions of similar affinity as the counter-ion establish an equilibrium distribution based on the relative affinity of the ions for the ion-exchanger. The equilibrium can either provide or not provide the uptake of ions from solution.
• the counter-ion can be almost any ion including chloride, hydroxide, acetate, formate, bromide, sulfate, nitrate, phosphate or any other organic or inorganic anion.
• the choice of counter-ion can be influenced by the nature of the ions in solution that are to be removed.
• a counter-ion can be selected that has a significantly lower affinity for the ion- exchange core relative to the ion in solution, thus providing exchange with the ion in solution.
• Neutralization using a cation exchange resin in a mixed bed can drive the uptake of an ion from solution. This can be the case even if the affinity of the cation for the resin is lower than the affinity for the counter-ion.
• Counter-ions for cation- exchange particles include hydronium, sodium, potassium, ammonium, calcium, magnesium, or any other organic or inorganic cation.
• Polyelectrolyte-coated ion-exchange particles can be prepared in any ionic form.
• mixture refers to more than one polyelectrolyte-coated 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 polyelectrolyte-coated cation-exchange particles, polyelectrolyte-coated anion- exchange particles, uncoated cation-exchange particles, uncoated anion-exchange particles, inerts, or any 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.
• the mixture can be any proportion including stoichiometric equivalent amounts.
• a mixture of particles can provide size-based removal with desalting of the solution.
• An example is a polyelectrolyte-coated ion-exchange particle in the hydroxide form in a mixed bed with cation- exchange particles in a hydronium fonn.
• a mixture of particles can provide size-based removal of small ions without desalting the solution.
• An example is a polyelectrolyte-coated ion- exchange particle in the chloride or acetate form (or any other anion other than hydroxide), and no cation exchange material.
• the choice of counter-ionic form used for the polyelectrolyte- coated ion-exchange particles can be based on the application for which they are to be implemented.
• coating and grammatical variations thereof as used herein refer to less than a monolayer, a monolayer, or multiple layers of a polyelectrolyte with the same charge, or multiple layers of varied polyelectrolytes with opposite charges covering the particle.
• Smaller molecules such as, for example, inorganic buffer ions, and nucleotides can penetrate or permeate through the coating and can be retained by or ion-exchanged with the particle.
• the coating can prevent larger molecules, such as, for example, nucleic acids, from penetrating or permeating through the coating and reacting with the particle.
• polymer polymerization
• polymerize cross-linked product
• cross-linking cross-link
• cross-link and other like terms as used herein are meant to include both polymerization products and methods, and cross-linked products and methods wherein the resultant product has a three-dimensional structure, as opposed to, for example, a linear polymer.
• polymer also refers to oligomers, homopolymers, and copolymers. Polymerization can be initiated thermally, photochemically, ionically, or by any other means known to those skilled in the art of polymer chemistry.
• the polymerization can be condensation (or step) polymerization, ring-opening polymerization, high energy electron-beam initiated polymerization, free-radical polymerization, including atomic- transfer radical addition (ATRA) polymerization, atomic-transfer radical polymerization (ATRP), reversible addition fragmentation chain transfer (RAFT) polymerization, or any other living free-radical polymerization.
• ATRA atomic- transfer radical addition
• ATRP atomic-transfer radical polymerization
• RAFT reversible addition fragmentation chain transfer
• (meth)acryl refers to methacryl and acryl.
• N-methyl (meth)acrylamide refers to N-methyl methacrylamide and N-methyl acrylamide
• 2-hydroxyethyl (meth)acrylate refers to 2-hydroxyethyl methacrylate and 2-hydroxyethyl
• DNA refers to any nucleic acid, including RNA, PNA, and others as understood to one skilled in the art of molecular biology.
• polyelectrolyte-coated particles can have many uses such as, for example, in the separation of biomolecules.
• polyelectrolyte-coated particles can provide separation of biomolecules by restricting the ability of large molecules to interact with ion-exchange active sites of the particle. Small molecules that can penetrate into the polyelectrolyte-coated particle can interact with the ion-exchange active sites and can be retained on those sites. Larger, highly charged species can be restricted from interacting with the ion-exchange core by the coating or by the pore size of the core particle. Such larger, highly charged species can remain in solution rather than bind to the ion-exchange particle.
• the small molecules can be eluted from polyelectrolyte-coated particles.
• large molecules can include single stranded DNA (ssDNA) fragments, and double stranded DNA (dsDNA) fragments, and small molecules can include nucleotides, short fragments of ssDNA, short fragments of dsDNA, and small ions such as chloride, acetate, and surfactants.
• a polyelectrolyte-coated particle can be provided by exposing an ion-exchange core to an excess of polyelectrolyte.
• the core surface can become coated with the polyelectrolyte.
• the polyelectrolyte can be a biopolymer, including a naturally occurring biopolymer such as DNA, or a synthetic polymer as described herein.
• a polyelectrolyte-coated particle can be provided by exposing an ion-exchange core to a polyelectrolyte containing charges opposite to that of the core.
• the coated particle After the coating of the first polyelectrolyte, the coated particle is exposed to another polyelectrolyte containing charges opposite to that of the first polyelectrolyte.
• the coating process can be repeated to provide a polyelectrolyte-coated particle with multiple layers of alternative polyanion and polycation.
• a coating including polyelectrolyte can decrease the interaction of large molecules, including ssDNA, with the core by a size sieving effect.
• the coating can cover the outer surface of an ion-exchange core, decreasing interaction of large molecules with the surface.
• the coating can create a size-exclusion barrier decreasing penetration of large molecules into the interior of the core particle.
• the chemical properties of the polyelectrolyte can determine the sieving properties that the polyelectrolyte-coated particle displays.
• the polyelectrolyte coating can be crosslinked in a later step to obtain desirable physical properties and size-exclusion characteristics.
• a polyelectrolyte-coated particle can function as a size-excluded ion-exchanger by exploiting the inherent porosity of the ion-exchange core.
• Ion- exchange cores can be obtained with a wide variety of pore sizes, such as 5 angstroms (microporous) and 1000 angstroms or greater (macroporous).
• An ion-exchange core can be selected based on pore size such that it excludes molecules of a given size based on the requirements of the application.
• the polyelectrolyte coating can be large enough to be excluded from the pores of the ion-exchange core, thereby coating the exterior surface with substantially decreased coating of the interior of the pores.
• the polyelectrolyte coating can decrease the interaction of large molecules, such as ssDNA with the surface of the ion-exchange core by blocking a substantial amount of the surface ion- exchange sites.
• the pore size of the ion-exchange core bead can be small enough to decrease
• the surface ion- exchange sites can be substantially blocked and the inner ion-exchange sites can become less accessible, such that the polyelectrolyte-coated particle retains significantly less large molecules, such as dsDNA.
• smaller ions such as chloride, acetate, phosphate, pyrophosphate, small oligonucleotides, and nucleotides can enter the pores of the ion- exchange core and interact with interior ion-exchange sites.
• the coating can decrease the interaction of small ions, like the large molecules, with the surface ion-exchange sites because the surface sites are occupied by the polyelectrolyte coating.
• a coating can be formed on an ion-exchange core such that the coating has a thickness of from less than an equivalent monolayer to multiple layers.
• the thickness of the coating can vary over the surface of the ion-exchange core, or the thickness of the coating can be uniform over the entire surface of the ion-exchange core.
• the coating can at least partially cover the ion-exchange core.
• the coating material can at least partially fill one or more pore or surface feature, for example, pores, cracks, crevices, pits, channels, holes, recesses, or grooves, of the ion-exchange core.
• an ion-exchange core can be coated on all internal and external surfaces with a polyelectrolyte suitable for forming a coating.
• Fig. 1 illustrates polyelectrolyte-coated particle 30 which can include ion-exchange core 12 with pores 32 coated with a polyelectrolyte layer 20 composed of a biopolymer 300.
• Fig. 2 illustrates polyelectrolyte- coated particle 30 which can include ion-exchange core 12 with pores 32 coated with a polyelectrolyte layer 20 composed of a synthetic polymer 310.
• Small ionic particles (not shown) can sieve and/or enter pores 32 to bind to ion-exchange sites illustrated by positive charges, as in the case of anion-exchange core.
• the polyelectrolyte coating can substantially decrease the amount of large molecules illustrated by large ssDNA fragments that bind to the
• the ion-exchange core can be an anionic or cationic material.
• the ion-exchange core can be a polymer, cross-linked polymer, or inorganic material, for example, silica.
• the ion-exchange core can be a solid core material capable of ion- exchange, or a solid core material treated with an ion-exchange resin.
• the ion-exchange core can be surface-activated.
• the ion-exchange core can be non-magnetic, paramagnetic, or magnetic. Exemplary ion-exchange core materials include those listed below.
• ion-exchange material for the core can include anion-exchange resins such as Macro-Prep High Q, Macro-Prep 25Q, Aminex A-27, AG 1-X2, AG 1-X4, AG 1-X8, and AG 2-X8 (Bio-Rad, Hercules, CA, USA), Chromalite 30 SBG (Purolite Company, Bala Cynwyd, PA, USA), POROS HQ 20 (Applied Biosystems, Framingham, MA, USA), CA08Y and CA08S (Mitsubishi Chemical America, White Plains, NY, USA), Powdex PAO (Graver Technologies, Glasgow, DE, USA), Nucleosil SB (Alltech Associates, Inc., Deerfield, IL, USA), Fractogel TMAE (EM Science, Gibbstown, NJ, USA), IE 1-X8 (Spectrum Chromatography, Houston, TX, USA), Super Q-650S (TosoHaas Bioscience, Montgomeryville,
• anion-exchange resins
• the cores material can include PMMA, PS-DNB, silica, and/or cellulose.
• ion-exchange cores can include cation-exchange resins provided by manufacturers similar to those for anion-exchange resins including Chromalite 30 SAG (Purolite International Ltd., UK), AG 50WX8 and Macro-Prep High S (Bio-Rad, Hercules, CA, USA). Other cation and anion resins that can be used as ion-exchange cores will be apparent to one of ordinary skill in the art of ion-exchange resins.
• the ion-exchange core includes a solid core material capable of ion-exchange
• 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 those of ordinary skill in the art of ion-exchange separation, or a combination thereof.
• the solid core material can have various surface features, including, for example, pores, cracks, crevices, pits, channels, holes, recesses, or grooves.
• the solid core material can include sodium oxide, silicon dioxide, sodium borate, or a combination thereof.
• the solid core material can be surface-activated to be capable of ion- exchange, for example, modification to be capable of 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.
• surface can include external surfaces and/or internal surfaces. Internal surfaces can be, for example, the surfaces of voids or pores within the solid core material.
• the solid core material can be surface-activated to include one or more of quaternized functional groups, carboxylic acid groups, sulfonic acid groups, other cationic or anionic functional groups known to those of ordinary skill in the art of ion-exchange separation, or a combination thereof, on the surface of the solid core material.
• the biopolymer polyelectrolyte can be a naturally-occurring biopolymer such as DNA. Examples of naturally-occurring DNA include sheared salmon sperm DNA, plasmid DNA, restriction digests of plasmid DNA, herring sperm DNA, calf thymus DNA, and other naturally derived DNA.
• an ion-exchange core can be coated with a synthetic-polymer polyelectrolyte.
• the ion-exchange core can be coated with a water-soluble, or at least slightly water-soluble, polyanion.
• polyanion containing anionic functional groups can be used for coating.
• the anionic functional group can include carboxylic, boric, sulfonic, sulf ⁇ nic,
• the water- soluble, or at least slightly water-soluble, polyanion can be prepared by copolymerization of an acid- or phenolic-containing monomer, for example, acrylic acid, methacrylic acid, 4- acetoxystyrene that can be hydrolyzed to give phenolic group, 4-styrenesulfonic acid, styrylacetic acid, or maleic anhydride, with a water soluble, at least slightly water-soluble or water-insoluble, co-monomer.
• the synthetic polymer can be can be a homopolymer, a copolymer, a terpolymer, or another polymer.
• the synthetic polymer can include monomers including: (meth)acrylamide, N-methyl (methyl)acrylamide, N,N-dimethyl (methyl)acrylamide, N-ethyl (meth)acrylamide, N- ⁇ -propyl (meth)acrylamide, N-t_.o-propyl (meth)acrylamide, N-ethyl-N-methyl (meth)acrylamide, N,N-diethyl (meth)acrylamide, N- hydroxymethyl (meth)acrylamide, N-(3-hydroxypropyl) (methy)acrylamide, N- vinylformamide, N-vinylacetamide, N-methyl-N-vinylacetamide, vinyl acetate that can be hydrolyzed to give vinylalcohol after polymerization,
• the synthetic-polymer polyelectrolyte can be poly(acrylic acid-co-N,N- dimethylacrylamide) or poly(N,N-dimethyl acrylamide-co-styrene sulfonic acid).
• the ion-exchange can be coated with a water- soluble, or at least slightly water-soluble, polycation.
• polycation containing cationic functional groups can be used for coating.
• the cationic functional group can include protonated primary, secondary, and tertiary amine.
• the water-soluble, or at least slightly water-soluble, polycation can be prepared by copolymerization of a positively charged monomer with a water-soluble, at least slightly water-soluble, or water-insoluble comomer.
• polycation can include allyl amine hydrochloride, (3-acrylamidopropyl)trismethylammonium chloride, N-(3- aminopropyl)methacrylamide hydrochloride, and N- vinyl amides that can be hydrolyzed to
• the synthetic polymer can be poly(N-(3 -aminopropyl)methacrylamide-co-N,N-dimethylacrylamide) .
• Fig. 2a illustrates the monomeric subunits for those synthetic polymers listed.
• the abbreviations include acrylic acid (AA), acrylamide (AAm), N.N-dimethylacrylamide (DMA), (polyethylene oxide)monoacrylate (PEOacrylate), and vinyl sulfonic acid (NSA).
• the preparation of synthetic polymer can provide weight average molecular weights (M w ) ranging from 50 kiloDaltons to 15.0 megaDaltons, or 200 kiloDaltons to 4.0 megaDaltons, or 500 kiloDaltons to 3.0 megaDaltons.
• the molar percentage of the negatively or positively charged comonomer can contribute from 0.01 percent to 100 percent, or 0.1 percent to 20.0 percent, or 1.0 percent to 10.0 percent.
• other synthetic polymers can include homopolymers of styrene sulfonic acid, homopolymers and copolymers of acrylic acid, methacrylic acid, vinyl sulfonic acid, styrene sulfonic acid, 4-acetoxystyrene (precursor of 4- hydroxystyrene), and vinylphosphonic acid.
• other synthetic polymers can include homopolymers and copolymers of allyl amine hydrochloride, (3-acrylamidopropyl)trimethylammonium chloride, ⁇ -(3-aminopropyl)methacrylamide
• the comonomers can include acrylamide, met acrylamide, vinyl acetate that can be converted into vinyl alcohol in a subsequent step, NN-dimethylacrylamide, N- ethylacrylamide, N-propylacrylamide, N-vinyl-N-methyl acetamide, 2-hydroxyethyl acrylate, and vinyl methyl ether.
• the polyelectrolyte can include polyanions such as poly(styrenephosphoric acid), poly(phosphoric acid), homo-polymers and copolymers of maleic acid, derivatives thereof and the like, homo-polymers and co-polymers of fumaric acid, derivatives thereof and the like, peptide-type synthetic polyanions such as poly(aspartic acid), poly(galactronic acid), poly(glutamic acid), nucleic acid type synthetic polyanions such as poly(adenylic acid), poly(inosinic acid), poly(uridylic acid), and natural polyanions such as polysaccharides.
• polyanions such as poly(styrenephosphoric acid), poly(phosphoric acid), homo-polymers and copolymers of maleic acid, derivatives thereof and the like, homo-polymers and co-polymers of fumaric acid, derivatives thereof and the like
• peptide-type synthetic polyanions such as poly(aspartic acid), poly(galact
• the ion-exchange core can be coated with multiple layers of polyelectrolyte.
• the polyelectrolyte layers can include alternating layers of polyanions and polycations. Such alternating layers can provide strength durability and means to tailor the permeability of the coating.
• the outer most polyelectrolyte of the multiple-layer structure is negatively charged, DNA fragments will not be immobilized from its solution.
• Small anions such as, for example, chloride and primers can penetrate or permeate through the multiple-layer structure to be coupled to the core.
• the polyelectrolyte-coated particles can be provided as a mixture. The mixture can be incorporated in bed or column.
• the polyelectrolyte-coated particles can be provided in a bulk mode. A well-formed chromatographic bed of polyelectrolyte-coated particles is not necessarily required.
• the polyelectrolyte-coated particles can be provided in a device.
• the device can be a microfluidic device having one or more pathway, wherein at least a portion of at least one pathway includes polyelectrolyte-coated particles.
• the device can have an inlet and an outlet in fluid communication with the polyelectrolyte- coated particles.
• the particles can be present in, for example, a column.
• a column can be in a horizontal or vertical orientation, or in any position between a horizontal and a vertical orientation.
• the column can include a receptacle such as a cavity, chamber, reservoir, well, reaction region, bed, recess, or other receptacle suitable for containing or retaining polyelectrolyte-coated particles and the reaction products.
• the column can contain a plurality of polyelectrolyte-coated particles.
• the outlet of the device can be in fluid communication with a receptacle, such as a purified sample well, a tube, a glass plate, or another means of collecting a purified sample.
• the plurality of polyelectrolyte-coated particles can be a mixture.
• a device for separating the reaction products can be provided.
• the device can include a mixture of polyelectrolyte-coated particles.
• the method can include adding the particles to the column of the device.
• the reaction products can be placed in or introduced to the inlet of the device.
• the reaction products can travel from the inlet through the column including polyelectrolyte-coated particles and optional additional material.
• the sample can be subjected to a combination of size-exclusion separation and ion-exchange resulting in a filtration and/or purification of the reaction products.
• the filtered and/or purified solution can be eluted or removed from the column through the outlet, and can be directed to a receptacle for analysis and/or further processing.
• the reaction products can be moved through the column by centripetal force.
• the plurality of polyelectrolyte-coated particles can be mixed with reaction products in bulk.
• the plurality of polyelectrolyte-coated particles form a first volume and the reaction products form a second volume where the first volume is less than or
• separation of the reaction products using polyelectrolyte-coated particles can be achieved using a volume of polyelectrolyte-coated particles that is sufficient to provide adequate ion-exchange capacity, such as, ion-exchange of at least 80%, at least 90%, or at least 95% of the reaction products.
• the separation can occur in ten minutes or less, five minutes or less, or two minutes or less.
• the separation can include contacting the reaction products with the polyelectrolyte-coated particles for a period of time sufficient for the polyelectrolyte-coated particles to ion-exchange with the reaction products, and removing the purified reaction products from the polyelectrolyte-coated
• separating the purified reaction products from the particles can include removing the purified reaction products from the polyelectrolyte- coated particles, removing the particles from the purified reaction products, and/or sampling the purified reaction products from the mixture of particles and purified reaction products.
• An example of sampling the purified reaction products from the mixture of particles and purified reaction products can include analyzing the product in a tube by dipping a capillary directly into the tube and injecting into an instrument for further analysis.
• separations for the polyelectrolyte-coated particles can include, for example, separation of polymerase chain reaction (PCR) products, separation of DNA sequencing reaction mixtures, and purification of RNA.
• PCR polymerase chain reaction
• Polyelectrolyte- coated 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. Polyelectrolyte-coated particles can also be used for desalting of biological products or
• the selectivity of the polyelectrolyte-coated particles can depend on at least one of these criteria: (1) the molecular weight of the polyelectrolyte in the coating, (2) the chemical nature of the charged functionality and the charge density or molar percent of the charge of the polyelectrolyte in the coating, (3) the pore size of the ion-exchange core, (4) the nature of co-monomer in the coating polymer, and (5) the ratio of various co-monomers in the polymer.
• Each separation can provide different desirable features for at least one of the above criteria.
• PCR products include materials that can interfere with downstream analysis.
• PCR products can include amplified target sequences (amplicons), buffer salts, surfactants, metal ions, enzymes (e.g. polymerase), nucleotides, oligonucleotide primers, and other components in solution.
• the target sequences of double-stranded DNA dsDNA
• dsDNA can be analyzed, or used in subsequent enzymatic reactions that can be sensitive to at least some of the other PCR products.
• free nucleotides and oligonucleotide primers can interfere with downstream enzymatic reactions.
• a polyelectrolyte- coated particle can separate nucleotides, oligonucleotide primers, and buffer salts from the target sequences of dsDNA.
• the resulting 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 separating larger double stranded DNA (dsDNA) from smaller ssDNA, and dsDNA (e.g. primer-dimer, an unwanted side reaction product which is a dsDNA, or other non-specifically amplified fragments), free nucleotides, and salts.
• dsDNA double stranded DNA
• dsDNA e.g. primer-dimer, an unwanted side reaction product which is a dsDNA, or other non-specifically amplified fragments
• PCR products for removal include nucleotides, primers with 45 nucleotides of ssDNA, primer- dimer with 60 bp of dsDNA, and dsDNA fragments smaller than 200 bp.
• primers, primer-dimer, and DNA fragments smaller than 100 bp can be captured by polyelectrolyte-coated particles.
• primers, primer-dimer, and DNA fragments smaller than 300 bp can be captured by polyelectrolyte- coated particles.
• separation of larger dsDNA from other PCR products can include desalting.
• separation of larger dsDNA from other PCR products does not include desalting. There are circumstances when desalting can interfere with downstream processing such as separation and detection of the
• the polyelectrolyte-coated particles can be subjected to the same PCR conditions as the PCR reactants prior to PCR termination and purification.
• the polyelectrolyte-coated particles can be subjected to temperature cycling from 65 to 95 °C.
• the polyelectrolyte-coated particles can be bundled into a device that provides PCR and subsequent purification.
• an ion-exchange core with a pore size in the range of 100 Angstroms to 2000 Angstroms, coated with a polyelectrolyte of M w in the range of 1.0 megaDaltons to 3.0 megaDaltons provides PCR purification.
• an ion-exchange core with a pore size of 1000 Angstroms, coated with a polyelectrolyte of M of 1.7 megaDaltons to 2.6 megaDaltons provides PCR purification.
• DNA sequencing reaction products can include material that can interfere with downstream analysis.
• the quality of separation for purification sequencing reaction products can be evaluated by analyzing at least one of these criteria: (1) residual dye artifacts ("blobs") that appear as broad peaks superimposed over the sequence data, (2) peak intensity balance between large or small fragments, and (3) desalting of the sequencing reaction products.
• blobs residual dye artifacts
• DNA sequencing reaction products can include dye-labeled target sequences (the "sequencing ladder"), buffer salts, phosphate and pyrophosphate ions, metal ions, enzymes (e.g. polymerases), nucleotides, oligonucleotide primers, dye-labeled oligonucleotide primers, and other components such as residual, unincorporated dye-labeled-dideoxynucleotides ("dye terminators").
• the dye-labeled target sequences can be subjected to electrophoretic analysis and DNA sequencing ("basecalling") that can be sensitive to at least some of the other sequencing reaction products resulting in "blobs” that can cause errors in "basecalling.”
• capillary sequencers can use electrokinetic injection to introduce sequencing reaction products into the capillary for electrophoretic separation. The presence of salts in the sequencing reaction products can affect their introduction into the capillary, where a reduced salt concentration can enhance injection into the capillary.
• polyelectrolyte-coated particles can remove
• sequencing reaction products purified with polyelectrolyte-coated particles can have a salt concentration of less than or equal to 100 ⁇ M or less than or equal to 50 ⁇ M.
• a sample solution purified by polyelectrolyte-coated particles can be suitable for electrokinetic capillary injection.
• the polyelectrolyte-coated particle can have a size-exclusion limit of, for example, less than 10 nucleotides ssDNA, and can be able to remove small ions such as salts and dye-labeled primers from a sample solution while leaving ssDNA free in solution.
• Sequencing reaction purification using polyelectrolyte- coated particles can be used to separate ssDNA, for example, having a size of from 10 nucleotides to 1500 nucleotides or larger in size, from smaller components such as, for example, dye-labeled primers and salts.
• separation of DNA sequencing reaction products can include separating primers, dye-labeled primers, and salts from dye-labeled ssDNA targets by substantially excluding dye-labeled ssDNA fragments having greater than 45 nucleotides.
• purification of a sequencing reaction sample can remove dye-labeled dideoxynucleotides and salts from the sequencing reaction products by allowing such components to pass through the coating and react with the ion-exchange core, leaving a purified sample containing an amount of ssDNA relative to the pre-filtered amount, in an amount of 70% or more, 80% or more, 90% or more, or 95% or more.
• an ion-exchange core with a pore size in the range of 5 Angstroms to 1000 Angstroms, coated with a polyelectrolyte of M in the range of 1000 -Daltons to 6.0 megaDaltons provides sequencing reaction purification.
• an ion-exchange core with a pore size of 10 Angstroms to 50 Angstroms, coated with a polyelectrolyte of M of 2.4 megaDaltons to 4.9 megaDaltons provides sequencing reaction purification.
• a method for purifying DNA sequencing reaction products can include providing a plurality of polyelectrolyte-coated particles, and contacting the DNA sequencing reaction products to separate dye-labeled ssDNA fragments.
• the method can include removing residual dye artifacts such as dye-labeled primers that can result in blobs in the sequencing analysis.
• the method can include maintaining dye-labeled ssDNA fragment lengths.
• the ion-exchange resin was converted to an ion-exchange by washing a lOOuL volume of resin with lOOOuL of 1M salt, acid, or base solution. The mixture was vortexed for 5 minutes, and spun down in a centrifuge. The supernatant was removed and another lOOOuL aliquot of salt, acid, or base solution was added. This was repeated three times. The ion- exchange core was then washed and spun five times using lOOOuL aliquots of DI water.
• the ion-exchange cores were coated with DNA by repeated washings with lOOuL aliquots of lmg/mL sheared salmon sperm DNA (Eppendorf AG, Hamburg, Germany). Coating was performed by washing a lOOuL volume of resin with lOOuL of lmg/mL sheared salmon sperm DNA. The mixture was vortexed for 5 minutes, and spun down in a centrifuge. The supernatant was removed and another lOOuL aliquot of lmg/mL sheared salmon sperm DNA was added. This was repeated two times. The SEIE particles were then washed and spun three times using lOOOuL aliquots of DI water. ⁇ ATTNC PARTTCT FS WTTTT SYNTTTF TC POT,YM ,H.
• Poly(AA-co-DMA) polyelectrolyte for coating particles was provided by free radical polymerization of 0.32 g (4.44 mmol) of acrylic acid with 8.04 g (81.07 mmol) of N,N-dimethylacrylamide in 200 mL of DI water at 45 °C for 15 hours, using ammonium persulfate as an initiator and N,N,N'N'-tetramethylethylenediamine as a catalyst.
• the anion-exchange resin Prior to coating, the anion-exchange resin was first converted into hydroxide anion-exchange core in the same manner as the previous example. A 1000 ⁇ L aliquot of DI water was added to 0.20-0.25 mL volume of an anion-exchange core in chloride form. The mixture was vortexed for 2 minutes, and spun down in a centrifuge. The supernatant was removed and this DI water washing was repeated two times. A 1000 ⁇ L aliquot of 2.0 M of ammonium hydroxide was added to the washed resin. The mixture was vortexed for 2 minutes, let standing for 5 minutes at ambient temperature, vortexed one minute, and spun down in a centrifuge.
• a sample was prepared containing 400 uL dRhodamine Terminator Ready Reaction Mix (Applied Biosystems, Foster City, CA, USA), 50 uL Ml 3 universal reverse primer (3.2 pmol/uL), 25 uL template-amplicon ( ⁇ 100 ng/uL), and 525 uL DI water. This solution was aliquoted into wells in a thermal cycler plate at a volume of 20uL/well. The mixture was subjected to 25 cycles of heating, wherein each cycle included heating at 95°C for 10 seconds, heating at 50°C for 5 seconds, and heating at 60°C for 120 seconds.
• the mixed beds were washed with lmg/mL sheared salmon sperm DNA as described above and washed with DI water. 2uL of the each of the coated mixed beds were added to a small MicroAmp® tube (Applied Biosystems, Foster City, CA, USA). A mixed bed prepared from uncoated Aminex A-27 and Chromalite 30 SAG was prepared as a control. [00077] To perform the purification, luL of the above described sequencing reaction was added to each MicroAmp tube containing 2uL of the DNA-coated mixed bed ion exchange resins. The tubes were vortexed for 5 minutes after which 5uL DI water was added to each tube and mixed with a pipette.
• Figs. 3a-3d illustrate the results from this purification.
• Figs. 3a-3c illustrate that the biopolymer-polyelectrolyte-coated particles provided desirable purification (Fig. 3a illustrating DNA-coated Aminex A-27, Fig. 3b illustrating DNA-coated Bio-Rad AG 1-X8, and Fig.
• FIG. 3c illustrating DNA-coated Bio-Rad AG 2-X8, all three in a cationic-anionic mixed bed).
• Fig. 3d illustrates that the uncoated mixed bed (Aminex A-27 mixed with Chromalite 30 SAG) did not provide desirable purification. Loss of most of the DNA fragments was
• each cycle included heating at 96°C for 30 seconds, then at 60°C for 120 seconds.
• MicroAmp tube containing 2uL of the DNA-coated mixed bed ion exchange resins.
• the microcentrifuge tube was spun at 5000x g and 5 ⁇ L of supernatant was removed,
• FIGs. 4 and 5 illustrate the results from this purification as analyzed using
• Fig. 4a illustrates the PCR reaction solution prior to purification.
• Fig. 4b illustrates the PCR reaction solution purified and desalted by polyelectrolyte-coated particles.
• Fig. 4a illustrates peaks from 4000-5000 scans, labeled 330, generated by the primer, while the peak at 15,500 scans, labeled 320, was generated by the 550bp amplicon.
• the presence of PCR primer can often interfere with subsequent analyses of the PCR product or with subsequent reactions. Removal of the primer from PCR product can be desirable.
• Fig. 4b illustrates the resulting data when the PCR product is purified as described in the previous paragraph using polyelectrolyte-coated particles with biopolymer.
• the 550bp amplicon, labeled 320 is still visible, but the primer has been reduced to a less than detectable amount.
• Figs. 5a and 5b are a magnified view of a region of the electrophorograms illustrated in Figs. 4a and 4b, focusing on the region of the PCR primer, labeled 330.
• An internal standard was added to the injection solution after the purification step, but prior to analysis on the ABI Prism® 3100.
• the internal standard a synthetic ROX-labeled oligonucleotide at 20nM concentration, was used to measure the relative injection efficiency from the two solutions.
• the internal standard was observed in both electrophorograms, labeled 340.
• Fig. 5 illustrates that the injection efficiency from the two solutions is similar.
• the reduction in primer is 100%, while the loss of PCR product is 22%.
• This example illustrates that contact with the polyelectrolyte-coated particles can substantially decrease the primer from the PCR solution while leaving 78% of the PCR product in solution. Contact of the same solution with uncoated ion exchange beads resulted in loss of most of the primer as well as all of the PCR product (not shown).
• a sample was prepared using a fluorescently-labeled primer so that the sequencing reaction product could be analyzed on a fluorescent capillary sequencer.
• a solution was prepared containing 400 uL dRhodamine Terminator Ready Reaction Mix (Applied Biosystems, Foster City, California, 50 uL Ml 3 universal reverse primer (3.2 pmol/uL), 25 uL template-amplicon (-100 ug/uL), and 525 uL DI water. This master solution was aliquoted into wells in a thermal cycler plate at a volume
• Fig. 6 illustrated the results from this purification.
• Fig. 6 illustrates that the synthetic polymer-polyelectrolyte-coated particles provided desirable purification.
• poly(AA-co-DMA) polyelectrolyte-coated particles provided substantial removal of dye blob (residual dye-labeled ddNTPs) and relatively high signal strength indicating a
• FIG. 7a illustrates the purification of sequencing reaction products by Bio- Rad AG 1-X8 ion-exchange resin coated with poly(AA-co-DMA) prepared as described herein
• Fig. 7b illustrates the purification of sequencing reaction products by Aminex A-27 ion-exchange resin coated with poly(AA-co-DMA) prepared as described herein
• the polyelectrolyte-coated particles provided substantial removal of dye blob (residual dye- labeled ddNTPs) and relatively high signal strength indicating a desirable desalting of the sample.
• Other ion-exchange resins including Nucleosil, Isolute, Chromalite 30 SBG, Purolite-Cliromalite, Macro-Prep Hi-Q, Bio-Rad AG 2-X8,
• Fig. 8 illustrates the purification of sequencing reaction products purified by polyelectrolyte-coated particles coated with poly(AA-co-DMA).
• the sequencing reaction products were purified with different molecular weights of poly(AA-co-DMA) that increase from bottom to top electrophorograms for both the first column and second column. Sizing discrimination of polyelectrolyte-coated particles was evaluated using an assay based on the GeneScan® 500 ROX reagent (Applied Biosystems). The size standard consists of 16 dsDNA fragments ranging in size from 35bp to 500bp. The assay was performed by adding 5uL of the GeneScan® 500 ROX reagent to 5 ul of polyelectrolyte-coated particles. The mixture was agitated or vortexed for 5 minutes and the liquid is separated from the polyelectrolyte-coated particles.
• Fig. 8 represents electrophorograms after separation by coated resins with pore sizes of 10 Angstroms to 15 Angstroms.
• the second column represents electrophorograms after separation by coated resins of pore size of 1000 Angstroms.
• the largest peak observed early in the electrophorogram is a primer peak and represents a fragment of 25 nucleotides.
• Electrophorograms in the second column of Fig. 8 shows the elimination of the earlier peaks (smaller fragments) after separation with a lower molecular weight coating.
• FIG. 9a and 9b illustrates the purification of sequencing reaction products purified by polyelectrolyte-coated particles including Powdex-PAO ion-exchange resin coated with poly(AA-co-DMA).
• Fig. 9a illustrates an electrophorogram taken before purification
• Fig. 9b illustrates an electrophorogram taken after purification.
• the polyelectrolyte-coated particles removed LIZ® dye dTDP (2PP) and LIZ® dye dTTP (3PP) from the sequencing reaction products as labeled on Fig. 9a.
• the polyelectrolyte-coated particles removed other anions from the sequencing reaction products and improved electrokinetic injection of the sequencing reaction products, resulting in a factor often increase in signal strength.
• PCR reaction product purification was provided by polyelectrolyte-coated particles with synthetic polymer.
• Macro-Prep HQ ion-exchange resin was coated with poly(AA-co-DMA).
• Fig. 10 illustrates varying molar percentage of acrylic acid and molecular weigh of the poly(AA-co-DMA). The molecular weight and molar percentage increase from bottom to top from the bottom electrophorogram representing separation with resin coated with 1.1 mol% acrylic acid and 98.9 mol% DMA to the top electrophorogram representing separation with resin coated with 100 molar percent of acrylic acid or poly(AA) without N,N-dimethylacrylamide (DMA).
• Fig. 11 illustrates the removal of oligonucleotide primers, primer-dimer and DNA fragments by non-desalting Macro-Prep 50 HQ (chloride form) ion-exchange resin coated with poly(AA-co-DMA) in the ranges described above. Lane 1 of Fig.
• lane 11 was loaded with the size standard
• lane 2 was loaded with the one microliter of raw (no separation with polyelectrolyte coated ion-exchange particles) PCR product with 20 micromolar of primer
• lanes 3-7 were loaded with one microliter of PCR product after separation with polyelectrolyte-coated ion-exchange particles
• lanes 8-12 were loaded with two microliters of PCR product after separation with polyelectrolyte-coated ion-exchange particles.
• Lane 2 shows the unseparated PCR products such as primers, primer-dimer, etc. as a diffuse band below the main band. Lanes 3-12 do not have such as corresponding band.
• FIG. 12 illustrates the size-based removal of primer-dimer and non-specifically amplified dsDNA from PCR products by non-desalting Macro-Prep 50 HQ (chloride form) ion- exchange resin coated with poly(AA-co-DMA) in the ranges described above.
• the upper electrophorogram shows PCR products with no separation with polyelectrolyte coated ion- exchange particles.
• the lower electrophorogram shows PCR products separated with polyelectrolyte coated ion-exchange particles. The difference illustrates that dsDNA fragments smaller than lOObp were separated from larger fragments that remain in solution after separation with polyelectrolyte coated ion-exchange particles.
• Surfactants can be added to DNA sequencing reactions prior to purification using size-based purification methods. For example, prior to purification of DNA sequencing reactions using size-exclusion spin columns, sodium dodecyl sulfate (SDS) can be added to assist in purification. Further, the DNA sequencing reaction including SDS can be heated prior to purification using size-exclusion spin columns.
• SDS sodium dodecyl sulfate
• anionic surfactants can be replaced with non-ionic surfactants in DNA sequencing reaction purified by polyelectrolyte-coated ion-exchange particles.
• non-ionic surfactant refers to non-ionic surfactants, zwitter-ionic surfactants, or other surfactants that do not substantially contribute to the conductivity of the solution.
• non-ionic surfactants include Brij 35 and Brij 58 (CalBiochem, San Diego, CA), Triton X- 100 (Sigma, St.
• non-ionic surfactants can be activatable without heating in order to facilitate the removal of unico ⁇ orated dye-terminators from the DNA sequencing reaction.
• a sample was prepared using fluorescently-labeled dideoxy nucleotide terminators so that the sequencing reaction product could be analyzed on a fluorescent capillary sequencer.
• a solution was prepared containing 20 uL using a 8 uL of Big Dye Terminator® v.3.1 Ready Reaction Mix (Applied Biosystems, Foster City, California).
• the sequencing primer was Ml 3 universal reverse primer (3.2 pmol/uL).
• the template was a plasmid, pGEM (200 ng/uL).
• This master solution was aliquoted into wells in a GeneAmp® 9700 thermal cycler plate. The mixture was subjected to 25 cycles of heating, wherein each cycle included heating at 96°C for 10 seconds, heating at 50°C for 5 seconds, and heating at 60°C for 240 seconds.
• surfactant solutions were used: (1) 10 uL of Brij 35 aqueous solution at three percent by volume, final concentration of Brij 35 in entire solution was 0.67%; (2) and 10 uL of Triton X-100 aqueous solution at 10 percent by volume, final concentration of Triton X-100 in entire solution was 2.2%. Controls for each of the surfactants replaced the surfactant with 10 uL of water.
• surfactants can be effective at concentrations ranging from 0.01% to 10%. In various embodiments, surfactants can be effective at concentrations ranging from 0.1 % to 5%.
• Fig. 15 A purified without Brij 35 surfactant, dye artifacts appeared, for example, in the region from 15-60 nucleotides of the sequence.
• Fig. 15B purified with Brij 35 surfactant, dye artifacts in that same region did not appear.
• Figs. 16A and 16B illustrate DNA sequencing purification with and without Triton X-100 surfactant, respectively.
• Fig. 16 A purified without Triton X-100 surfactant, dye artifacts appeared, for example, in the region from 15-60 nucleotides of the sequence.
• Fig. 16B purified with Triton X-100 surfactant, dye artifacts in that same region did not appear.
• the surfactant was able to facilitate removal of the dye artifacts without heating and without interfering with direct electrokinetic injection or polyelectrolyte-coated ion-exchange particle purification.
• Poly(AA-co-DMA) polyelectrolyte was prepared as described above. Poly(allylamine hydrochloride), or "PAH,” M w 0.07 MDa and ⁇ oly(acrylic acid), or "PAA,” M w 4.00 MDa (Aldrich Chemicals) were readily available. The following polyelectrolyte solutions (0.5 wt%) were prepared by adding the appropriate amount of polymers to the deionized waster and then tumbled overnight at ambient temperature: poly(AA-co-DMA) 0.2286g /39.915g H 2 O; PAH 0.2268g /40.033g H 2 O; PAA 0.1192 g / 19.99g H 2 O.
• the anion-exchange resin (Macro-Prep High Q 50, Bio-Rad) was washed with de-ionized water three times. A slurry solution of resin (3 mL, 0.35g of wet resin) and 20 mL of de-ionized water were added to a 50 mL tube. To coat the resin, the polyelectrolyte solution (0.750 mL) was added. The tube was vortexed for one minute, spun at 1760 ⁇ m for three minutes, and the supernatant was decanted. The wet resin was dried under high vacuum oven at 70 degrees centigrade for two hours.
• Fig. 13 illustrates the multiple polyelectrolyte coatings applied to the resin in one of the examples.
• PAA M w 4.00 MDa (Aldrich Chemicals) and Fe 3 O 4 (EMG 1111) (Ferrotec Co ⁇ .) were readily available.
• a solution of PAA (0.750 mL) was added to a slurry solution of resin (0.2g of wet resin) and 10 mL de-ionized water. The mixture was vortexed for one minute, incubated at ambient temperature for five minutes, spun at 1760 ⁇ m for three minutes, and the supernatant was decanted.
• the resin was washed once with de-ionized water (10 mL). The suspension was spun at 1760 ⁇ m for three minutes and water was decanted.
• PCR reaction polyelectrolyte-coated ion- exchange particles can provide purification for sequence detection applications (both end-point and real-time), PCR cloning applications, etc.
• DNA sequencing reaction polyelectrolyte- coated ion-exchange particles can provide purification for any type of nucleic acid sequencing, for example, sequencing, fragment analysis, single nucleotide polymo ⁇ hism identification, etc.
• sequences can be separated from the purified supernatant liquid before sequencing the nucleic acids. Sequencing can be achieved by, for example, capillary electrophoresis. Isolating the purified supernatant liquid from the polyelectrolyte-coated ion-exchange particles can add steps such as pipetting, sample transfers, centrifugation, magnetic removal and/or thermal cycling between purification and sequencing. It is desirable to eliminate such steps to increase efficiency and speed, and reduce cost and hands-on time for the user between purification and sequencing.
• isolation of the polyelectrolyte-coated ion- exchange particles from the purified supernatant liquid can be achieved by filtration to retain the particles or centrifugation to collect the particles followed by aspiration of the supernatant liquid. It is desirable to eliminate the step of removing the polyelectrolyte-coated ion-exchange particles from the purified supernatant liquid by loading the sample for capillary electrophoresis directly from the supernatant liquid.
• the polyelectrolyte-coated ion-exchange particles can interfere with loading by inhibiting electrokinetic injection and causing random disruption in sequencing data or by clogging the capillary when polyelectrolyte-coated ion- exchange particles are crushed by the capillary against the bottom of the sample tube.
• the present teachings provide a method for loading the capillary from the bottom of the sample well, tube, or chamber.
• the polyelectrolyte- coated ion-exchange particles can be moved to the sides or the top of the well. Figs.
• FIG. 17A-17C illustrate a well 170 where the polyelectrolyte-coated ion-exchange particles 172 have been isolated from the supernatant liquid 174.
• the teachings provide positioning the polyelectrolyte- coated ion-exchange particles 172 at the top of the well 170 as illustrated in Fig. 17B or positioning the polyelectrolyte-coated ion-exchange particles 172 at the sides of well 170 as illustrated in Fig. 17C.
• the polyelectrolyte-coated ion-exchange particles can be positioned at the top of well by floating with a higher density solvent.
• the supernatant liquid's density can be increased such that it has greater density than the polyelectrolyte-coated ion-exchange particles.
• the polyelectrolyte-coated ion-exchange particles Upon increasing the density of the supernatant liquid, the polyelectrolyte-coated ion-exchange particles float to the top of the well.
• the capillary and electrode can then be immersed in the well below the layer of polyelectrolyte- coated ion-exchange particles.
• the loading of the capillary can be free of the polyelectrolyte- coated ion-exchange particles.
• the addition of the solvent eliminates centrifugation step in sample preparation and can be automated by an array pipettor.
• the polyelectrolyte-coated ion-exchange particles can be positioned at the sides of the well by magnetic attraction.
• the polyelectrolyte-coated ion-exchange particles can be coated with Fe 3 O 4 to provide magnetization.
• the polyelectrolyte-coated ion-exchange particles can be attracted to the sides of the well by a magnetic field on a side of the well or around the well provided by a magnet or an electrically induced magnetic field.
• the capillary and electrode can then be immersed in the center of the well clear of the concentric layer or beside the layer of polyelectrolyte-coated ion- exchange particles.
• the loading of the capillary can be free of the polyelectrolyte-coated ion- exchange particles.
• the magnetization of the polyelectrolyte-coated ion-exchange particles eliminates centrifugation step in sample preparation.
• the present teachings provide a method for loading the capillary from the top of the sample well, tube, or chamber.
• the polyelectrolyte- coated ion-exchange particles can be moved to the bottom of the well.
• Fig. 17A illustrates a well 170 where the polyelectrolyte-coated ion-exchange particles 172 have been isolated from the supernatant liquid 174.
• the particles can be moved to the bottom by centrifugation or magnetization as discussed above.
• a sequencer instrument can be calibrated to load the sample from a higher point in the well free of the polyelectrolyte-coated ion-exchange particles.
• Modification of the capillary position for supernatant loading can be provided by increasing the distance between the capillary tip to the bottom of the tube. For standard microtiter trays, an example of such a distance is 4.5 mm. This corresponds to a well volume for a 96-well tray of 30 uL.
• the volume of the supernatant can be increased to insure that the capillary is immersed in supernatant for good electrokinetic injection. This can be done by increasing the volume of the supernatant by addition of de-ionized water.
• the mixture of the de-ionized water and the supernatant can be provided by vortexing the mixture for 30 minutes.
• the sequencer instrument can be calibrated to load the sample from a higher point in the well by mechanically changing the tray or frame into which the tray mates so that the tray sits lower in the instrument.
• the sequencer instrument can be calibrated to load the sample from a higher point in the well by modifying the firmware initialization file to change the z-axis point-of-reference.
• the sequencer instrument can be calibrated to load the sample from a higher point in the well by programming software to retract the capillary from the bottom loading position to a point in the supernatant liquid layer above the polyelectrolyte- coated ion-exchange particles. Figs.
• FIGS. 18A-18B illustrate nucleic acid sequencing from supernatant using Big Dye Terminator® chemistry (Fig. 18A) and dRhodamine terminator chemistry (Fig. 18B) using a sequencer instrument calibrated to load the sample from a higher point in the well by modifying the firmware initialization file to change the z-axis point-of-

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