US20030008320A1 - Isolation of nucleic acids - Google Patents

Isolation of nucleic acids Download PDF

Info

Publication number
US20030008320A1
US20030008320A1 US10/232,135 US23213502A US2003008320A1 US 20030008320 A1 US20030008320 A1 US 20030008320A1 US 23213502 A US23213502 A US 23213502A US 2003008320 A1 US2003008320 A1 US 2003008320A1
Authority
US
United States
Prior art keywords
acid
bis
nucleic acid
gly
ionisable
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.)
Abandoned
Application number
US10/232,135
Inventor
Matthew Baker
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.)
Life Technologies Corp
Original Assignee
Baker Matthew John
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 GBGB9725839.6A external-priority patent/GB9725839D0/en
Priority claimed from GBGB9815541.9A external-priority patent/GB9815541D0/en
Priority claimed from PCT/GB1998/003602 external-priority patent/WO1999029703A2/en
Priority to US10/232,135 priority Critical patent/US20030008320A1/en
Application filed by Baker Matthew John filed Critical Baker Matthew John
Publication of US20030008320A1 publication Critical patent/US20030008320A1/en
Assigned to INVITROGEN CORPORATION reassignment INVITROGEN CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BAKER, MATTHEW JOHN, DNA RESEARCH INNOVATIONS LIMITED
Priority to US11/761,956 priority patent/US20070231892A1/en
Priority to US12/137,125 priority patent/US20080305528A1/en
Assigned to Life Technologies Corporation reassignment Life Technologies Corporation MERGER (SEE DOCUMENT FOR DETAILS). Assignors: INVITROGEN CORPORATION
Priority to US13/361,815 priority patent/US20120196944A1/en
Priority to US13/447,130 priority patent/US20120197009A1/en
Priority to US13/898,400 priority patent/US20130338245A1/en
Assigned to Life Technologies Corporation reassignment Life Technologies Corporation CORRECTIVE ASSIGNMENT TO CORRECT THE APPLICATION NO 09452626 PREVIOUSLY RECORDED ON REEL 023882 FRAME 0551. ASSIGNOR(S) HEREBY CONFIRMS THE MERGER SHOULD NOT HAVE BEEN RECORDED AGAINST THIS PATENT APPLICATION NUMBER. Assignors: INVITROGEN CORPORATION
Abandoned legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads

Definitions

  • Samples for use for DNA identification or analysis can be taken from a wide range of sources such as biological material such as animal and plant cells, faeces, tissue etc. also samples can be taken from soil, foodstuffs, water etc.
  • EP0707077A2 describes a synthetic water soluble polymer to precipitate nucleic acids at acid pH and release at alkaline pH The redissolving of the nucleic acids is performed at extremes of pH, temperature and/or high salt concentrations where the nucleic acids, especially RNA, can become denatured, degraded or require further purification or adjustments before storage and analysis.
  • WO 96/09116 discloses mixed mode resins for recovering a target compound, especially a protein, from aqueous solution at high or low ionic strength, using changes in pH.
  • the resins have a hydrophobic character at the pH of binding of the target compound and a hydrophilic and/or electrostatic character at the pH of desorption of the target compound.
  • a method for the extraction of biomolecules from biological material comprises contacting the biological material with a solid phase which is able to bind the biomolecules to it at a first pH and then extracting the biomolecules bound to the solid phase by elution using an elution solvent at a second pH.
  • a method for extracting nucleic acid from a sample containing nucleic acid comprises: contacting the sample with said solid phase at a first pH at which the solid phase has a positive charge and will bind negatively charged nucleic acid; and then releasing the nucleic acid at a higher pH at which the solid phase possesses a neutral, negative or less positive charge than at the first pH.
  • the solid phase will possess an overall positive charge, that is the sum of all positive and negative charges on the solid phase as a whole is positive. It is possible (though not preferred), however, that the solid phase as a whole could be negatively charged, but have areas of predominantly positive charge to which the nucleic acid can bind. Such solid phases are within the scope of the invention.
  • charge switching The change in the charge of the solid phase is referred to herein as “charge switching” and is accomplished by the use of a “charge switch material” in, on or as the solid phase.
  • the charge switch material comprises an ionisable group, which changes charge to according to the ambient conditions.
  • the charge switch material is chosen so that the pKa of the ionisable group is appropriate to the conditions at which it is desired to bind nucleic acid to and release nucleic acid from the solid phase.
  • nucleic acid will be bound to the charge switch material at a pH below or roughly equal to the pKa, when the charge switch material is positively charged, and will be released at a higher pH (usually above the pKa), when the charge switch material is less positively charged, neutral, or negatively charged.
  • the present invention is more particularly directed to the use of charge switch materials which allow binding and/or releasing (especially releasing) of the nucleic acid to occur under mild conditions of temperature and/or pH and/or ionic strength.
  • the charge switch material will change charge because of a change in charge on a positively ionisable group from positive to less positive or neutral, as the pH is increased in a range spanning or close to the pKa of the positively ionisable group. This may also be combined with a change of charge on a negatively ionisable group from neutral or less negative to more negative.
  • the charge switch material comprises a material which is positively charged at both pH values (such as a metal oxide) and a negatively ionisable group, the charge of which becomes more negative as the pH is increased in a range spanning or close to its pKa.
  • the charge switch material may comprise an ionisable group having a pKa between about 3 and 9.
  • the pKa is more preferably at least about 4.5, 5.0, 5.5, 6.0 or 6.5 and/or at most about 8.5, 8.0, 7.5 or 7.0.
  • a particularly preferred pKa for a positively ionisable group is between about 5 and 8; even more preferred is a pKa between about 6.0 and 7.0, more preferably between about 6.5 and 7.0.
  • the pKa for negatively ionisable groups is preferably between about 3 and 7, still more preferably between about 4 and 6, further preferably approximately at the pH at which it is desired to bind nucleic acid.
  • Materials having more than one pKa value may also be suitable for use as charge switch materials in accordance with the invention, provided that at a first (lower) pH the material(s) possess(es) a positive charge and that at a higher pH the charge is less positive, neutral or negative.
  • charge switch effects can also be achieved by changing the pH in a range close to, but not spanning, the pKa of an ionisable group.
  • a negatively ionisable group such as a carboxy group (pKa typically around 4)
  • 50% of such groups will be in the ionised form (e.g. COO ⁇ ) and 50% in the neutral form (e.g. COOH).
  • the pH increases, an increasing proportion of the groups will be in the negative form.
  • the binding step is carried out at a pH of below the pKa of the ionisable group, or (though this is not preferred) within about 1 pH unit above the pKa.
  • the releasing step is carried out at a pH above the pKa of the ionisable group, preferably at a pH between 1 and 3 pH units above the pKa.
  • Prior art methods such as those disclosed in EP0707077, often use high pH to release the nucleic acid, for example using strong bases such as NaOH.
  • strong bases such as NaOH.
  • Such high pH can cause depurination of nucleic acid, leading to the problems of imperfect replication, which can impede subsequent use of the nucleic acid, e.g. in detection and/or amplification techniques such as Southern or northern blotting or PCR.
  • pKa value(s) in accordance with the invention allows the step of releasing DNA from the solid phase to be performed under mild conditions, unlike in the prior art.
  • mild conditions generally means conditions under which nucleic acid is not denatured and/or not degraded and/or not depurinated, and/or conditions which are substantially physiological.
  • the releasing step is performed at a pH of no greater than about pH 10.5, more preferably no greater than about pH 10,0, 9.8, 9.6, 9.4, 9.2, 9.0, 8.9, 8.8, 8.7, 8.6 or 8.5.
  • the releasing step may even be performed at lower pH values, such as 8.0, 7.5 or 7.0.
  • the releasing step is carried out in the substantial absence of NaOH, preferably also the substantial absence of other alkali metal hydroxides, more preferably the substantial absence of strong mineral bases. Substantial absence may mean that the concentration is less than 25 mM, preferably less than 20 mM, more preferably less than 15 mM or 10 mM.
  • the desired change in pH can be achieved by altering the ionic strength of the solution and/or the temperature, since pH is dependent on both these factors.
  • neither high temperature nor high ionic strength are generally compatible with the desired mild conditions, and accordingly, the change in pH is preferably not achieved by large changes in ionic strength or temperature.
  • increasing ionic strength increases competition of charged species with the nucleic acid for binding to the solid phase, so can assist in releasing the nucleic acid. Small changes of ionic strength are therefore acceptable and may be used in conjunction with the change in pH to release the nucleic acid, preferably within the limits and ranges given below.
  • the temperature at which the releasing step performed is no greater than about 70° C., more preferably no greater than about 65° C., 60° C., 55° C., 50° C., 45° C. or 40° C. More preferably, such temperatures apply to the entire process.
  • the releasing step, or the entire process may even be performed at lower temperatures, such as 35° C., 30° C. or 25° C.
  • the releasing step preferably occurs under conditions of low ionic strength, suitably less than 1M or 500 mM, preferably less than 400 mM, 300 mM, 200 mM, 100 mM, 75 mM, 50 mM, 40 mM, 30 mM, 25 mM, 20 mM or 15 mM. It may even be below 10 mM
  • the ionic strength maybe at least about 5 mM, more preferably at least about 10 mM.
  • these ionic strengths also apply to the binding step.
  • PCR is sensitive to pH and the presence of charged contaminants.
  • the releasing step is performed using reagents suitable for storing nucleic acid (such as a commercially available storage buffer, e.g. 10 mM Tris.HCl, pH8.0-8.5, optionally in the presence of 1 mM EDTA), or using reagents suitable for use in a procedure to which the nucleic acid is to be subjected (such as a PCR buffer, e.g. 10 mM Tris.HCl, 50 mM KCl, pH 8.5).
  • a PCR buffer e.g. 10 mM Tris.HCl, 50 mM KCl, pH 8.5
  • nucleic acid extraction processes require a step of diluting the elution product containing nucleic acid, to make the solution suitable for e.g. PCR.
  • the present invention substantially avoids diluting the released nucleic acid.
  • the step of binding DNA occurs under mild conditions, suitably at a pH of no less than 3.0, preferably no less than 3.5, 4.0, 4.5 or 5.0.
  • Previous methods have used high concentrations of chaotropic agents, such as 8M guanidine. Such conditions may not be necessary in the practice of the present invention, in which the binding step preferably occurs in solution having a total concentration of 1M or less. More preferred temperatures and ionic strengths are as detailed above for the releasing step.
  • nucleic acid of interest is especially useful for extracting small quantities of nucleic acid, as the extracted DNA or RNA can be added directly to a reaction or storage tube without further purification steps (e.g. steps necessitated by the use of high ion concentrations in prior art methods), and without the need to dilute high ionic strength (as is the case with prior art methods using high ionic strength to elute the nucleic acid). Therefore loss of nucleic acid through changing the container, imperfect recovery during purification steps, degradation, or denaturation, and dilution of small amounts of nucleic acid can be avoided. This is particularly advantageous when a nucleic acid of interest is present in a sample (or is expected to be present) at a low copy number, such as in certain detection and/or amplification methods.
  • preferred chemical species for use as charge switch materials in accordance with the invention comprise a positively ionisable nitrogen atom, and at least one, but preferably more than one, electronegative group (such as a hydroxy, carboxy, carbonyl, phosphate or sulphonic acid group) or double bond (e.g. C ⁇ C double bond), which is sufficiently close to the nitrogen atom to lower its pKa. It has been found that such molecules tend to have suitable pKa values for the extraction of nucleic acid under mild conditions according to the present invention.
  • at least one (but more preferably more than one) electronegative group is separated from the ionisable nitrogen by no more than two atoms (usually carbon atoms).
  • Hydroxyl groups are particularly preferred electronegative groups (particularly when several hydroxyl groups are present, e.g. in polyhydroxyl amines, such as Tris (C(CH 2 OH) 3 —NH 2 ) or Bis-Tris (see below)), as they (1) lower the pKa of the nitrogen atom (e.g. amine group, e.g. from about 10 or 11) to a suitable value around neutral (i.e. pKa of about 7), (2) allow the species to remain soluble/hydrophilic above the pKa, when the nitrogen atom of the amine group loses its positive charge, (3) provide a site for covalent linkage to a solid substrate, e.g.
  • Tris C(CH 2 OH) 3 —NH 2
  • Bis-Tris see below
  • a polycarboxylated polymer such as polyacrylic acid
  • (4) are uncharged at pH values suitable for the releasing step and at which procedures such as PCR are performed (typically pH 8.5); the presence of charged species can interfere with PCR especially.
  • chemical species having an ionisable nitrogen atom and at least 2, 3, 4, 5 or 6 hydroxyl groups.
  • chemical species comprising ionisable groups can be immobilised onto solid supports (e.g. beads, particles, tubes, wells, probes, dipsticks, pipette tips, slides, fibers, membranes, papers, celluloses, agaroses, glass or plastics) in a monomeric or polymeric form via adsorption, ionic or covalent interactions, or by covalent attachment to a polymer backbone which is in turn immobilised onto the solid support.
  • solid supports e.g. beads, particles, tubes, wells, probes, dipsticks, pipette tips, slides, fibers, membranes or plastics.
  • Solid phase materials especially beads and particles, may be magnetisable, magnetic or paramagnetic. This can aid removal of the solid phase from a solution containing the released nucleic acid, prior to further processing or storage of the nucleic acid.
  • the weakly basic buffers are biological buffers, i.e. buffers from the class of buffers commonly used in biological buffer solutions.
  • biological buffers may be found in commercial chemical catalogues, such as the Sigma catalogue.
  • the releasing step takes place in a buffer solution containing the same biological buffer that is used in, as or on the charge switch material.
  • N-2-acetamido-2-aminoethanesulfonic acid ⁇ (ACES), pKa 6.8;
  • imidazole*, pKa 6.9, and derivatives* thereof i.e. imidazoles, especially derivatives containing hydroxyl groups**;
  • di/tri/oligo amino acids** for example Gly-Gly, pKa 8.2; and Ser-Ser, Gly-Gly-Gly, and Ser-Gly, the latter three having pKa values in the range 7-9.
  • the buffers marked above with an asterisk are not considered to be biological buffers for the purposes of the invention (whether or not they are designated as such in any chemical catalogue).
  • those marked with two asterisks (**) are also not considered to be biological buffers.
  • Preferred biological buffers are marked with a dagger ( ⁇ ), more preferred buffers are marked with two daggers ( ⁇ ), still more preferred buffers are marked with a double dagger ( ⁇ ) and most preferred buffers are marked with two double daggers ( ⁇ ).
  • These and other chemical species comprising ionisable groups may be coated as monomers onto a solid phase support using covalent, ionic or adsorption interactions. Additionally or alternatively, they may be coated onto such solid phase supports in polymeric form (preferably following condensation polymerization), for example by adsorption onto a negatively charged surface (e.g. a surface having exposed COOH or SO 3 groups) , or by covalent attachment. Additionally or alternatively, the chemical species containing ionisable groups may be attached to a polymer (see below) which is then attached to a solid support, e.g. by adsorption or covalent attachment
  • the chemical species or polymer backbones are covalently coupled to the solid support via a hydroxyl group or other group so that the ionisable group having the desired pKa value (usually, but not limited to, a nitrogen atom) remains capable of binding and releasing nucleic acid.
  • a hydroxyl group or other group so that the ionisable group having the desired pKa value (usually, but not limited to, a nitrogen atom) remains capable of binding and releasing nucleic acid.
  • Biological buffers and other chemical species comprising positively ionisable groups may be used in conjunction with a chemical species containing a negatively ionisable group which has a suitable pKa, preferably in the ranges described above.
  • a biological buffer having one or more positively ionisable nitrogen atoms
  • a polymer or other solid phase material which has exposed carboxy groups even after attachment of the biological buffer.
  • Such a material may bind nucleic acids at a low pH when few of the carboxy groups are negatively charged (i.e. few are in the COO ⁇ form, most being in the COOH form) and most of the ionisable nitrogen atoms are positively charged.
  • the negative charge is stronger (i.e. a greater proportion of carboxy groups are in the COO ⁇ form) and/or the positive charge is weaker, and the nucleic acid is repelled from the solid phase.
  • Chemical species containing ionisable groups can be attached to a polymer backbone using known chemistries.
  • a chemical species containing a hydroxyl group can be attached using carbodiimide chemistry to a carboxylated polymer backbones.
  • Other chemistries include can be employed by someone skilled in the art using other polymer backbones (e.g. based on polyethylene glycol (PEG) or carbohydrate) using a range of standard coupling chemistries (see e.g. Immobilised Affinity Ligand Techniques, Greg T. Hermanson, A. Krishna Mallia and Paul K. Smith, Academic Press, Inc., San Diego, Calif., 1992, ISBN 0123423309, which is incorporated herein by reference in its entirety.)
  • the chemical species containing ionizable groups can he polymerised without a backbone polymer, using cross-linking agents, for example reagents that couple via a hydroxy group (e.g. carbonyldiimidazole, butanediol diglycidyl ether, dialdehydes, diisothiocyanates).
  • cross-linking agents for example reagents that couple via a hydroxy group (e.g. carbonyldiimidazole, butanediol diglycidyl ether, dialdehydes, diisothiocyanates).
  • Polymers may also be formed by simple condensation chemistries to generate polymeric amino acids with the appropriate pKa e.g. Gly-Gly.
  • such immobilisation, attachment and/or polymerisation of the chemical species containing the ionisable group does not affect the pKa of the ionisable group, or leaves it in the desired ranges given above.
  • a preferred polymeric material is a dimer or oligomer of Bis-Tris, or a material formed by attaching a plurality of Bis-Tris molecules to a polyacrylic acid backbone, e.g. by reacting Bis-Tris monomer with polyacrylic acid using 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC).
  • EDC 1-ethyl-3-dimethylaminopropyl carbodiimide
  • the polymer can then be easily separated from the reactants using dialysis against a suitable reagent or water.
  • the polyacrylic acid has molecular weight of between about 500 and 5 million or more, More preferably it has a molecular weight of between 100,000 and 500,000.
  • the nature of the resultant Bis-Tris/polyacrylic acid molecule will depend on the ratio of the coupled components, since the polymer will have different properties depending on the proportion of the acrylic acid groups that are modified with Bis-Tris, for example it is desirable for some carboxy groups to remain unmodified, as the presence of these will not prevent the Bis-Tris from binding nucleic acid at low pH (especially if the Bis-Tris is in excess), but their negative charge at higher pHs will assist with release of the nucleic acid.
  • the molar ratio of Bis-Tris:carboxy groups (before attachment) is preferably between 5:1 and 1:5, more preferably between 3:1 and 1:3, still more preferably between 2:1 and 1:2, further preferably between 1.5:1 and 1:1.5, and most preferably about 1:1.
  • Preferred materials for use in the invention possess minimal residual positive charge (preferably minimal residual charge) at the pH at which the nucleic acid is released, and/or at pHs 8-8.5 making interference with or inhibition of downstream processes unlikely.
  • Patent application PCT/GB00/02211 discloses certain methods within the scope of the present invention and is incorporated herein by reference in its entirety as exemplification of the present invention (in all its aspects—see below for other aspects of the invention).
  • a method for the extraction of biomolecules from biological material which method comprises contacting the biological material with a solid phase which incorporates histidine or a polyhistidine which will tend to bind nucleic acids at low pH and then extracting the biomolecules bound to the solid phase by elution using an elution solvent which will then release the bound nucleic acids when the pH is increased.
  • An alternative embodiment of the present invention uses a material which is positively charged across a wide pH range, such as 0-12 or 0-14 (e.g. an electropositive substance such as a metal oxide, metal, strong or weak base, which lacks a pKa value, or for which the pKa value is at an extreme of high pH.
  • a positively charged material is combined with negatively ionisable material having a pKa intermediate between the pH values at which it is desired to bind and release nucleic acid, or slightly below the pH at which it is desired to bind nucleic acid.
  • This combination of materials allows nucleic acid to be bound at certain pH values, around and below the pKa of the negatively ionisable material, when there are fewer negatively charged groups, but allows the nucleic acid to be released when the pH is increased and a greater number of the ionisable groups are negatively charged.
  • the combination of iron II,IXX oxide and polycarboxylates (see Examples) binds nucleic acid at pH 4, when a relative scarcity of negative charges allowing the positively charged iron oxides to bind the nucleic acid.
  • the pH is increased to around 8
  • a large proportion of the carboxy groups become negatively charged and, despite the remaining presence of positive charges on the iron oxides, the reduction in overall positive charge allows the nucleic acid to be released.
  • charge switching molecules for nucleic acid purification are based on detergents or surfactants that have a hydrophobic portion and a hydrophilic portion which comprises a positively ionisable group with a suitable pKa, e.g. decyl methyl imidazole or dodecyl-Bis-Tris.
  • a suitable pKa e.g. decyl methyl imidazole or dodecyl-Bis-Tris.
  • These detergents/surfactants can be adsorbed onto surfaces e.g. plastic via their hydrophobic portions and the hydrophilic (ionisable) portions can be used to capture nucleic acid.
  • nucleic acids Another family of suitable materials for capture and easy release of nucleic acids are carbohydrates e.g. glucosamine, polyglucosamine (including chitosans), kanamycins and their derivatives i.e. sugar ring based structures containing one or more nitrogen atoms surrounded by hydroxyl groups which may also contain other groups such as acetate or sulphate groups to provide a suitable pKa for binding and release of nucleic acids.
  • carbohydrates e.g. glucosamine, polyglucosamine (including chitosans), kanamycins and their derivatives i.e. sugar ring based structures containing one or more nitrogen atoms surrounded by hydroxyl groups which may also contain other groups such as acetate or sulphate groups to provide a suitable pKa for binding and release of nucleic acids.
  • nucleic acid bases e.g. cytidine (pKa 4.2). These can be immobilised via hydroxy groups to a polymer or solid phase carboxy group using carbodiimides.
  • a still further group of materials having members with suitable pKa values are heterocyclic nitrogen-containing compounds.
  • Such compounds may be aromatic or aliphatic and may be monomers, oligomers or polymers, such as morpholine-, pyrrole-, pyrrolidine-, pyridine-, pyridinol-, pyridone-, pyrroline-, pyrazole-, pyridazine-, pyrazine-, piperidone-, piperidine-, or piperazine-containing compounds, e.g. polyvinylpyridine.
  • Such compounds may be substituted with electronegative groups to bring the pKa value(s) of the ionisable nitrogen atom(s) into an acceptable range, e.g. as defined above. However, in some compounds this may not be necessary, the pKa already being in such a range.
  • Preferred materials for use in accordance with the invention are hydrophilic, for example comprising charge switch materials which are (or which comprise chemical species which before immobilisation or polymerisation are) water soluble.
  • a suitable solid phase comprising a charge switch material
  • repeated capture and release of nucleic acids can be performed by adjusting the pH up or down.
  • sequential reactions or analyses can be performed on the nucleic acids using the same solid phase.
  • DNA can be isolated from a biological sample using a PCR tube comprising a charge switch material. Then, following PCR, the amplified DNA product may be isolated from the buffer constituents or primers by adjusting the pH in the same tube.
  • Particularly preferred solid phase materials are non-porous.
  • Porous supports are commonly used for isolating proteins, which can be trapped in the pores of the support.
  • nucleic acids tend to be too big to enter into pores of commonly used such supports, and will therefore become bound to the surface of the support, potentially trapping impurities in the pores.
  • the method can be used to separate single stranded RNA or DNA from double stranded DNA, because of the different charge densities on single and double stranded molecules, by appropriate manipulation of the pH or salt concentration. Typically, single stranded molecules will be released from binding to the solid phase at a lower pH than double stranded molecules.
  • the method of the invention may comprise a prior step of converting double stranded nucleic acid in the sample to single stranded nucleic acid (preferably using a strong base, e.g. 100 mM NaOH, or a weak base at high temperature, e.g. 60-100° C.).
  • the solid phase material is preferably then added simultaneously with a buffer which changes the pH of the sample to the pH for binding single stranded nucleic acid (typically a pH of 4-7).
  • the materials described herein may also be employed to capture nucleic acids in the liquid phase where binding leads to a cross-linked lattice large enough to separated from the liquid phase, e.g. by filtration or centrifugation.
  • the present invention provides a method for extracting nucleic acid from a sample containing nucleic acids, which method comprises: contacting the sample with a charge switch material at a first pH at which the charge switch material has a positive charge and will bind negatively charged nucleic acid; and then releasing the nucleic acid at a second, higher pH at which the charge is neutral, negative or less positive than at the first pH, wherein the charge switch material is soluble at said first pH, and wherein the combination of the charge switch material and the bound nucleic acid is insoluble at or above said first pH and below said second pH.
  • the charge switch materials will be soluble at the second pH, and will remain in solution with the nucleic acid upon release of the nucleic acid; the use of a weakly basic buffer (optionally bound to a soluble backbone, e.g. polyacrylic acid) as the charge switch material can avoid problems of contamination as described above.
  • a weakly basic buffer optionally bound to a soluble backbone, e.g. polyacrylic acid
  • the methods of the invention preferably include one or more washing steps between the binding and releasing steps.
  • Such (a) washing step(s) will generally be carried out at said first pH, or a pH above said first pH but lower then said second pH, such that the nucleic acid is substantially not released during the washing step(s).
  • the methods of the invention are particularly suitable for extracting nucleic acid which is then stored or further processed (e.g. by PCR), particularly when the charge switch material is in the form of e.g. a tube or well in which such storage and/or processing can occur.
  • the releasing step and any subsequent storage or processing need not be carried out as discrete steps, but can coincide, when said storage or processing occurs at a pH at which release of the nucleic acid occurs.
  • the method of the invention includes binding nucleic acid to a charge switch material coated on or otherwise provided by a PCR tube, washing the bound nucleic acid, and then without a separate releasing step commencing the PCR reaction using a PCR buffer which causes release of the nucleic acid.
  • the present invention provides novel charge switch materials for use in the methods of the receding aspects. It further comprises the use of such charge switch materials in such methods. All preferred features of the charge switch materials described in above in the context of the methods apply equally and independently to the present aspect of the invention (i.e. preferred combinations of features may be different in relation to this aspect from the preferred combinations in relation to the method aspects).
  • the present invention provides a container (preferably a PCR or storage tube or well, or a pipette tip) coated with, comprising or formed from a charge switch material, preferably a charge switch material comprising a biological buffer.
  • a container preferably a PCR or storage tube or well, or a pipette tip coated with, comprising or formed from a charge switch material, preferably a charge switch material comprising a biological buffer.
  • nucleic acid from blood, but applies also to the extraction of nucleic acid from any liquid sample, particularly biological samples or samples produced during laboratory techniques, such as PCR.
  • the method is particularly useful if the biological material is blood, but the method can be used for a range of applications substances such as plasmid and vector isolation and plant DNA extraction.
  • the cells in the blood are lysed to release nucleic acids and known lysing agents and methods can be used, such as contacting with ionic and non ionic detergents, hypotonic solutions of salts, proteases, chaotropic agents, solvents, using pH changes or heat.
  • lysing agents and methods can be used, such as contacting with ionic and non ionic detergents, hypotonic solutions of salts, proteases, chaotropic agents, solvents, using pH changes or heat.
  • a method of lysing cells to isolate nucleic acid is described in WO 96/00228.
  • the samples can optionally be diluted with water or other diluent in order to make it easier to manipulate and to process.
  • Dilutions up to ten times can be used and in general more dilution can be better and it is a feature of the present invention that it allows low dilution of blood to be possible.
  • the solid phase with which the blood is contacted can be a formed of a material which has a natural affinity for nucleic acids or it can be formed of a material which has its surface treated with an agent which will cause nucleic acids to bind to it or increase its affinity for nucleic acids.
  • Suitable materials include controlled pore glass, polysaccharide (agarose or cellulose), other types of silica/glass, ceramic materials, porous plastic materials such as porous plastic plugs which in a single moulded part or as an insert in a standard tube, polystyrene beads para magnetic beads etc.
  • the size and porosity is not critical and can vary and be selected for particular applications.
  • Suitable means for treating the surface of the solid phase or for derivatising it include treating it with a substance which can introduce a charge e.g. a positive charge on the surface or a hydrophilic or hydrophobic surface on the solid phase e.g. hydroxyl groups, nitrate groups, autoreactive groups, dyes and other aromatic compounds.
  • a substance which can introduce a charge e.g. a positive charge on the surface or a hydrophilic or hydrophobic surface on the solid phase e.g. hydroxyl groups, nitrate groups, autoreactive groups, dyes and other aromatic compounds.
  • the solid phase will cause DNA to be bound to it at one pH in preference to contaminants in the blood sample and will allow the bound nucleic acid to be released when it is contacted with an eluant at a different pH.
  • This system can be used with a solid phase which incorporates histidine or a polyhistidine which will tend to bind nucleic acids at low pH e.g. less than 6 and will then release the bound nucleic acids when the pH is increased e.g. to greater than 8.
  • the nucleic acids are bound at substantially neutral pH to an aminated surface and released at very high pH.
  • a plastic moulding can incorporate a binding agent e.g. in a well in a plate etc. so that the binding agent is incorporated in the surface, the blood sample is then contacted with the surface so as to cause nucleic acids to be bound to the surface. The blood sample is then removed and the surface treated with an eluting agent to release the bound nucleic acids.
  • a binding agent e.g. in a well in a plate etc.
  • the total system can be readily adapted for rapid large scale sampling and extraction techniques.
  • Binding agents which can be used include charge switchable ion exchange resins using a positively charged solid phase that can be reversed or made neutral by changing the pH above its pKa. e.g. nucleotides, polyamines, imidazole groups and other similar reagents with a suitable pKa value.
  • nucleic acids can be bound by intercalation using a variety of intercalating compounds incorporated into the solid phase e.g. actinomycin D, ethidium bromide etc.
  • a plastic surface can be modified to include functional groups.
  • the plastic can be any plastic used for containing samples e.g. polypropylene.
  • the functional groups can be positively or negatively charged so as to bind the nucleic acids in the correct buffer solution.
  • the functional groups can be chemical groups capable of covalent coupling to other ligands or polymers.
  • the surface characteristics of the plastic can be suitably modified for use in the present invention by including or adding the appropriate chemicals in the moulding compound e.g. as in an injection moulding compound.
  • the tubes or wells can be used to isolate and immobilise small quantities of DNA or RNA generating a pure template for subsequent PCR or other genetic analysis and manipulation.
  • the plastic is polypropylene e.g. it is in the form of a thin walled PCR tube
  • the polypropylene surface can be modified by oxidising the surface with an oxidising agent such as potassium permanganate and sulphuric acid to create a carboxylated surface (COOH groups).
  • an oxidising agent such as potassium permanganate and sulphuric acid
  • This tube can then be used to improve the isolation of DNA from solutions or from crude samples e.g. blood.
  • pH, di-electric constant, solubility or ionic strength the DNA or RNA can be immobilised on the walls of the tube, washed free of contaminants, ready for PCR or other analytical techniques.
  • the carboxy groups can be further modified by covalently coupling an anionic group such as imidazole or polyhistidine or any strong or weak ion exchanger, to allow binding of nucleic acids by a charge interaction.
  • an anionic group such as imidazole or polyhistidine or any strong or weak ion exchanger
  • This tube could then be used to improve the isolation of DNA from solutions or from crude samples e.g. of blood. Again by adjusting the pH, di-electric constant, or ionic strength the DNA or RNA can be immobilised on the walls of the tube, washed free of contaminants, ready for PCR or other analytical techniques.
  • the nucleic acids can be eluted with in a low salt buffer so that it is ready for PCR or other analysis.
  • the solid phase can be contacted with a blood sample by mixing with the solid phase in a mixing/stirring device, by passing the blood sample over the solid phase or the solid phase can be paramagnetic and manipulated by a magnetic field.
  • the invention is particularly suitable for the separation or isolation of nucleic acids from blood it can be used with a range of biomolecules particularly those that require removal of cell wall debris or insoluble particles.
  • the solid phase is in granular form in a column and the blood sample is drawn up through the column by means of a pressure differential being applied through the column, the blood sample is drawn up with air and the granular solid material can become fluidised thus increasing the mixing and contacting rates and minimising clogging.
  • the method of the invention is suitable for use in a multi-well format when a series of extractions from different samples can take place substantially simultaneously and this will facilitate the automation of the extraction process allowing rapid high throughput extraction to take place and to allow combinational chemistry to be performed. This will enable there to be a high throughput in a standard well array e.g. an eight by twelve array so that a large number of sample types can be treated automatically at the same time.
  • a charge switchable ion-exchanger was prepared by covalently coupling polyhistidine to 100 (m glass beads using glutaldehyde by mixing 1 gram of the aminated glass beads with 0.01%(v/v) glutaldehyde in 0.1M sodium bicarbonate at pH8 containing 20 mg polyhistidine. After overnight incubation the beads were washed exhaustively to remove non-covalently bound material and stored in 10 mM MES, pH5 containing 0.1% (v/v) Tween 20.
  • a blood sample was incubated with an equal volume of 10 mM MES pHS, containing 1% Tween 20, proteases (200 (g/ml) and 1 mM EDTA. After digestion is complete the blood was sucked up the column containing the glass beads and the DNA became immobilised allowing the contaminating proteins to pass through to waste,
  • the glass beads containing the immobilised DNA were washed with a buffer comprising 10 mM MES pH5, containing 1% Tween 20, and 1 mM EDTA and this was repeated until the wash solution was colourless.
  • the beads were dried with air and DNA eluted with a small quantity of 10 mM Tris HCl, pH 8.5 and collected in a sterile tube ready for analysis. Thus the DNA were separated from the blood.
  • the buffer etc. can be suitably modified.
  • the magnetic beads were also used to extract DNA directly from whole blood using a detergent based digestion reagent containing proteinase K.
  • Bis-Tris monomer was converted into a polymer by mixing together 160 mg of polyacrylic acid with a molecular weight of 240,000, 1.6 g of Bis-Tris and 1.6 g of EDC in 50 mM imidazole pH6.0. Following an overnight incubation, the mixture was dialysed in water. The purified polymer was then coated onto magnetic COOH beads or used in the liquid phase to bind genomic DNA from blood. A 5 ml blood sample was centrifuged to obtain the nuclei and WBC population and the resulting pellet digested with 1% SDS.
  • the cleared supernatant was mixed with either 25 mg of magnetic-Bis-Tris or about 250 ⁇ g of poly-Bis-Tris as a liquid. In both cases the captured DNA could be separated, washed in water and then redissolved in 10 mM Tris HCl pH8.5 in a pure form.
  • an insoluble polymer was made with inherent charge switching properties by mixing 80 mg of polyacrylic acid with 800 mg of Tris HCl and 800 mg of EDC in 50 mM Imidazole pH6.
  • the insoluble precipitate that formed generated a particulate solid phase that was used to capture DNA and release it in a similar manner to that described in example 4 for genomic DNA.
  • a blood sample was prepared as described in Example 4 and to the resulting supernatant decyl imidazole was added at pH 4 causing precipitation of the DNA.
  • the DNA pellet was collected by centrifugation and redissolved in 10 mM Tris pH 8.5.
  • Decyl imidazole was adsorbed onto a 200 ul plastic pipette tip by soaking in a 1% solution at pH4 in 0.1M sodium acetate.
  • a blood sample was prepared as described in Example 3 and the tips were used to bind the DNA by repeated pumping and sucking. After a wash with water, about 50 ng of DNA was recovered in water at pH10.
  • a solution of genomic DNA was prepared as described in example 3. To this sample 2 mg of Kanmycin was added at a concentration of 10 mg/ml. The resulting precipitate of DNA was filtered, washed in water at pH5 and re-dissolved in water at pH10.
  • Plasmid mini-prep was prepared using standard alkaline lysis reagents to generate a cleared lysate with a potassium acetate composition of 0.5M pH4.
  • a potassium acetate composition of 0.5M pH4 To this cleared supernatant, 2.5 mg of commercially available 1 ⁇ m carboxylated polystyrene magnetisable particles were added to bind the plasmid DNA The particles were washed with water at pH4 and then the DNA eluted using 10 mM Tris HCl at pH 8.5.
  • Typical UV ratios at 260 and 280 nm were 1.7-2.0, indicating pure nucleic acids with a single band observed with standard gel electrophoresis.
  • a solution of DNA at 100 ⁇ g per ml in 0.1M Potassium Acetate pH4 was allowed to stand for 1 hour in a 300 ⁇ l flat bottomed microtitre plastic plate, the plastic plate contained titanium oxide which was incorporated as a powder in the plastic when the plate was formed, After washing at pH4, the DNA was recovered with water at pH10 and 2 ml measured at 260 nm versus a plain polystyrene plate with no titanium oxide. Approximately, 50 ng of DNA was recovered per 300 ⁇ l well for the plate incorporating the titanium oxide compared to zero for the plain polystyrene plate.
  • a solution of tRNA and sheared genomic DNA was prepared at 30 ⁇ g per ml in 50 mM Potassium acetate buffer pH6.5 with 1M sodium chloride. Approximately 4 mg of magnetic polyhistidine beads were mixed with 1 ml of the nucleic acid solution for one minute until binding was complete. The beads were then thoroughly washed with water at pH5. To elute the bound material, the beads were mixed with 300 ⁇ l of 10 mM Tris.HCl, 10 mM NaCl, pH8.5. Gel analysis showed that most of the tRNA remained in solution and was not bound to the beads. The eluted material contained mostly genomic DNA with little or no tRNA.
  • extracted DNA was analysed by one or more of the following:

Abstract

A method for extracting nucleic acids from a biological material such as blood comprises contacting the mixture with a material at a pH such that the material is positively charged and will bind negatively charged nucleic acids and then eluting the nucleic acids at a pH when the said materials possess a neutral or negative charge to release the nucleic acids. The nucleic acids can be removed under mildly alkaline conditions to the maintain integrity of the nucleic acids and to allow retrieval of the nucleic acids in reagents that are immediately compatible with either storage or analytical testing.

Description

  • This application is a continuation-in-part of U.S. Ser. No. 09/586009, filed Jun. 2, 2000, which derives from PCT/GB98/03602, filed Dec. 4, 1998, which claims priority from UK patent application numbers 9725839.6, filed Dec. 6, 1997, and 9815541.9, filed Jul. 17, 1998. The entire disclosure of the '009 application is incorporated by reference herein. The present invention relates to a method for extracting nucleic acids and other biomolecules from biological materials, particularly blood and other liquid samples.[0001]
  • There is a very large demand for DNA analysis for a range of purposes and this has lead to the requirement for quick, safe, high throughput methods for the isolation and purification of DNA and other nucleic acids. [0002]
  • Samples for use for DNA identification or analysis can be taken from a wide range of sources such as biological material such as animal and plant cells, faeces, tissue etc. also samples can be taken from soil, foodstuffs, water etc. [0003]
  • Existing methods for the extraction of DNA include the use of phenol/chloroform, salting out, the use of chaotropic salts and silica resins, the use of affinity resins, ion exchange chromatography and the use of magnetic beads. Methods are described in U.S. Pat. Nos. 5,057,426, 4,923,978, EP Patents 0512767 A1 and EP 0515484B and WO 95/13368, WO 97/10331 and WO 96/18731. These patents and patent applications disclose methods of adsorbing nucleic acids on to a solid support and then isolating the nucleic acids. The previously used methods use some type of solvent to isolate the nucleic acids and these solvents are often flammable, combustible or toxic. [0004]
  • EP0707077A2 describes a synthetic water soluble polymer to precipitate nucleic acids at acid pH and release at alkaline pH The redissolving of the nucleic acids is performed at extremes of pH, temperature and/or high salt concentrations where the nucleic acids, especially RNA, can become denatured, degraded or require further purification or adjustments before storage and analysis. [0005]
  • WO 96/09116 discloses mixed mode resins for recovering a target compound, especially a protein, from aqueous solution at high or low ionic strength, using changes in pH. The resins have a hydrophobic character at the pH of binding of the target compound and a hydrophilic and/or electrostatic character at the pH of desorption of the target compound. [0006]
  • Blood is one of the most abundant sample sources for DNA analysis as blood samples are routinely taken for a wide range of reasons. However because of the viscous and proteinaceous nature of blood using known DNA extraction methods it has proved difficult to accomplish using automation due to the difficulties of handling large volumes containing relatively small amounts of DNA. Hitherto nucleic acid extraction has been partially automated only by using hazardous reagents and slow processing times. [0007]
  • I have now devised an improved method for the extraction of nucleic acids and other biomolecules from blood and other biological materials, and other samples containing nucleic acid [0008]
  • According to the invention there is provided a method for the extraction of biomolecules from biological material which method comprises contacting the biological material with a solid phase which is able to bind the biomolecules to it at a first pH and then extracting the biomolecules bound to the solid phase by elution using an elution solvent at a second pH. [0009]
  • In particular there is provided a method for extracting nucleic acid from a sample containing nucleic acid, which method comprises: contacting the sample with said solid phase at a first pH at which the solid phase has a positive charge and will bind negatively charged nucleic acid; and then releasing the nucleic acid at a higher pH at which the solid phase possesses a neutral, negative or less positive charge than at the first pH. [0010]
  • Generally the solid phase will possess an overall positive charge, that is the sum of all positive and negative charges on the solid phase as a whole is positive. It is possible (though not preferred), however, that the solid phase as a whole could be negatively charged, but have areas of predominantly positive charge to which the nucleic acid can bind. Such solid phases are within the scope of the invention. [0011]
  • The change in the charge of the solid phase is referred to herein as “charge switching” and is accomplished by the use of a “charge switch material” in, on or as the solid phase. [0012]
  • The charge switch material comprises an ionisable group, which changes charge to according to the ambient conditions. The charge switch material is chosen so that the pKa of the ionisable group is appropriate to the conditions at which it is desired to bind nucleic acid to and release nucleic acid from the solid phase. Generally, nucleic acid will be bound to the charge switch material at a pH below or roughly equal to the pKa, when the charge switch material is positively charged, and will be released at a higher pH (usually above the pKa), when the charge switch material is less positively charged, neutral, or negatively charged. [0013]
  • The present invention is more particularly directed to the use of charge switch materials which allow binding and/or releasing (especially releasing) of the nucleic acid to occur under mild conditions of temperature and/or pH and/or ionic strength. [0014]
  • Generally the charge switch material will change charge because of a change in charge on a positively ionisable group from positive to less positive or neutral, as the pH is increased in a range spanning or close to the pKa of the positively ionisable group. This may also be combined with a change of charge on a negatively ionisable group from neutral or less negative to more negative. In an alternative embodiment (described below), however, the charge switch material comprises a material which is positively charged at both pH values (such as a metal oxide) and a negatively ionisable group, the charge of which becomes more negative as the pH is increased in a range spanning or close to its pKa. [0015]
  • The charge switch material may comprise an ionisable group having a pKa between about 3 and 9. For positively ionisable groups, the pKa is more preferably at least about 4.5, 5.0, 5.5, 6.0 or 6.5 and/or at most about 8.5, 8.0, 7.5 or 7.0. A particularly preferred pKa for a positively ionisable group is between about 5 and 8; even more preferred is a pKa between about 6.0 and 7.0, more preferably between about 6.5 and 7.0. The pKa for negatively ionisable groups is preferably between about 3 and 7, still more preferably between about 4 and 6, further preferably approximately at the pH at which it is desired to bind nucleic acid. [0016]
  • Materials having more than one pKa value (e.g. having different ionisable groups), or combinations of materials having different pKa values, may also be suitable for use as charge switch materials in accordance with the invention, provided that at a first (lower) pH the material(s) possess(es) a positive charge and that at a higher pH the charge is less positive, neutral or negative. [0017]
  • Generally a charge switch will be achieved by changing the pH from a value below to a value above the pKa of the or an ionisable group. However, it will be appreciated that when the pH is the same as the pKa value of a particular ionisable group, 50% of the individual ionisable groups will be charged and 50% neutral. Therefore, charge switch effects can also be achieved by changing the pH in a range close to, but not spanning, the pKa of an ionisable group. For example, at the pKa of a negatively ionisable group, such as a carboxy group (pKa typically around 4), 50% of such groups will be in the ionised form (e.g. COO[0018] ) and 50% in the neutral form (e.g. COOH). As the pH increases, an increasing proportion of the groups will be in the negative form.
  • Preferably the binding step is carried out at a pH of below the pKa of the ionisable group, or (though this is not preferred) within about 1 pH unit above the pKa. Generally the releasing step is carried out at a pH above the pKa of the ionisable group, preferably at a pH between 1 and 3 pH units above the pKa. [0019]
  • Prior art methods, such as those disclosed in EP0707077, often use high pH to release the nucleic acid, for example using strong bases such as NaOH. Such high pH can cause depurination of nucleic acid, leading to the problems of imperfect replication, which can impede subsequent use of the nucleic acid, e.g. in detection and/or amplification techniques such as Southern or northern blotting or PCR. [0020]
  • The use of strong bases, or weak bases in combination with heating, again as in EP0707077, can also lead to degradation of RNA (especially at pH values of 10 or above), and denaturation of double stranded DNA (i.e. irreversible conversion of DNA from the double stranded form at least partially into the single stranded form), which can lead to a lack of specific binding in PCR. [0021]
  • The appropriate choice of pKa value(s) in accordance with the invention allows the step of releasing DNA from the solid phase to be performed under mild conditions, unlike in the prior art. As used herein, the term “mild conditions” generally means conditions under which nucleic acid is not denatured and/or not degraded and/or not depurinated, and/or conditions which are substantially physiological. [0022]
  • Preferably the releasing step is performed at a pH of no greater than about pH 10.5, more preferably no greater than about pH 10,0, 9.8, 9.6, 9.4, 9.2, 9.0, 8.9, 8.8, 8.7, 8.6 or 8.5. Depending on the pka(s) of the charge switch material, the releasing step may even be performed at lower pH values, such as 8.0, 7.5 or 7.0. Preferably the releasing step is carried out in the substantial absence of NaOH, preferably also the substantial absence of other alkali metal hydroxides, more preferably the substantial absence of strong mineral bases. Substantial absence may mean that the concentration is less than 25 mM, preferably less than 20 mM, more preferably less than 15 mM or 10 mM. [0023]
  • The desired change in pH can be achieved by altering the ionic strength of the solution and/or the temperature, since pH is dependent on both these factors. However, neither high temperature nor high ionic strength are generally compatible with the desired mild conditions, and accordingly, the change in pH is preferably not achieved by large changes in ionic strength or temperature. Moreover, increasing ionic strength increases competition of charged species with the nucleic acid for binding to the solid phase, so can assist in releasing the nucleic acid. Small changes of ionic strength are therefore acceptable and may be used in conjunction with the change in pH to release the nucleic acid, preferably within the limits and ranges given below. [0024]
  • Preferably the temperature at which the releasing step performed is no greater than about 70° C., more preferably no greater than about 65° C., 60° C., 55° C., 50° C., 45° C. or 40° C. More preferably, such temperatures apply to the entire process. The releasing step, or the entire process, may even be performed at lower temperatures, such as 35° C., 30° C. or 25° C. [0025]
  • Furthermore, the releasing step preferably occurs under conditions of low ionic strength, suitably less than 1M or 500 mM, preferably less than 400 mM, 300 mM, 200 mM, 100 mM, 75 mM, 50 mM, 40 mM, 30 mM, 25 mM, 20 mM or 15 mM. It may even be below 10 mM The ionic strength maybe at least about 5 mM, more preferably at least about 10 mM. [0026]
  • More preferably, these ionic strengths also apply to the binding step. [0027]
  • PCR is sensitive to pH and the presence of charged contaminants. In particularly preferred embodiments, the releasing step is performed using reagents suitable for storing nucleic acid (such as a commercially available storage buffer, e.g. 10 mM Tris.HCl, pH8.0-8.5, optionally in the presence of 1 mM EDTA), or using reagents suitable for use in a procedure to which the nucleic acid is to be subjected (such as a PCR buffer, e.g. 10 mM Tris.HCl, 50 mM KCl, pH 8.5). [0028]
  • Common previously known nucleic acid extraction processes require a step of diluting the elution product containing nucleic acid, to make the solution suitable for e.g. PCR. Preferably the present invention substantially avoids diluting the released nucleic acid. [0029]
  • Preferably the step of binding DNA occurs under mild conditions, suitably at a pH of no less than 3.0, preferably no less than 3.5, 4.0, 4.5 or 5.0. Previous methods have used high concentrations of chaotropic agents, such as 8M guanidine. Such conditions may not be necessary in the practice of the present invention, in which the binding step preferably occurs in solution having a total concentration of 1M or less. More preferred temperatures and ionic strengths are as detailed above for the releasing step. [0030]
  • The use of such mild conditions for the release of nucleic acid is especially useful for extracting small quantities of nucleic acid, as the extracted DNA or RNA can be added directly to a reaction or storage tube without further purification steps (e.g. steps necessitated by the use of high ion concentrations in prior art methods), and without the need to dilute high ionic strength (as is the case with prior art methods using high ionic strength to elute the nucleic acid). Therefore loss of nucleic acid through changing the container, imperfect recovery during purification steps, degradation, or denaturation, and dilution of small amounts of nucleic acid can be avoided. This is particularly advantageous when a nucleic acid of interest is present in a sample (or is expected to be present) at a low copy number, such as in certain detection and/or amplification methods. [0031]
  • Broadly speaking, preferred chemical species for use as charge switch materials in accordance with the invention comprise a positively ionisable nitrogen atom, and at least one, but preferably more than one, electronegative group (such as a hydroxy, carboxy, carbonyl, phosphate or sulphonic acid group) or double bond (e.g. C═C double bond), which is sufficiently close to the nitrogen atom to lower its pKa. It has been found that such molecules tend to have suitable pKa values for the extraction of nucleic acid under mild conditions according to the present invention. Preferably at least one (but more preferably more than one) electronegative group is separated from the ionisable nitrogen by no more than two atoms (usually carbon atoms). Hydroxyl groups are particularly preferred electronegative groups (particularly when several hydroxyl groups are present, e.g. in polyhydroxyl amines, such as Tris (C(CH[0032] 2OH)3—NH2) or Bis-Tris (see below)), as they (1) lower the pKa of the nitrogen atom (e.g. amine group, e.g. from about 10 or 11) to a suitable value around neutral (i.e. pKa of about 7), (2) allow the species to remain soluble/hydrophilic above the pKa, when the nitrogen atom of the amine group loses its positive charge, (3) provide a site for covalent linkage to a solid substrate, e.g. a polycarboxylated polymer (such as polyacrylic acid), and (4) are uncharged at pH values suitable for the releasing step and at which procedures such as PCR are performed (typically pH 8.5); the presence of charged species can interfere with PCR especially. Especially preferred are chemical species having an ionisable nitrogen atom and at least 2, 3, 4, 5 or 6 hydroxyl groups.
  • Many standard, weakly basic, buffers are ideal chemical species to provide the ionisable groups of charge switch materials, as they have pKa values close to neutral (i.e. 7). [0033]
  • For use as a charge switch material, chemical species comprising ionisable groups can be immobilised onto solid supports (e.g. beads, particles, tubes, wells, probes, dipsticks, pipette tips, slides, fibers, membranes, papers, celluloses, agaroses, glass or plastics) in a monomeric or polymeric form via adsorption, ionic or covalent interactions, or by covalent attachment to a polymer backbone which is in turn immobilised onto the solid support. Alternatively, they can be incorporated into solid, insoluble forms (with or without attachment to a polymer backbone) which inherently exhibit charge switching, e.g. beads, particles, tubes, wells, probes, dipsticks, pipette tips, slides, fibers, membranes or plastics. [0034]
  • Solid phase materials, especially beads and particles, may be magnetisable, magnetic or paramagnetic. This can aid removal of the solid phase from a solution containing the released nucleic acid, prior to further processing or storage of the nucleic acid. [0035]
  • Preferably the weakly basic buffers are biological buffers, i.e. buffers from the class of buffers commonly used in biological buffer solutions. Examples of biological buffers may be found in commercial chemical catalogues, such as the Sigma catalogue. [0036]
  • Leaching (i.e. transfer from the solid phase into solution in the liquid phase) of chemical species used to provide ionisable groups in ion exchange resins is a virtually inevitable phenomenon to some extent, especially when the species are attached to the solid phase by adsorption. Such leaching typically causes impurity in the resultant product, which can lead to significant problems, particularly if the resultant product is intended to be used in PCR (and especially when the species are charged). The use of biological buffers to provide the ionisable groups in charge switch materials can avoid this problem, since leaching of such buffers into the liquid phase will generally not significantly affect the nucleic acid, nor any downstream processes such as PCR to which it might be subjected. Indeed, many biological buffers are routinely used in PCR buffers, storage buffers and other buffer solutions [0037]
  • In a particularly preferred embodiment, the releasing step takes place in a buffer solution containing the same biological buffer that is used in, as or on the charge switch material. [0038]
  • Examples of suitable biological buffers for use in charge switch materials in accordance with the invention, and their pKa values, are as follows: [0039]
  • N-2-acetamido-2-aminoethanesulfonic acid ‡‡ (ACES), pKa 6.8; [0040]
  • N-2-acetamido-2-iminodiacetic acid ‡‡ (ADA), pKa 6.6; [0041]
  • amino methyl propanediol † (AMP), pKa 8.8; [0042]
  • 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid † (AMPSO), pKa 9.0, [0043]
  • N,N-bis-2-hydroxyethyl-2-aminoethanesulfonic acid †† (BES), pKa 7.1; [0044]
  • N,N-bis-2-hydroxyethylglycine † (BICINE), pKa 8.3; [0045]
  • bis-2-hydroxyethyliminotrishydroxymethylmethane †† (Bis-Tris), pKa 6.5; [0046]
  • 1,3-bistrishydroxymethylmethylaminopropane ‡‡ (BIS-TRIS Propane), pKa 6.8; [0047]
  • 4-cyclohexylamino-1-butane sulfonic acid (CABS), pKa 10.7; [0048]
  • 3-cyclohexylamino-1-propane sulfonic acid (CAPS), pKa 10.4; [0049]
  • 3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO), pKa 9.6; [0050]
  • 2-N-cyclohexylaminoethanesulfonic acid (CHES) pKa 9.6; [0051]
  • 3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid ‡‡ (DIPSO), pKa 7.6; [0052]
  • N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid †† (EPPS or HEPPS), pKa 8.0; [0053]
  • N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid † (HEPBS), pKa 8.3; [0054]
  • N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid †† (HEPES), pKa 7.5; [0055]
  • N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid †† (HEPPSO), pKa 7.8; [0056]
  • 2-N-morpholinoethanesulfonic acid ‡ (MES), pKa 6.1; [0057]
  • 4-N-morpholinobutanesulfonic acid †† (MOBS), pKa 7.6; [0058]
  • 3-N-morpholinopropanesulfonic acid †† (MOPS), pKa 7.2; [0059]
  • 3-N-morpholino-2-hydroxypropanesulfonic acid †† (MOPSO), pKa 6.9; [0060]
  • piperazine-N-N-bis-2-ethanesulfonic acid †† (PIPES), pKa 6.8; [0061]
  • piperazine-N-N-bis-2-hydroxypropanesulfonic acid †† (POPSO), pKa 7.8; [0062]
  • N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid † (TABS), pKA 8.9; [0063]
  • N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid †† (TAPS), pKa 8.4; [0064]
  • 3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid †† (TAPSO), pKa 7.4; [0065]
  • N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid †† (TES), pKa 7.4; [0066]
  • N-trishydroxymethylmethylglycine † (TRICINE), pKa 8.1; and [0067]
  • trishydroxymethylaminomethane † (TRIS), pKa 8.1; [0068]
  • histidine*, pKa 6.0, and polyhistidine ††; [0069]
  • imidazole*, pKa 6.9, and derivatives* thereof (i.e. imidazoles), especially derivatives containing hydroxyl groups**; [0070]
  • triethanolamine dimers**, oligomers** and polymers**; and [0071]
  • di/tri/oligo amino acids**, for example Gly-Gly, pKa 8.2; and Ser-Ser, Gly-Gly-Gly, and Ser-Gly, the latter three having pKa values in the range 7-9. [0072]
  • In a preferred embodiment, the buffers marked above with an asterisk (*) are not considered to be biological buffers for the purposes of the invention (whether or not they are designated as such in any chemical catalogue). In a more preferred embodiment, those marked with two asterisks (**) are also not considered to be biological buffers. Preferred biological buffers are marked with a dagger (†), more preferred buffers are marked with two daggers (††), still more preferred buffers are marked with a double dagger (‡) and most preferred buffers are marked with two double daggers (‡‡). [0073]
  • These and other chemical species comprising ionisable groups may be coated as monomers onto a solid phase support using covalent, ionic or adsorption interactions. Additionally or alternatively, they may be coated onto such solid phase supports in polymeric form (preferably following condensation polymerization), for example by adsorption onto a negatively charged surface (e.g. a surface having exposed COOH or SO[0074] 3 groups) , or by covalent attachment. Additionally or alternatively, the chemical species containing ionisable groups may be attached to a polymer (see below) which is then attached to a solid support, e.g. by adsorption or covalent attachment
  • Preferably the chemical species or polymer backbones are covalently coupled to the solid support via a hydroxyl group or other group so that the ionisable group having the desired pKa value (usually, but not limited to, a nitrogen atom) remains capable of binding and releasing nucleic acid. [0075]
  • Biological buffers and other chemical species comprising positively ionisable groups may be used in conjunction with a chemical species containing a negatively ionisable group which has a suitable pKa, preferably in the ranges described above. For example a biological buffer (having one or more positively ionisable nitrogen atoms) may be attached to a polymer or other solid phase material which has exposed carboxy groups even after attachment of the biological buffer. Such a material may bind nucleic acids at a low pH when few of the carboxy groups are negatively charged (i.e. few are in the COO[0076] form, most being in the COOH form) and most of the ionisable nitrogen atoms are positively charged. At higher pH the negative charge is stronger (i.e. a greater proportion of carboxy groups are in the COO form) and/or the positive charge is weaker, and the nucleic acid is repelled from the solid phase.
  • Chemical species containing ionisable groups (such as the biological buffers listed above) can be attached to a polymer backbone using known chemistries. For example a chemical species containing a hydroxyl group can be attached using carbodiimide chemistry to a carboxylated polymer backbones. Other chemistries include can be employed by someone skilled in the art using other polymer backbones (e.g. based on polyethylene glycol (PEG) or carbohydrate) using a range of standard coupling chemistries (see e.g. Immobilised Affinity Ligand Techniques, Greg T. Hermanson, A. Krishna Mallia and Paul K. Smith, Academic Press, Inc., San Diego, Calif., 1992, ISBN 0123423309, which is incorporated herein by reference in its entirety.) [0077]
  • Alternatively, the chemical species containing ionizable groups can he polymerised without a backbone polymer, using cross-linking agents, for example reagents that couple via a hydroxy group (e.g. carbonyldiimidazole, butanediol diglycidyl ether, dialdehydes, diisothiocyanates). Polymers may also be formed by simple condensation chemistries to generate polymeric amino acids with the appropriate pKa e.g. Gly-Gly. [0078]
  • Preferably such immobilisation, attachment and/or polymerisation of the chemical species containing the ionisable group does not affect the pKa of the ionisable group, or leaves it in the desired ranges given above. For example it is generally preferred not to couple or polymerise the chemical species via a positively ionisable nitrogen atom (in constrast for example to WO97/2982). In the practice of the invention, it is especially preferred to immobilise, attach and/or polymerise the chemical species via an hydroxyl group. [0079]
  • A preferred polymeric material is a dimer or oligomer of Bis-Tris, or a material formed by attaching a plurality of Bis-Tris molecules to a polyacrylic acid backbone, e.g. by reacting Bis-Tris monomer with polyacrylic acid using 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC). The polymer can then be easily separated from the reactants using dialysis against a suitable reagent or water. Preferably the polyacrylic acid has molecular weight of between about 500 and 5 million or more, More preferably it has a molecular weight of between 100,000 and 500,000. [0080]
  • The nature of the resultant Bis-Tris/polyacrylic acid molecule will depend on the ratio of the coupled components, since the polymer will have different properties depending on the proportion of the acrylic acid groups that are modified with Bis-Tris, for example it is desirable for some carboxy groups to remain unmodified, as the presence of these will not prevent the Bis-Tris from binding nucleic acid at low pH (especially if the Bis-Tris is in excess), but their negative charge at higher pHs will assist with release of the nucleic acid. For use in the present invention, the molar ratio of Bis-Tris:carboxy groups (before attachment) is preferably between 5:1 and 1:5, more preferably between 3:1 and 1:3, still more preferably between 2:1 and 1:2, further preferably between 1.5:1 and 1:1.5, and most preferably about 1:1. [0081]
  • The presence of high residual charge (i.e. charged species present in solution along with the extracted nucleic acid) may adversely affect the analysis of nucleic acids by PCR, or interfere with the binding of primers, dNTPs or polymerase to the nucleic acid, or to the sequestration of Mg[0082] 2+ ions, which are essential to PCR It is particularly preferable to avoid residual positive charge.
  • Preferred materials for use in the invention, such as the biological buffers described above, possess minimal residual positive charge (preferably minimal residual charge) at the pH at which the nucleic acid is released, and/or at pHs 8-8.5 making interference with or inhibition of downstream processes unlikely. [0083]
  • Patent application PCT/GB00/02211, of the same inventor, discloses certain methods within the scope of the present invention and is incorporated herein by reference in its entirety as exemplification of the present invention (in all its aspects—see below for other aspects of the invention). In particular, it discloses a method for the extraction of biomolecules from biological material which method comprises contacting the biological material with a solid phase which incorporates histidine or a polyhistidine which will tend to bind nucleic acids at low pH and then extracting the biomolecules bound to the solid phase by elution using an elution solvent which will then release the bound nucleic acids when the pH is increased. [0084]
  • An alternative embodiment of the present invention uses a material which is positively charged across a wide pH range, such as 0-12 or 0-14 (e.g. an electropositive substance such as a metal oxide, metal, strong or weak base, which lacks a pKa value, or for which the pKa value is at an extreme of high pH. Such a positively charged material is combined with negatively ionisable material having a pKa intermediate between the pH values at which it is desired to bind and release nucleic acid, or slightly below the pH at which it is desired to bind nucleic acid. This combination of materials allows nucleic acid to be bound at certain pH values, around and below the pKa of the negatively ionisable material, when there are fewer negatively charged groups, but allows the nucleic acid to be released when the pH is increased and a greater number of the ionisable groups are negatively charged. For example, the combination of iron II,IXX oxide and polycarboxylates (see Examples) binds nucleic acid at pH 4, when a relative scarcity of negative charges allowing the positively charged iron oxides to bind the nucleic acid. When the pH is increased to around 8, a large proportion of the carboxy groups become negatively charged and, despite the remaining presence of positive charges on the iron oxides, the reduction in overall positive charge allows the nucleic acid to be released. [0085]
  • Further examples of charge switching molecules for nucleic acid purification are based on detergents or surfactants that have a hydrophobic portion and a hydrophilic portion which comprises a positively ionisable group with a suitable pKa, e.g. decyl methyl imidazole or dodecyl-Bis-Tris. These detergents/surfactants can be adsorbed onto surfaces e.g. plastic via their hydrophobic portions and the hydrophilic (ionisable) portions can be used to capture nucleic acid. [0086]
  • Another family of suitable materials for capture and easy release of nucleic acids are carbohydrates e.g. glucosamine, polyglucosamine (including chitosans), kanamycins and their derivatives i.e. sugar ring based structures containing one or more nitrogen atoms surrounded by hydroxyl groups which may also contain other groups such as acetate or sulphate groups to provide a suitable pKa for binding and release of nucleic acids. [0087]
  • Another group of materials with suitable pKa values are nucleic acid bases, e.g. cytidine (pKa 4.2). These can be immobilised via hydroxy groups to a polymer or solid phase carboxy group using carbodiimides. [0088]
  • A still further group of materials having members with suitable pKa values are heterocyclic nitrogen-containing compounds. Such compounds may be aromatic or aliphatic and may be monomers, oligomers or polymers, such as morpholine-, pyrrole-, pyrrolidine-, pyridine-, pyridinol-, pyridone-, pyrroline-, pyrazole-, pyridazine-, pyrazine-, piperidone-, piperidine-, or piperazine-containing compounds, e.g. polyvinylpyridine. Such compounds may be substituted with electronegative groups to bring the pKa value(s) of the ionisable nitrogen atom(s) into an acceptable range, e.g. as defined above. However, in some compounds this may not be necessary, the pKa already being in such a range. [0089]
  • Preferred materials for use in accordance with the invention are hydrophilic, for example comprising charge switch materials which are (or which comprise chemical species which before immobilisation or polymerisation are) water soluble. [0090]
  • Once a suitable solid phase has been prepared, comprising a charge switch material, repeated capture and release of nucleic acids can be performed by adjusting the pH up or down. Thus sequential reactions or analyses can be performed on the nucleic acids using the same solid phase. For example, DNA can be isolated from a biological sample using a PCR tube comprising a charge switch material. Then, following PCR, the amplified DNA product may be isolated from the buffer constituents or primers by adjusting the pH in the same tube. [0091]
  • Particularly preferred solid phase materials are non-porous. Porous supports are commonly used for isolating proteins, which can be trapped in the pores of the support. However, nucleic acids tend to be too big to enter into pores of commonly used such supports, and will therefore become bound to the surface of the support, potentially trapping impurities in the pores. [0092]
  • The method can be used to separate single stranded RNA or DNA from double stranded DNA, because of the different charge densities on single and double stranded molecules, by appropriate manipulation of the pH or salt concentration. Typically, single stranded molecules will be released from binding to the solid phase at a lower pH than double stranded molecules. [0093]
  • In some circumstances, for example for the construction of gene chips, and for the preparation of probes, it may be desirable to produce single stranded DNA. Manipulation of pH and/or ionic strength can assist in purification and release of single stranded nucleic acid. The method of the invention may comprise a prior step of converting double stranded nucleic acid in the sample to single stranded nucleic acid (preferably using a strong base, e.g. 100 mM NaOH, or a weak base at high temperature, e.g. 60-100° C.). The solid phase material is preferably then added simultaneously with a buffer which changes the pH of the sample to the pH for binding single stranded nucleic acid (typically a pH of 4-7). [0094]
  • The materials described herein may also be employed to capture nucleic acids in the liquid phase where binding leads to a cross-linked lattice large enough to separated from the liquid phase, e.g. by filtration or centrifugation. [0095]
  • Accordingly, in a second aspect, the present invention provides a method for extracting nucleic acid from a sample containing nucleic acids, which method comprises: contacting the sample with a charge switch material at a first pH at which the charge switch material has a positive charge and will bind negatively charged nucleic acid; and then releasing the nucleic acid at a second, higher pH at which the charge is neutral, negative or less positive than at the first pH, wherein the charge switch material is soluble at said first pH, and wherein the combination of the charge switch material and the bound nucleic acid is insoluble at or above said first pH and below said second pH. [0096]
  • Preferred features of the method are as set out above, with the exception of the charge switch material being formed into, immobilised on, or attached to, a solid phase material. [0097]
  • Usually the charge switch materials will be soluble at the second pH, and will remain in solution with the nucleic acid upon release of the nucleic acid; the use of a weakly basic buffer (optionally bound to a soluble backbone, e.g. polyacrylic acid) as the charge switch material can avoid problems of contamination as described above. [0098]
  • The methods of the invention preferably include one or more washing steps between the binding and releasing steps. Such (a) washing step(s) will generally be carried out at said first pH, or a pH above said first pH but lower then said second pH, such that the nucleic acid is substantially not released during the washing step(s). [0099]
  • As has been indicated previously, the methods of the invention are particularly suitable for extracting nucleic acid which is then stored or further processed (e.g. by PCR), particularly when the charge switch material is in the form of e.g. a tube or well in which such storage and/or processing can occur. For the avoidance of doubt, however, it is emphasised that the releasing step and any subsequent storage or processing need not be carried out as discrete steps, but can coincide, when said storage or processing occurs at a pH at which release of the nucleic acid occurs. For example, the method of the invention includes binding nucleic acid to a charge switch material coated on or otherwise provided by a PCR tube, washing the bound nucleic acid, and then without a separate releasing step commencing the PCR reaction using a PCR buffer which causes release of the nucleic acid. [0100]
  • In a further aspect, the present invention provides novel charge switch materials for use in the methods of the receding aspects. It further comprises the use of such charge switch materials in such methods. All preferred features of the charge switch materials described in above in the context of the methods apply equally and independently to the present aspect of the invention (i.e. preferred combinations of features may be different in relation to this aspect from the preferred combinations in relation to the method aspects). [0101]
  • In a further aspect, the present invention provides a container (preferably a PCR or storage tube or well, or a pipette tip) coated with, comprising or formed from a charge switch material, preferably a charge switch material comprising a biological buffer. [0102]
  • The following description is directed particularly to the extraction of nucleic acid from blood, but applies also to the extraction of nucleic acid from any liquid sample, particularly biological samples or samples produced during laboratory techniques, such as PCR. [0103]
  • The method is particularly useful if the biological material is blood, but the method can be used for a range of applications substances such as plasmid and vector isolation and plant DNA extraction. [0104]
  • Preferably the cells in the blood are lysed to release nucleic acids and known lysing agents and methods can be used, such as contacting with ionic and non ionic detergents, hypotonic solutions of salts, proteases, chaotropic agents, solvents, using pH changes or heat. A method of lysing cells to isolate nucleic acid is described in WO 96/00228. [0105]
  • When the biological material consists of blood the samples can optionally be diluted with water or other diluent in order to make it easier to manipulate and to process. [0106]
  • Dilutions up to ten times can be used and in general more dilution can be better and it is a feature of the present invention that it allows low dilution of blood to be possible. [0107]
  • The solid phase with which the blood is contacted, can be a formed of a material which has a natural affinity for nucleic acids or it can be formed of a material which has its surface treated with an agent which will cause nucleic acids to bind to it or increase its affinity for nucleic acids. Suitable materials include controlled pore glass, polysaccharide (agarose or cellulose), other types of silica/glass, ceramic materials, porous plastic materials such as porous plastic plugs which in a single moulded part or as an insert in a standard tube, polystyrene beads para magnetic beads etc. The size and porosity is not critical and can vary and be selected for particular applications. [0108]
  • Suitable means for treating the surface of the solid phase or for derivatising it include treating it with a substance which can introduce a charge e.g. a positive charge on the surface or a hydrophilic or hydrophobic surface on the solid phase e.g. hydroxyl groups, nitrate groups, autoreactive groups, dyes and other aromatic compounds. [0109]
  • In a preferred embodiment of the invention the solid phase will cause DNA to be bound to it at one pH in preference to contaminants in the blood sample and will allow the bound nucleic acid to be released when it is contacted with an eluant at a different pH. This system can be used with a solid phase which incorporates histidine or a polyhistidine which will tend to bind nucleic acids at low pH e.g. less than 6 and will then release the bound nucleic acids when the pH is increased e.g. to greater than 8. Alternatively the nucleic acids are bound at substantially neutral pH to an aminated surface and released at very high pH. [0110]
  • In another embodiment of the invention a plastic moulding can incorporate a binding agent e.g. in a well in a plate etc. so that the binding agent is incorporated in the surface, the blood sample is then contacted with the surface so as to cause nucleic acids to be bound to the surface. The blood sample is then removed and the surface treated with an eluting agent to release the bound nucleic acids. When the surface is part of a well in a multi-well plate, the total system can be readily adapted for rapid large scale sampling and extraction techniques. [0111]
  • Binding agents which can be used include charge switchable ion exchange resins using a positively charged solid phase that can be reversed or made neutral by changing the pH above its pKa. e.g. nucleotides, polyamines, imidazole groups and other similar reagents with a suitable pKa value. [0112]
  • Also, nucleic acids can be bound by intercalation using a variety of intercalating compounds incorporated into the solid phase e.g. actinomycin D, ethidium bromide etc. [0113]
  • In a further embodiment of the invention a plastic surface can be modified to include functional groups. The plastic can be any plastic used for containing samples e.g. polypropylene. The functional groups can be positively or negatively charged so as to bind the nucleic acids in the correct buffer solution. [0114]
  • Alternatively the functional groups can be chemical groups capable of covalent coupling to other ligands or polymers. [0115]
  • When the plastic is used in a plastic moulding e.g. in a well in a plate, or as a polymerase chain reaction (PCR) tube, the surface characteristics of the plastic can be suitably modified for use in the present invention by including or adding the appropriate chemicals in the moulding compound e.g. as in an injection moulding compound. [0116]
  • When this is used in a PCR tube or in a deep well plate the tubes or wells can be used to isolate and immobilise small quantities of DNA or RNA generating a pure template for subsequent PCR or other genetic analysis and manipulation. [0117]
  • When the plastic is polypropylene e.g. it is in the form of a thin walled PCR tube the polypropylene surface can be modified by oxidising the surface with an oxidising agent such as potassium permanganate and sulphuric acid to create a carboxylated surface (COOH groups). This tube can then be used to improve the isolation of DNA from solutions or from crude samples e.g. blood. By adjusting the pH, di-electric constant, solubility or ionic strength the DNA or RNA can be immobilised on the walls of the tube, washed free of contaminants, ready for PCR or other analytical techniques. [0118]
  • The carboxy groups can be further modified by covalently coupling an anionic group such as imidazole or polyhistidine or any strong or weak ion exchanger, to allow binding of nucleic acids by a charge interaction. This tube could then be used to improve the isolation of DNA from solutions or from crude samples e.g. of blood. Again by adjusting the pH, di-electric constant, or ionic strength the DNA or RNA can be immobilised on the walls of the tube, washed free of contaminants, ready for PCR or other analytical techniques. [0119]
  • The nucleic acids can be eluted with in a low salt buffer so that it is ready for PCR or other analysis. [0120]
  • The solid phase can be contacted with a blood sample by mixing with the solid phase in a mixing/stirring device, by passing the blood sample over the solid phase or the solid phase can be paramagnetic and manipulated by a magnetic field. Although the invention is particularly suitable for the separation or isolation of nucleic acids from blood it can be used with a range of biomolecules particularly those that require removal of cell wall debris or insoluble particles. [0121]
  • In a preferred embodiment of the invention the solid phase is in granular form in a column and the blood sample is drawn up through the column by means of a pressure differential being applied through the column, the blood sample is drawn up with air and the granular solid material can become fluidised thus increasing the mixing and contacting rates and minimising clogging. [0122]
  • The method of the invention is suitable for use in a multi-well format when a series of extractions from different samples can take place substantially simultaneously and this will facilitate the automation of the extraction process allowing rapid high throughput extraction to take place and to allow combinational chemistry to be performed. This will enable there to be a high throughput in a standard well array e.g. an eight by twelve array so that a large number of sample types can be treated automatically at the same time. [0123]
  • The invention, in its various aspects, will now be described in detail, by way of example only. [0124]
  • EXAMPLE 1 Extraction of Nucleic Acids from Whole Blood
  • A charge switchable ion-exchanger was prepared by covalently coupling polyhistidine to 100 (m glass beads using glutaldehyde by mixing 1 gram of the aminated glass beads with 0.01%(v/v) glutaldehyde in 0.1M sodium bicarbonate at pH8 containing 20 mg polyhistidine. After overnight incubation the beads were washed exhaustively to remove non-covalently bound material and stored in 10 mM MES, pH5 containing 0.1% (v/v) Tween 20. [0125]
  • About 300 mg of the 100 (m derivitised glass beads were added to a 1 ml plastic column enclosed at both ends. [0126]
  • A blood sample was incubated with an equal volume of 10 mM MES pHS, containing 1% Tween 20, proteases (200 (g/ml) and 1 mM EDTA. After digestion is complete the blood was sucked up the column containing the glass beads and the DNA became immobilised allowing the contaminating proteins to pass through to waste, [0127]
  • The glass beads containing the immobilised DNA were washed with a buffer comprising 10 mM MES pH5, containing 1% Tween 20, and 1 mM EDTA and this was repeated until the wash solution was colourless. [0128]
  • After washing, the beads were dried with air and DNA eluted with a small quantity of 10 mM Tris HCl, pH 8.5 and collected in a sterile tube ready for analysis. Thus the DNA were separated from the blood. [0129]
  • For different biomolecules, the buffer etc. can be suitably modified. [0130]
  • EXAMPLE 2
  • One gram of carboxylated paramagnetic beads were washed in 50 mM Imidazole buffer pH6 and then mixed with 100 mg of polyhistidine in 50 ml of 50 mM Imidazole buffer pH 6. A chemical coupling agent was added (EDC) at a final concentration of 5 mg per ml and mixed overnight. The beads were washed in water, 0.5M sodium chloride, 1% Tween 20, 100 mM Tris HCl pH 8 and stored in 10 mM MES, 0.1% Tween 20 pH5. [0131]
  • To extract DNA from blood, 1 mg of beads were mixed with blood diluted in 10% Tween 20 with 25 mM MES, 1 mM EDTA pH 5. The beads were separated with a magnet and washed by resuspending in 1 mM MES, 0.1% Tween 20. To elute the DNA the beads were resuspended in 10 mM Tris HCl pH 8.5 and separated with magnet leaving the DNA in solution. [0132]
  • EXAMPLE 3 Bis-Tris Solid Phase Magnetic Beads
  • 200 mg of carboxylated 1 μm magnetic particles were reacted in a one step procedure with 100 mg of Bis-Tris and 100 mg of the carbodiimide, EDC, in 50 mM imidazole buffer pH6.0. Following an overnight incubation, the magnetic particles were washed and used to isolate Plasmid DNA. [0133]
  • An alkaline lysis method was used to prepare a cleared 5 ml bacterial lysate generating a supernatant containing the plasmid in 0.5M potassium acetate, pH5, To the supernatant, 2.5 mg of magnetic particles were added and mixed for 1 minute. After magnetic separation and washing with water pH5, the pure plasmid DNA was eluted off in 200 μl of 10 mM Tris.HCl pH 8.5. [0134]
  • The magnetic beads were also used to extract DNA directly from whole blood using a detergent based digestion reagent containing proteinase K. [0135]
  • EXAMPLE 4 Tricine on Solid Phase Magnetic Beads
  • 50 mg of carboxylated 1 μm magnetic particles were reacted in a one step procedure with 50 mg of Tricine and 100 mg of the carbodiimide, EDC, in 50 mM imidazole buffer pH6.0. Following an overnight incubation, the magnetic particles were washed and used to isolate Plasmid DNA. An alkaline lysis method was used to prepare a cleared 5 ml bacterial lysate generating a supernatant containing the plasmid in 0.5M potassium acetate, pH5. To the supernatant, 2.5 mg of magnetic particles were added and mixed for 1 minute. After magnetic separation and washing with water pH5, the pure nucleic acids were eluted off in 200 μl of 10 mM Tris.HCl pH 8.5. [0136]
  • EXAMPLE 5 Bis-Tris Solid Phase Polystyrene Beads
  • 1 gram of carboxylated 60 μm polystyrene particles were reacted in a one step procedure with 500 mg of Bis-Tris and 500 mg of the carbodiimide, EDC, in 50 mM imidazole buffer pH6.0. Following an overnight incubation, the particles were washed and used to isolate plasmid nucleic acids as described above. [0137]
  • EXAMPLE 6 Bis-Tris Polymer
  • Bis-Tris monomer was converted into a polymer by mixing together 160 mg of polyacrylic acid with a molecular weight of 240,000, 1.6 g of Bis-Tris and 1.6 g of EDC in 50 mM imidazole pH6.0. Following an overnight incubation, the mixture was dialysed in water. The purified polymer was then coated onto magnetic COOH beads or used in the liquid phase to bind genomic DNA from blood. A 5 ml blood sample was centrifuged to obtain the nuclei and WBC population and the resulting pellet digested with 1% SDS. Following precipitation with potassium acetate the cleared supernatant was mixed with either 25 mg of magnetic-Bis-Tris or about 250 μg of poly-Bis-Tris as a liquid. In both cases the captured DNA could be separated, washed in water and then redissolved in 10 mM Tris HCl pH8.5 in a pure form. [0138]
  • EXAMPLE 7 Insoluble Tris HCl Polymer
  • In this example an insoluble polymer was made with inherent charge switching properties by mixing 80 mg of polyacrylic acid with 800 mg of Tris HCl and 800 mg of EDC in 50 mM Imidazole pH6. The insoluble precipitate that formed generated a particulate solid phase that was used to capture DNA and release it in a similar manner to that described in example 4 for genomic DNA. [0139]
  • EXAMPLE 8 Immobilised poly Bis-Tris on Tips
  • A solution of poly Bis-Tris at 1 mg/ml, prepared as in Example 2, in 0.1M sodium bicarbonate pH8 incubated at 60° C. for 8 hours with twenty 200 μl polyproplylene pipette tips. The tips were then rinsed and used to capture about 150 ng of plasmid DNA from a cleared bacterial lysate by pumping up and down ten times. After a quick wash with water pH5, the DNA was eluted in 50 μl of 10 mM Tris pH 8.5. [0140]
  • EXAMPLE 9 Immobilised Poly Bis-Tris on PCR Tubes
  • A solution of poly Bis-Tris at 1 mg/ml, prepared as in Example 2, in 0.1M sodium bicarbonate pH8 incubated at 60° C. for 8 hours in a 200 μl PCR plate of 8×12 tubes. After rinsing, the tubes were used to bind genomic DNA from a sample prepared according to example 4. About 50 ng of DNA was subsequently eluted off per tube using 10 mM Tris HCl pH8.5. [0141]
  • EXAMPLE 10 Charge Switch Detergents in Liquid Phase
  • A blood sample was prepared as described in Example 4 and to the resulting supernatant decyl imidazole was added at pH 4 causing precipitation of the DNA. The DNA pellet was collected by centrifugation and redissolved in 10 mM Tris pH 8.5. [0142]
  • EXAMPLE 11 Charge Switch Detergents on Solid Phase
  • Decyl imidazole was adsorbed onto a 200 ul plastic pipette tip by soaking in a 1% solution at pH4 in 0.1M sodium acetate. A blood sample was prepared as described in Example 3 and the tips were used to bind the DNA by repeated pumping and sucking. After a wash with water, about 50 ng of DNA was recovered in water at pH10. [0143]
  • EXAMPLE 12 Polyglucosamines
  • 10 mg of low molecular weight Chitosan was dissolved in acidified water and then 50 mM imidazole pH5.5, this was mixed with 100 mg of carboxy 1 μm magnetic beads and with 20 mg of the carbodiimide EDC in 50 mM imidazole pH5.5. Following an overnight incubation, the beads were washed and resuspended in 10 mM MES pH5. To bind genomic DNA, 2 mg of magnetic particles were added to a supernatant prepared by methods described earlier in Example 1, after magnetic separation, the DNA was eluted using 100 mM Tris.HCl pH 9.5. [0144]
  • EXAMPLE 13 Kanamycin
  • A solution of genomic DNA was prepared as described in example 3. To this sample 2 mg of Kanmycin was added at a concentration of 10 mg/ml. The resulting precipitate of DNA was filtered, washed in water at pH5 and re-dissolved in water at pH10. [0145]
  • EXAMPLE 14 Magnetisable Iron Oxides in Carboxylated Polystrene
  • A 5 ml Plasmid mini-prep was prepared using standard alkaline lysis reagents to generate a cleared lysate with a potassium acetate composition of 0.5M pH4. To this cleared supernatant, 2.5 mg of commercially available 1 μm carboxylated polystyrene magnetisable particles were added to bind the plasmid DNA The particles were washed with water at pH4 and then the DNA eluted using 10 mM Tris HCl at pH 8.5. Typical UV ratios at 260 and 280 nm were 1.7-2.0, indicating pure nucleic acids with a single band observed with standard gel electrophoresis. [0146]
  • EXAMPLE 15 Titanium Dioxide in Polystyrene Microtitre Plates
  • A solution of DNA at 100 μg per ml in 0.1M Potassium Acetate pH4 was allowed to stand for 1 hour in a 300 μl flat bottomed microtitre plastic plate, the plastic plate contained titanium oxide which was incorporated as a powder in the plastic when the plate was formed, After washing at pH4, the DNA was recovered with water at pH10 and 2 ml measured at 260 nm versus a plain polystyrene plate with no titanium oxide. Approximately, 50 ng of DNA was recovered per 300 μl well for the plate incorporating the titanium oxide compared to zero for the plain polystyrene plate. [0147]
  • EXAMPLE 16 Cytidine Coupled to Magnetic Beads
  • 1 gram of carboxylated 1 μm magnetic particles were reacted in a one step procedure with 500 mg of Cytidine and 500 mg of the carbodiimide, EDC, in 50 mM imidazole buffer pH6.0. After thorough washing, the beads were used to bind nucleic acids from a plasmid preparation as described in example 1 and recovering the pure nucleic acids in water at pH10. [0148]
  • EXAMPLE 17 Polyvinyl Pyridine (PVP)
  • 20 mg of commercially available PVP beads was mixed with the supernatant containing genomic DNA from a 5 ml blood extraction described in example 4. After allowing the DNA to bind, the beads were washed with water at pH5 and the DNA recovered using water at pH10. Ultra violet analyisis at 260 and 280 nm indicated a purity ratio of 1.65. [0149]
  • EXAMPLE 18 Separation of RNA and DNA
  • A solution of tRNA and sheared genomic DNA was prepared at 30 μg per ml in 50 mM Potassium acetate buffer pH6.5 with 1M sodium chloride. Approximately 4 mg of magnetic polyhistidine beads were mixed with 1 ml of the nucleic acid solution for one minute until binding was complete. The beads were then thoroughly washed with water at pH5. To elute the bound material, the beads were mixed with 300 μl of 10 mM Tris.HCl, 10 mM NaCl, pH8.5. Gel analysis showed that most of the tRNA remained in solution and was not bound to the beads. The eluted material contained mostly genomic DNA with little or no tRNA. [0150]
  • EXAMPLE 19 DNA Analysis
  • In all previous examples, extracted DNA was analysed by one or more of the following: [0151]
  • (1): ultra violet (UV) analysis at 260 nm and 280 nm, to provide a measure of nucleic acid concentration; [0152]
  • (2): Gel electrophoresis using 1% agarose in TBE buffer run at 60V for 20 minutes vs a commercial preparation of DNA as control, with ethidium bromide staining to measure molecular size and to provide an estimate of quantity of the nucleic acid; or [0153]
  • (3): PCR using primers specific for actin or other ubiquitous genes, to test integrity of the nucleic acid. [0154]
  • The results are presented as (1): direct readings from the instrument; and (2); and (3): gel pictures. [0155]
  • In all cases, the examples demonstrated effective extraction of nucleic acid which was not significantly damaged. [0156]

Claims (41)

1. A method for extracting nucleic acid from a sample containing nucleic acid, which method comprises:
at a first pH, bringing the sample into contact with a material which comprises an ionisable group, wherein the material has a positive charge at said first pH, such that nucleic acid is bound to the material; and
releasing the nucleic acid at a second, higher, pH at which the charge on the material is negative, neutral or less positive,
wherein the release of the nucleic acid occurs under mild conditions.
2. A method according to claim 1, wherein the mild conditions are conditions at which said nucleic acid is not denatured and/or not degraded and/or not depurinated and/or substantially physiological conditions.
3. A method according to claim 1, wherein the releasing step occurs at a pH of no more than about 10.5, preferably no more than about 9.0.
4. A method according to claim 1, wherein the releasing step occurs at an ionic strength of no more than about 500 mM, preferably no more than about 100 mM.
5. A method according to claim 1, wherein the releasing step occurs at a temperature of no more than about 70° C., preferably no more than about 50° C.
6. A method according to claim 5, wherein the releasing step occurs at about room temperature.
7. A method according to claim 1, wherein the releasing step comprises contacting the bound nucleic acid with a buffer solution to release the nucleic acid, the buffer solution being suitable for the storage or further processing of the released nucleic acid.
8. A method according to claim 7, wherein the buffer solution is a buffer solution suitable for PCR.
9. A method according to claim 1, wherein the pKa of said ionisable group is between about 3.0 and 9.0, preferably between about 4.0 and 9.0.
10. A method according to claim 9, wherein the material comprises a positively ionisable group, the pKa of which is between about 5.0 and 8.0, preferably between about 6.0 and 7.0.
11. A method according to claim 10, wherein the material comprises a weak base.
12. A method according to claim 10, wherein the material comprises a biological buffer,
13. A method according to claim 10, wherein the material comprises a positively ionisable nitrogen atom and one or more electronegative groups capable of lowering the pKa of the positively ionisable nitrogen atom.
14. A method according to claim 10, wherein the material comprises a chemical species selected from the group consisting of:
N-2-acetamido-2-aminoethanesulfonic acid (ACES);
N-2-acetamido-2-iminodiacetic acid (ADA);
amino methyl propanediol (AMP);
3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO);
N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES);
N,N-bis-2-hydroxyethylglycine (BICINE);
bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris);
1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris Propane);
4-cyclohexylamino-1-butane sulfonic acid (CABS);
3-cyclohexylamino-1-propane sulfonic acid (CAPS);
3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO);
2-N-cyclohexylaminoethanesulfonic acid (CHES);
3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO);
N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS);
N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);
2-N-morpholinoethanesulfonic acid (MES);
4-N-morpholinobutanesulfonic acid (MOBS);
3-N-morpholinopropanesulfonic acid (MOPS);
3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
piperazine-N-N-bis-2-ethanesulfonic acid (PIPES);
piperazine-N-N-bis-2-hydroxypropanesulfonic acid (POPSO);
N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS);
N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS);
3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO);
N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES);
N-trishydroxymethylmethylglycine (TRICINE);
trishydroxymethylaminomethane (Tris);
polyhydroxylated amines;
histidine, and polyhistidine;
imidazole, and derivatives thereof (i.e. imidazoles), especially derivatives containing hydroxyl groups;
triethanolamine dimers and polymers; and
di/tri/oligo amino acids, for example; Ala-Ala; Gly-Gly, pKa 8.2; Ser-Ser; Gly-Gly-Gly, Ser-Gly;
a detergent, such as decylmethylimidazole or dodecyl-Bis-Tris;
a carbohydrate containing nitrogen and electronegative groups, such as a glucosamine, a polyglucosamine (e.g. a chitosan), a kanamycin or derivative thereof;
a nucleic acid base, such as cytidine; and
a monomeric, oligomeric or polymeric compound containing an aliphatic or aromatic nitrogen-containing heterocyclic ring, such as morpholine-, pyrrole-, pyrrolidine-, pyridine-, pyridinol-, pyridone-, pyrroline-, pyrazole-, pyridazine-, pyrazine-, piperidone-, piperidine-, or piperazine-containing compounds, e.g. polyvinylpyridine, said ring optionally being substituted with one or more electronegative groups.
15. A method according to claim 14, wherein the chemical species is selected from the group consisting of
Tris;
Bis-Tris;
Bis-Tris Propane;
Tricine;
Bicine;
polyhydroxylated amines; and
polyhistidine.
16. A method according to claim 9, wherein the material comprises:
a negatively ionisable group, the pKa of which is between about 3.0 and 7.0;
and a group which is positively charged at said first pH, and optionally also at said second pH.
17. A method according to claim 16 wherein said negatively ionisable group is a carboxy group.
18. A method according to claim 16 wherein said group which is positively charged is a metal oxide, such as iron II,III oxide.
19. A method according to claim 1, wherein the material comprises an ionizable group having a pKa value, said pKa value being between the first and second pH, or within about 1.0 pH unit, preferably within about 0.5 pH unit, below said first pH.
20. A method according to claim 19, wherein said second pH is within about 3 pH units, preferably within about 2 pH units, above the pKa value.
21. A method according to claim 1, wherein the method is for separating single stranded nucleic acid from double stranded nucleic acid.
22. A method according to claim 1, wherein the method is for extracting single stranded nucleic acid, said method comprising a prior step of converting double stranded nucleic acid into single stranded nucleic acid.
23. A method according to claim 1, wherein the material is a solid phase material.
24. A method according to claim 1, wherein the binding step occurs in a solution having a concentration of 1M or less.
25. A solid phase product for use in a method of extracting nucleic acid from a sample, the product comprising a plurality of positively ionisable groups, the ionisable groups being provided by a chemical species selected from the list consisting of:
biological buffers;
polyhydroxylated amines;
histidine; and
polyhistidine.
26. A product according to claim 25 wherein the biological buffer is selected from the group consisting of;
N-2-acetamido-2-aminoethanesulfonic acid (ACES);
N-2-acetamido-2-iminodiacetic acid (ADA);
amino methyl propanediol (AMP);
3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO);
N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES);
N,N-bis2-hydroxyethylglycine (BICINE);
bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris);
1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris Propane);
4-cyclohexylamino-1-butane sulfonic acid (CABS);
3-cyclohexylamino-1-propane sulfonic acid (CAPS);
3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO);
2-N-cyclohexylaminoethanesulfonic acid (CHES);
3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO);
N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS);
N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);
2-N-morpholinoethanesulfonic acid (MES);
4-N-morpholinobutanesulfonic acid (MOBS);
3-N-morpholinopropanesulfonic acid (MOPS);
3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
piperazine-N-N-bis-2-ethanesulfonic acid (PIPES);
piperazine-N-N-bis-2-hydroxypropanesulfonic acid (POPSO);
N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS);
N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS);
3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO);
N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES);
N-trishydroxymethylmethylglycine (TRICINE);
trishydroxymethylaminomethane (Tris);
polyhistidine;
polyhydroxylated imidazoles;
triethanolamine dimers and polymers; and
di/tri/oligo amino acids, for example Gly-Gly, Ser-Ser, Gly-Gly-Gly, and Ser-Gly.
27. A product according to claim 25, wherein the plurality of ionisable groups are separately immobilised on a solid support by covalent or ionic bonding or by adsorption.
28. A product according to claim 25, wherein the plurality of ionisable groups are separately attached to a polymer, said polymer being immobilised on a solid support by covalent or ionic bonding or by adsorption.
29. A product according to claim 25, wherein the ionisable groups are polymerised, optionally by means of cross-linking reagents.
30. A product according to claim 29, wherein the polymer is immobilised on a solid support by covalent or ionic bonding or by adsorption.
31. A product according to claim 29, wherein the polymer is a solid.
32. A product according to claim 29 which is a container.
33. A container according to claim 32 which is a PCR or storage tube or well, or a pipette tip.
34. A water soluble product for use in a method of extracting nucleic acid from a sample, the product comprising a plurality of positively ionisable groups, the ionisable groups being provided by a chemical species selected from the list consisting of:
biological buffers;
polyhydroxylated amines;
histidine; and
polyhistidine.
35. A product according to claim 34 wherein the biological buffer is selected from the group consisting of:
N-2-acetamido-2-aminoethanesulfonic acid (ACES);
N-2-acetamido-2-iminodiacetic acid (ADA);
amino methyl propanediol (AMP);
3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO);
N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES);
N,N-bis-2-hydroxyethylglycine (BICINE);
bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris);
1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris Propane);
4-cyclohexylamino-1-butane sulfonic acid (CABS);
3-cyclohexylamino-1-propane sulfonic acid (CAPS);
3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO);
2-N-cyclohexylaminoethanesulfonic acid (CHES);
3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO);
N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS);
N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);
2-N-morpholinoethanesulfonic acid (MES);
4-N-morpholinobutanesulfonic acid (MOBS);
3-N-morpholinopropanesulfonic acid (MOPS);
3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
piperazine-N-N-bis-2-ethanesulfonic acid (PIPES);
piperazine-N-N-bis-2-hydroxypropanesulfonic acid (POPSO);
N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS);
N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS);
3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO);
N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES);
N-trishydroxymethylmethylglycine (TRICINE);
trishydroxymethylaminomethane (Tris);
polyhistidine;
polyhydroxylated imidazoles;
triethanolamine dimers and polymers; and
di/tri/oligo amino acids, for example Gly-Gly, Ser-Ser, Gly-Gly-Gly, and Ser-Gly.
36. A product according to claim 34, wherein the plurality of ionisable groups are separately attached to a polymer.
37. A product according to claim 34, wherein the ionisable groups are polymerised, optionally by means of cross-linking reagents.
38. A product for use in a method of extracting nucleic acid from a sample, wherein the product possesses a positive charge at both a first pH at which it is desired to bind nucleic acid and a second higher pH at which it is desired to release nucleic acid, the product comprising a plurality of negatively ionisable groups, the combined charge of which becomes more negative between said first pH and said second pH, such that the product is capable of binding nucleic acid at said first pH, which bound nucleic acid is released from the product at said second pH.
39. A product according to claim 38, wherein the negatively ionisable group has a pKa between about 3 and 7, preferably between about 4 and 7.
40. A product according to claim 38, wherein the negatively ionisable is a carboxy group.
41. A product according to claim 38 wherein said positive charge is provided by a metal or metal oxide, preferably iron II,III oxide.
US10/232,135 1997-12-06 2002-08-29 Isolation of nucleic acids Abandoned US20030008320A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US10/232,135 US20030008320A1 (en) 1997-12-06 2002-08-29 Isolation of nucleic acids
US11/761,956 US20070231892A1 (en) 1997-12-06 2007-06-12 Isolation of nucleic acids
US12/137,125 US20080305528A1 (en) 1997-12-06 2008-06-11 Isolation of nucleic acids
US13/361,815 US20120196944A1 (en) 1997-12-06 2012-01-30 Isolation of nucleic acids
US13/447,130 US20120197009A1 (en) 1997-12-06 2012-04-13 Isolation of nucleic acids
US13/898,400 US20130338245A1 (en) 1997-12-06 2013-05-20 Isolation of nucleic acids

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
GB9725839.6 1997-12-06
GBGB9725839.6A GB9725839D0 (en) 1997-12-06 1997-12-06 Isolation of nucleic acids
GB9815541.9 1998-07-17
GBGB9815541.9A GB9815541D0 (en) 1998-07-17 1998-07-17 Isolation of nucleic acids
PCT/GB1998/003602 WO1999029703A2 (en) 1997-12-06 1998-12-04 Isolation of nucleic acids
US58600900A 2000-06-02 2000-06-02
US09/736,632 US6914137B2 (en) 1997-12-06 2000-12-14 Isolation of nucleic acids
US10/232,135 US20030008320A1 (en) 1997-12-06 2002-08-29 Isolation of nucleic acids

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US09/736,632 Division US6914137B2 (en) 1997-12-06 2000-12-14 Isolation of nucleic acids

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/761,956 Continuation US20070231892A1 (en) 1997-12-06 2007-06-12 Isolation of nucleic acids

Publications (1)

Publication Number Publication Date
US20030008320A1 true US20030008320A1 (en) 2003-01-09

Family

ID=24960633

Family Applications (8)

Application Number Title Priority Date Filing Date
US09/736,632 Expired - Lifetime US6914137B2 (en) 1997-12-06 2000-12-14 Isolation of nucleic acids
US10/232,135 Abandoned US20030008320A1 (en) 1997-12-06 2002-08-29 Isolation of nucleic acids
US10/232,971 Abandoned US20030054395A1 (en) 1997-12-06 2002-08-30 Isolation of nucleic acids
US11/761,956 Abandoned US20070231892A1 (en) 1997-12-06 2007-06-12 Isolation of nucleic acids
US12/137,125 Abandoned US20080305528A1 (en) 1997-12-06 2008-06-11 Isolation of nucleic acids
US13/361,815 Abandoned US20120196944A1 (en) 1997-12-06 2012-01-30 Isolation of nucleic acids
US13/447,130 Abandoned US20120197009A1 (en) 1997-12-06 2012-04-13 Isolation of nucleic acids
US13/898,400 Abandoned US20130338245A1 (en) 1997-12-06 2013-05-20 Isolation of nucleic acids

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US09/736,632 Expired - Lifetime US6914137B2 (en) 1997-12-06 2000-12-14 Isolation of nucleic acids

Family Applications After (6)

Application Number Title Priority Date Filing Date
US10/232,971 Abandoned US20030054395A1 (en) 1997-12-06 2002-08-30 Isolation of nucleic acids
US11/761,956 Abandoned US20070231892A1 (en) 1997-12-06 2007-06-12 Isolation of nucleic acids
US12/137,125 Abandoned US20080305528A1 (en) 1997-12-06 2008-06-11 Isolation of nucleic acids
US13/361,815 Abandoned US20120196944A1 (en) 1997-12-06 2012-01-30 Isolation of nucleic acids
US13/447,130 Abandoned US20120197009A1 (en) 1997-12-06 2012-04-13 Isolation of nucleic acids
US13/898,400 Abandoned US20130338245A1 (en) 1997-12-06 2013-05-20 Isolation of nucleic acids

Country Status (10)

Country Link
US (8) US6914137B2 (en)
EP (2) EP1473299A3 (en)
JP (1) JP4868697B2 (en)
AT (1) ATE283275T1 (en)
AU (1) AU2002216203A1 (en)
CA (1) CA2432075A1 (en)
DE (1) DE60107468T2 (en)
DK (1) DK1345952T3 (en)
ES (1) ES2233560T3 (en)
WO (1) WO2002048164A2 (en)

Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040197780A1 (en) * 2003-04-02 2004-10-07 Agencourt Bioscience Corporation Method for isolating nucleic acids
US20060024701A1 (en) * 2001-01-09 2006-02-02 Whitehead Institute For Biomedical Research Methods and reagents for the isolation of nucleic acids
US20060030056A1 (en) * 2004-08-03 2006-02-09 Becton, Dickinson And Company Use of magnetic material to fractionate samples
US20060084089A1 (en) * 2004-08-03 2006-04-20 Becton, Dickinson And Company Use of magnetic material to direct isolation of compounds and fractionation of multipart samples
US20060127887A1 (en) * 2004-12-10 2006-06-15 Lee Young-Sun Isolation and purification method of biomolecules using hydrogel
US20060177836A1 (en) * 2004-07-30 2006-08-10 Mckernan Kevin J Methods of isolating nucleic acids using multifunctional group-coated solid phase carriers
US20060199199A1 (en) * 2005-01-20 2006-09-07 Kim Kui-Hyun Method of removing nucleic acid amplification inhibitor from biological sample and PCR system
US20070031880A1 (en) * 2003-02-06 2007-02-08 Becton, Dickinson And Company Chemical treatment of biological samples for nucleic acid extraction and kits therefor
KR100785011B1 (en) 2006-04-07 2007-12-11 삼성전자주식회사 Method for increasing the specificity of nucleic acid hybridization using zwitterionic compounds
US20080023395A1 (en) * 2006-07-31 2008-01-31 Sigma Aldrich Co. Compositions and Methods for Isolation of Biological Molecules
US20080026375A1 (en) * 2006-07-31 2008-01-31 Sigma Aldrich Co. Compositions and Methods for Isolation of Biological Molecules
US20080026374A1 (en) * 2006-07-31 2008-01-31 Sigma Aldrich Co. Compositions and Methods for Isolation of Biological Molecules
EP1911844A1 (en) * 2006-10-10 2008-04-16 Qiagen GmbH Methods and kit for isolating nucleic acids
US20090061497A1 (en) * 2007-06-29 2009-03-05 Becton, Dickinson And Company Methods for Extraction and Purification of Components of Biological Samples
US20090130736A1 (en) * 2003-02-06 2009-05-21 Becton, Dickinson And Company Pretreatment method for extraction of nucleic acid from biological samples and kits therefor
US20100112576A1 (en) * 2008-10-03 2010-05-06 U.S. Genomics, Inc. Focusing chamber
US20100120101A1 (en) * 2007-01-08 2010-05-13 U.S. Genomics, Inc. Reaction chamber
US20100294665A1 (en) * 2007-07-12 2010-11-25 Richard Allen Method and system for transferring and/or concentrating a sample
WO2013062476A1 (en) * 2011-10-27 2013-05-02 Ge Healthcare Bio-Sciences Ab Purification of nucleic acid
WO2013159117A1 (en) 2012-04-20 2013-10-24 SlipChip, LLC Fluidic devices and systems for sample preparation or autonomous analysis
US8685708B2 (en) 2012-04-18 2014-04-01 Pathogenetix, Inc. Device for preparing a sample
US9006419B2 (en) 2009-10-22 2015-04-14 Industrial Technology Research Institute Method for isolating nucleic acids
US9803237B2 (en) 2012-04-24 2017-10-31 California Institute Of Technology Slip-induced compartmentalization
US9808798B2 (en) 2012-04-20 2017-11-07 California Institute Of Technology Fluidic devices for biospecimen preservation
US10875022B2 (en) 2007-07-13 2020-12-29 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
US10900066B2 (en) 2006-03-24 2021-01-26 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
US10913061B2 (en) 2006-03-24 2021-02-09 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using the same
US11060082B2 (en) 2007-07-13 2021-07-13 Handy Lab, Inc. Polynucleotide capture materials, and systems using same
US11078523B2 (en) 2003-07-31 2021-08-03 Handylab, Inc. Processing particle-containing samples
US11141734B2 (en) 2006-03-24 2021-10-12 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system
US11142785B2 (en) 2006-03-24 2021-10-12 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
US11266987B2 (en) 2007-07-13 2022-03-08 Handylab, Inc. Microfluidic cartridge
US11441171B2 (en) 2004-05-03 2022-09-13 Handylab, Inc. Method for processing polynucleotide-containing samples
US11453906B2 (en) 2011-11-04 2022-09-27 Handylab, Inc. Multiplexed diagnostic detection apparatus and methods
US11466263B2 (en) 2007-07-13 2022-10-11 Handylab, Inc. Diagnostic apparatus to extract nucleic acids including a magnetic assembly and a heater assembly
US11549959B2 (en) 2007-07-13 2023-01-10 Handylab, Inc. Automated pipetting apparatus having a combined liquid pump and pipette head system
US11788127B2 (en) 2011-04-15 2023-10-17 Becton, Dickinson And Company Scanning real-time microfluidic thermocycler and methods for synchronized thermocycling and scanning optical detection
US11806718B2 (en) 2006-03-24 2023-11-07 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system

Families Citing this family (214)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9425138D0 (en) 1994-12-12 1995-02-08 Dynal As Isolation of nucleic acid
US6048734A (en) 1995-09-15 2000-04-11 The Regents Of The University Of Michigan Thermal microvalves in a fluid flow method
US20060003447A1 (en) * 2003-12-30 2006-01-05 Richard Fike Dry powder cells and cell culture reagents and methods of production thereof
US7078224B1 (en) * 1999-05-14 2006-07-18 Promega Corporation Cell concentration and lysate clearance using paramagnetic particles
EP1147226B1 (en) * 1999-01-27 2013-01-23 Folim G. Halaka Materials and methods for the purification of polyelectrolytes
DE10006590B4 (en) * 2000-02-11 2007-10-18 Qiagen North American Holdings, Inc. Use of functionalized membranes or matrices for the purification of nucleic acids and corresponding methods
AU2002232406C1 (en) * 2000-11-06 2009-03-05 Invitrogen Corporation Dry powder cells and cell culture reagents and methods of production thereof
US6800453B2 (en) * 2001-01-23 2004-10-05 President And Fellows Of Harvard College Nucleic-acid programmable protein arrays
US6692700B2 (en) 2001-02-14 2004-02-17 Handylab, Inc. Heat-reduction methods and systems related to microfluidic devices
US7666588B2 (en) 2001-03-02 2010-02-23 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US7226739B2 (en) 2001-03-02 2007-06-05 Isis Pharmaceuticals, Inc Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations
US20030027135A1 (en) 2001-03-02 2003-02-06 Ecker David J. Method for rapid detection and identification of bioagents
WO2004060278A2 (en) 2002-12-06 2004-07-22 Isis Pharmaceuticals, Inc. Methods for rapid identification of pathogens in humans and animals
US7718354B2 (en) 2001-03-02 2010-05-18 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US20040121313A1 (en) 2002-12-06 2004-06-24 Ecker David J. Methods for rapid detection and identification of bioagents in organs for transplantation
US7323140B2 (en) 2001-03-28 2008-01-29 Handylab, Inc. Moving microdroplets in a microfluidic device
US7829025B2 (en) 2001-03-28 2010-11-09 Venture Lending & Leasing Iv, Inc. Systems and methods for thermal actuation of microfluidic devices
US7010391B2 (en) 2001-03-28 2006-03-07 Handylab, Inc. Methods and systems for control of microfluidic devices
US8895311B1 (en) 2001-03-28 2014-11-25 Handylab, Inc. Methods and systems for control of general purpose microfluidic devices
US6852287B2 (en) 2001-09-12 2005-02-08 Handylab, Inc. Microfluidic devices having a reduced number of input and output connections
US7217510B2 (en) 2001-06-26 2007-05-15 Isis Pharmaceuticals, Inc. Methods for providing bacterial bioagent characterizing information
US8073627B2 (en) 2001-06-26 2011-12-06 Ibis Biosciences, Inc. System for indentification of pathogens
AU2003202026A1 (en) 2002-01-16 2003-09-02 Dynal Biotech Asa Method for isolating nucleic acids and protein from a single sample
GB0212826D0 (en) * 2002-05-31 2002-07-10 Dna Res Innovations Ltd Materials and methods relating to polyions and substance delivery
US7597936B2 (en) * 2002-11-26 2009-10-06 University Of Utah Research Foundation Method of producing a pigmented composite microporous material
EP1576351A4 (en) * 2002-11-26 2010-06-23 Univ Utah Res Found Microporous materials, methods, and articles for localizing and quantifying analytes
GB0229287D0 (en) * 2002-12-16 2003-01-22 Dna Res Innovations Ltd Polyfunctional reagents
US8046171B2 (en) 2003-04-18 2011-10-25 Ibis Biosciences, Inc. Methods and apparatus for genetic evaluation
US8057993B2 (en) 2003-04-26 2011-11-15 Ibis Biosciences, Inc. Methods for identification of coronaviruses
US8158354B2 (en) 2003-05-13 2012-04-17 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US7964343B2 (en) * 2003-05-13 2011-06-21 Ibis Biosciences, Inc. Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
GB0317044D0 (en) * 2003-07-21 2003-08-27 Dna Res Innovations Ltd Nucleic acid isolation
WO2005024042A2 (en) * 2003-09-04 2005-03-17 The Regents Of The University Of California Aptamers and methods for their in vitro selection and uses thereof
US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US20120122103A1 (en) 2003-09-11 2012-05-17 Rangarajan Sampath Compositions for use in identification of bacteria
GB0323814D0 (en) * 2003-10-10 2003-11-12 Cobra Biolog Ltd Purification of biological macromolecules
US20050106576A1 (en) * 2003-11-17 2005-05-19 Hashem Akhavan-Tafti Methods of using cleavable solid phases for isolating nucleic acids
US20050106577A1 (en) * 2003-11-17 2005-05-19 Hashem Akhavan-Tafti Cleavable solid phases for isolating nucleic acids
US8163895B2 (en) 2003-12-05 2012-04-24 Ibis Biosciences, Inc. Compositions for use in identification of orthopoxviruses
US7666592B2 (en) 2004-02-18 2010-02-23 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
AU2005200670B2 (en) 2004-02-20 2007-05-03 F. Hoffmann-La Roche Ag Adsorption of nucleic acids to a solid phase
US8119336B2 (en) 2004-03-03 2012-02-21 Ibis Biosciences, Inc. Compositions for use in identification of alphaviruses
AU2005241003B2 (en) * 2004-04-14 2011-05-19 President And Fellows Of Harvard College Nucleic-acid programmable protein arrays
WO2005108620A2 (en) * 2004-05-03 2005-11-17 Handylab, Inc. Processing polynucleotide-containing samples
US20060010513A1 (en) * 2004-05-11 2006-01-12 Melville Mark W Oligonucleotide arrays to monitor gene expression and methods for making and using same
EP2458619B1 (en) 2004-05-24 2017-08-02 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US20050266411A1 (en) 2004-05-25 2005-12-01 Hofstadler Steven A Methods for rapid forensic analysis of mitochondrial DNA
US7811753B2 (en) 2004-07-14 2010-10-12 Ibis Biosciences, Inc. Methods for repairing degraded DNA
JP4990771B2 (en) 2004-07-28 2012-08-01 キヤノン ユー.エス. ライフ サイエンシズ, インコーポレイテッド Method for monitoring microbial genomic DNA
US7541060B2 (en) * 2004-08-17 2009-06-02 Xerox Corporation Bichromal balls
KR100613734B1 (en) * 2004-09-07 2006-08-22 굿젠 주식회사 Method for storing DNA by using chitosan, Method for analyzing the DNA stored and products using the methods
EP1650297B1 (en) * 2004-10-19 2011-04-13 Samsung Electronics Co., Ltd. Method and apparatus for the rapid disruption of cells or viruses using micro magnetic beads and laser
KR100647306B1 (en) 2004-12-23 2006-11-23 삼성전자주식회사 1 Method for isolating a nucleic acid using a material positively charged at the first pH and comprising an amino group and a carboxyl group
US7964380B2 (en) 2005-01-21 2011-06-21 Argylia Technologies Nanoparticles for manipulation of biopolymers and methods of thereof
EP1856282A4 (en) * 2005-02-15 2009-05-13 Univ Virginia Nucleic acid isolation methods and materials and devices thereof
AU2006214141B2 (en) * 2005-02-18 2010-07-15 Gen-Probe Incorporated Sample preparation method incorporating an alkaline shock
EP1871903B1 (en) * 2005-02-18 2011-12-21 Canon U.S. Life Sciences, Inc. Devices and methods for identifying genomic dna of organisms
US8084207B2 (en) 2005-03-03 2011-12-27 Ibis Bioscience, Inc. Compositions for use in identification of papillomavirus
CA2600184A1 (en) 2005-03-03 2006-09-08 Isis Pharmaceuticals, Inc. Compositions for use in identification of adventitious viruses
DE102005015005A1 (en) 2005-04-01 2006-10-05 Qiagen Gmbh Process for treating a sample containing biomolecules
KR100657957B1 (en) * 2005-04-12 2006-12-14 삼성전자주식회사 1 Method for isolating a nucleic acid using a material positively charged at the first pH and comprising an amino group and a carboxyl group and a solid material for a nucleic acid purification capable of being used for the method
US20070105181A1 (en) * 2005-05-04 2007-05-10 Invitrogen Corporation Identification of cancer biomarkers and phosphorylated pdroteins
KR100668338B1 (en) 2005-05-21 2007-01-12 삼성전자주식회사 Novel pH dependent ion exchange material a solid substrate having immobilized the material on the surface and a method for isolating a nucleic acid using the material and the solid substrate
KR100668337B1 (en) 2005-05-21 2007-01-12 삼성전자주식회사 PH dependent ion exchange material capable of selectively binding to nucleic acids in comparison with proteins a solid substrate having immobilized the material on the surface and a method for isolating a nucleic acid using the material and the solid substrate
EP1910389A4 (en) * 2005-05-31 2010-03-10 Life Technologies Corp Separation and purification of nucleic acid from paraffin-containing samples
JP2009500019A (en) * 2005-07-01 2009-01-08 プロメガ・コーポレーション Suspended particle network for the purification of biomolecules, and the use of buoyant particles or buoyant particle networks for the purification of biomolecules
US8026084B2 (en) 2005-07-21 2011-09-27 Ibis Biosciences, Inc. Methods for rapid identification and quantitation of nucleic acid variants
CN101300352B (en) 2005-09-01 2013-03-20 佳能美国生命科学公司 Method and molecular diagnostic device for detection, analysis and identification of genomic DNA
DE102005047736B4 (en) * 2005-09-29 2008-08-14 Aj Innuscreen Gmbh Method and system for isolating nucleic acids from any complex starting materials
WO2007047336A2 (en) * 2005-10-12 2007-04-26 University Of Virginia Patent Foundation Integrated microfluidic analysis systems
US8030034B2 (en) * 2005-12-09 2011-10-04 Promega Corporation Nucleic acid purification with a binding matrix
KR100829574B1 (en) * 2006-01-03 2008-05-14 삼성전자주식회사 Microarray substrate method of analyzing biomolecule using the microarray substrate and lab-on-a-chip including the microarray substrate
US20070190526A1 (en) * 2006-02-16 2007-08-16 Nexgen Diagnostics Llc Methods of extracting nucleic acids
WO2007097468A1 (en) * 2006-02-23 2007-08-30 Shino-Test Corporation Method of determining metal by colorimetry and determination reagent
US8088616B2 (en) 2006-03-24 2012-01-03 Handylab, Inc. Heater unit for microfluidic diagnostic system
WO2008054514A2 (en) 2006-04-21 2008-05-08 Wyeth Differential expression profiling analysis of cell culture phenotypes and the uses thereof
US8178316B2 (en) * 2006-06-29 2012-05-15 President And Fellows Of Harvard College Evaluating proteins
EP1882739A1 (en) * 2006-06-30 2008-01-30 Qiagen GmbH Nucleic acid extraction method
JP2009545317A (en) * 2006-08-01 2009-12-24 アプライド バイオシステムズ, エルエルシー Analyte and nucleic acid detection
JP5233176B2 (en) * 2006-08-04 2013-07-10 ソニー株式会社 Fuel cells and electronics
US9149473B2 (en) 2006-09-14 2015-10-06 Ibis Biosciences, Inc. Targeted whole genome amplification method for identification of pathogens
WO2008035991A2 (en) * 2006-09-19 2008-03-27 Michael Ronald Cook A nucleic acid extraction method
KR100862660B1 (en) 2006-09-25 2008-10-10 삼성전자주식회사 Method and apparatus for isolating and purifying nucleic acid by single bead
WO2008061165A2 (en) 2006-11-14 2008-05-22 Handylab, Inc. Microfluidic cartridge and method of making same
US7492312B2 (en) * 2006-11-14 2009-02-17 Fam Adly T Multiplicative mismatched filters for optimum range sidelobe suppression in barker code reception
US20080131954A1 (en) * 2006-11-30 2008-06-05 Canon U.S. Life Sciences, Inc. Method of Separating Target DNA from Mixed DNA
US20080131955A1 (en) * 2006-11-30 2008-06-05 Canon U.S. Life Sciences, Inc. Method of Separating Target DNA from Mixed DNA
JP2011503244A (en) * 2006-12-21 2011-01-27 インヴィトロジェン ダイナル エーエス Particles and their use in nucleic acid isolation methods or phosphorylated protein isolation methods
US8569464B2 (en) 2006-12-21 2013-10-29 Emd Millipore Corporation Purification of proteins
US8163886B2 (en) * 2006-12-21 2012-04-24 Emd Millipore Corporation Purification of proteins
WO2008104002A2 (en) 2007-02-23 2008-08-28 Ibis Biosciences, Inc. Methods for rapid forensic dna analysis
DE102007009347B4 (en) 2007-02-27 2009-11-26 Agowa Gmbh Method for isolating nucleic acids
CA2629589C (en) 2007-04-20 2016-03-29 F.Hoffmann-La Roche Ag Isolation and purification of nucleic acid molecules with a solid phase
WO2008151023A2 (en) 2007-06-01 2008-12-11 Ibis Biosciences, Inc. Methods and compositions for multiple displacement amplification of nucleic acids
JP5205818B2 (en) * 2007-06-05 2013-06-05 ソニー株式会社 Fuel cells and electronics
EP2157652A4 (en) 2007-06-13 2012-01-04 Sony Corp Fuel cell and electronic equipment
WO2008157299A2 (en) * 2007-06-15 2008-12-24 Wyeth Differential expression profiling analysis of cell culture phenotypes and uses thereof
US8133671B2 (en) 2007-07-13 2012-03-13 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
US20090136385A1 (en) 2007-07-13 2009-05-28 Handylab, Inc. Reagent Tube
US9618139B2 (en) 2007-07-13 2017-04-11 Handylab, Inc. Integrated heater and magnetic separator
USD621060S1 (en) 2008-07-14 2010-08-03 Handylab, Inc. Microfluidic cartridge
US20090048439A1 (en) * 2007-08-06 2009-02-19 Weisburg William G Isolation of nucleic acids molecules using modified solid supports
JP5298479B2 (en) * 2007-08-17 2013-09-25 ソニー株式会社 Fuel cells and electronics
EP2191012A1 (en) * 2007-09-21 2010-06-02 Streck, Inc. Nucleic acid isolation in preserved whole blood
US20090137024A1 (en) * 2007-11-28 2009-05-28 Canon U.S. Life Sciences, Inc. Method of Separating Target DNA from Mixed DNA
US20090186358A1 (en) * 2007-12-21 2009-07-23 Wyeth Pathway Analysis of Cell Culture Phenotypes and Uses Thereof
JP2009245920A (en) * 2008-03-11 2009-10-22 Sony Corp Fuel cell, electronic apparatus, and buffering liquid for fuel cell
JP2009245930A (en) * 2008-03-12 2009-10-22 Sony Corp Fuel cell and method of manufacturing the same, enzyme fixed electrode and method of manufacturing the same, and electronic device
USD618820S1 (en) 2008-07-11 2010-06-29 Handylab, Inc. Reagent holder
USD787087S1 (en) 2008-07-14 2017-05-16 Handylab, Inc. Housing
US8304185B2 (en) 2009-07-17 2012-11-06 Canon U.S. Life Sciences, Inc. Methods and systems for DNA isolation on a microfluidic device
WO2010009415A1 (en) 2008-07-18 2010-01-21 Canon U.S. Life Sciences, Inc. Methods and systems for microfluidic dna sample preparation
US10718909B2 (en) 2008-07-29 2020-07-21 Glenair, Inc. Expanded beam fiber optic connection system
GB0814570D0 (en) * 2008-08-08 2008-09-17 Diagnostics For The Real World Isolation of nucleic acid
EP2157181A1 (en) * 2008-08-13 2010-02-24 AGOWA Gesellschaft für molekularbiologische Technologie mbH Method for isolating nucleic acids and test kit
EP2315839A4 (en) * 2008-08-25 2012-03-21 Ge Healthcare Bio Sciences Simple load and elute process for purification of genomic dna
EP2331954B1 (en) 2008-08-27 2020-03-25 Life Technologies Corporation Apparatus for and method of processing biological samples
US11235323B2 (en) 2008-08-27 2022-02-01 Life Technologies Corporation Apparatus for and method of processing biological samples
KR101015502B1 (en) * 2008-09-09 2011-02-22 삼성전자주식회사 Material positively charged at first pH and negatively charged at second pH and method for isolating nucleic acid using the same
EP2349549B1 (en) 2008-09-16 2012-07-18 Ibis Biosciences, Inc. Mixing cartridges, mixing stations, and related kits, and system
US8148163B2 (en) 2008-09-16 2012-04-03 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US8534447B2 (en) 2008-09-16 2013-09-17 Ibis Biosciences, Inc. Microplate handling systems and related computer program products and methods
DE102008047790A1 (en) 2008-09-17 2010-04-15 Qiagen Gmbh Method for normalizing the content of biomolecules in a sample
KR101569832B1 (en) * 2008-11-19 2015-11-18 삼성전자주식회사 Method for separating genomic DNA and plasmid DNA from each other and kit for same
EP2370561B1 (en) 2008-12-16 2019-08-07 EMD Millipore Corporation Stirred tank reactor and method
DE102008063003A1 (en) * 2008-12-23 2010-06-24 Qiagen Gmbh Nucleic acid purification method
DE102008063001A1 (en) * 2008-12-23 2010-06-24 Qiagen Gmbh Nucleic acid purification method
EP2394152B1 (en) 2009-02-03 2019-05-01 ANDE Corporation Nucleic acid purification
WO2010093943A1 (en) 2009-02-12 2010-08-19 Ibis Biosciences, Inc. Ionization probe assemblies
GB2473868A (en) 2009-09-28 2011-03-30 Invitrogen Dynal As Apparatus and method of automated processing of biological samples
EP2256195A1 (en) * 2009-05-12 2010-12-01 Qiagen GmbH Nucleic acid purification method
US8950604B2 (en) 2009-07-17 2015-02-10 Ibis Biosciences, Inc. Lift and mount apparatus
EP2454000A4 (en) 2009-07-17 2016-08-10 Ibis Biosciences Inc Systems for bioagent identification
US8039613B2 (en) 2009-08-28 2011-10-18 Promega Corporation Methods of purifying a nucleic acid and formulation and kit for use in performing such methods
US8222397B2 (en) * 2009-08-28 2012-07-17 Promega Corporation Methods of optimal purification of nucleic acids and kit for use in performing such methods
AU2010298620B2 (en) 2009-09-24 2016-08-11 Qiagen Gaithersburg Inc. Compositions, methods, and kits for isolating and analyzing nucleic acids using an anion exchange material
DK2851399T3 (en) * 2009-09-29 2017-11-27 Covalon Tech Inc Medical device coating system and method
ES2628739T3 (en) 2009-10-15 2017-08-03 Ibis Biosciences, Inc. Multiple displacement amplification
EP2513335A4 (en) * 2009-12-14 2013-09-11 Betty Wu Method and materials for separating nucleic acid materials
EP2539450B1 (en) 2010-02-25 2016-02-17 Advanced Liquid Logic, Inc. Method of making nucleic acid libraries
SG10201804385YA (en) 2010-05-17 2018-06-28 Emd Millipore Corp Stimulus responsive polymers for the purification of biomolecules
EP2576779B1 (en) 2010-06-01 2017-08-09 Qiagen GmbH Method for isolating and/or purifying nucleic acid(s)
CN101956000A (en) * 2010-07-19 2011-01-26 博奥生物有限公司 Biomolecular controlled-release method and biomolecular controlled-release biochip
US20120034603A1 (en) 2010-08-06 2012-02-09 Tandem Diagnostics, Inc. Ligation-based detection of genetic variants
US20140342940A1 (en) 2011-01-25 2014-11-20 Ariosa Diagnostics, Inc. Detection of Target Nucleic Acids using Hybridization
US11203786B2 (en) 2010-08-06 2021-12-21 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US20130040375A1 (en) 2011-08-08 2013-02-14 Tandem Diagnotics, Inc. Assay systems for genetic analysis
US8700338B2 (en) 2011-01-25 2014-04-15 Ariosa Diagnosis, Inc. Risk calculation for evaluation of fetal aneuploidy
US11031095B2 (en) 2010-08-06 2021-06-08 Ariosa Diagnostics, Inc. Assay systems for determination of fetal copy number variation
US10167508B2 (en) 2010-08-06 2019-01-01 Ariosa Diagnostics, Inc. Detection of genetic abnormalities
US10533223B2 (en) 2010-08-06 2020-01-14 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US20130261003A1 (en) 2010-08-06 2013-10-03 Ariosa Diagnostics, In. Ligation-based detection of genetic variants
US9994897B2 (en) 2013-03-08 2018-06-12 Ariosa Diagnostics, Inc. Non-invasive fetal sex determination
US10131947B2 (en) 2011-01-25 2018-11-20 Ariosa Diagnostics, Inc. Noninvasive detection of fetal aneuploidy in egg donor pregnancies
US8756020B2 (en) 2011-01-25 2014-06-17 Ariosa Diagnostics, Inc. Enhanced risk probabilities using biomolecule estimations
US11270781B2 (en) 2011-01-25 2022-03-08 Ariosa Diagnostics, Inc. Statistical analysis for non-invasive sex chromosome aneuploidy determination
WO2013004710A2 (en) * 2011-07-04 2013-01-10 Qiagen Gmbh Reagent usable for the isolation and/or purification of nucleic acids
JP5924888B2 (en) * 2011-08-26 2016-05-25 関東化學株式会社 Nucleic acid extraction method, nucleic acid extraction reagent kit, and nucleic acid extraction reagent
US8712697B2 (en) 2011-09-07 2014-04-29 Ariosa Diagnostics, Inc. Determination of copy number variations using binomial probability calculations
JP5899731B2 (en) 2011-09-13 2016-04-06 ソニー株式会社 Nucleic acid purification method, nucleic acid extraction method, and nucleic acid purification kit
EP4159857A1 (en) 2011-09-26 2023-04-05 QIAGEN GmbH Rapid method for isolating extracellular nucleic acids
EP2760999A1 (en) 2011-09-26 2014-08-06 Qiagen GmbH Methods for separating nucleic acids by size
USD692162S1 (en) 2011-09-30 2013-10-22 Becton, Dickinson And Company Single piece reagent holder
KR102121853B1 (en) 2011-09-30 2020-06-12 벡톤 디킨슨 앤드 컴퍼니 Unitized reagent strip
CA2863637C (en) 2012-02-03 2021-10-26 Becton, Dickinson And Company External files for distribution of molecular diagnostic tests and determination of compatibility between tests
US11648561B2 (en) 2012-02-13 2023-05-16 Neumodx Molecular, Inc. System and method for processing and detecting nucleic acids
US9604213B2 (en) 2012-02-13 2017-03-28 Neumodx Molecular, Inc. System and method for processing and detecting nucleic acids
US9637775B2 (en) 2012-02-13 2017-05-02 Neumodx Molecular, Inc. System and method for processing biological samples
CN114134029A (en) 2012-02-13 2022-03-04 纽莫德克斯莫勒库拉尔公司 Microfluidic cartridge for processing and detecting nucleic acids
US10289800B2 (en) 2012-05-21 2019-05-14 Ariosa Diagnostics, Inc. Processes for calculating phased fetal genomic sequences
EP2875156A4 (en) 2012-07-19 2016-02-24 Ariosa Diagnostics Inc Multiplexed sequential ligation-based detection of genetic variants
US20140322706A1 (en) 2012-10-24 2014-10-30 Jon Faiz Kayyem Integrated multipelx target analysis
JP1628115S (en) 2012-10-24 2019-04-01
CN103789297A (en) * 2012-10-26 2014-05-14 上海医脉赛科技有限公司 Nucleic acid rapid-purifying method and kit
GB201304797D0 (en) 2013-03-15 2013-05-01 Diagnostics For The Real World Ltd Apparatus and method for automated sample preparation and adaptor for use in the apparatus
EP3520895A1 (en) 2013-03-15 2019-08-07 Genmark Diagnostics Inc. Fluid container with cantilevered lance
US9670479B2 (en) 2013-03-15 2017-06-06 F Cubed, LLC Sample preparation device and methods of use
USD881409S1 (en) 2013-10-24 2020-04-14 Genmark Diagnostics, Inc. Biochip cartridge
US9498778B2 (en) 2014-11-11 2016-11-22 Genmark Diagnostics, Inc. Instrument for processing cartridge for performing assays in a closed sample preparation and reaction system
US9719082B2 (en) 2013-10-31 2017-08-01 General Electric Company Substrates and associated methods for elution of nucleic acids
KR102207922B1 (en) 2014-03-06 2021-01-26 삼성전자주식회사 Primer set specific for a vancomycin resistant Enterococcus, composition comprising the same and method for detecting a vancomycin resistant Enterococcus in a sample
US11060149B2 (en) 2014-06-18 2021-07-13 Clear Gene, Inc. Methods, compositions, and devices for rapid analysis of biological markers
WO2016065218A1 (en) 2014-10-23 2016-04-28 Corning Incorporated Polymer-encapsulated magnetic nanoparticles
KR102287811B1 (en) 2014-10-31 2021-08-09 삼성전자주식회사 Method of bonding two surfaces and construct therefrom and microfluidic device containing the construct
US10005080B2 (en) 2014-11-11 2018-06-26 Genmark Diagnostics, Inc. Instrument and cartridge for performing assays in a closed sample preparation and reaction system employing electrowetting fluid manipulation
US9598722B2 (en) 2014-11-11 2017-03-21 Genmark Diagnostics, Inc. Cartridge for performing assays in a closed sample preparation and reaction system
US10364428B2 (en) 2014-11-14 2019-07-30 Corning Incorporated Methods and kits for post-IVT RNA purification
JP6709799B2 (en) 2015-03-24 2020-06-17 スリーエム イノベイティブ プロパティズ カンパニー Method for purifying biological composition and article therefor
AU2016277476B2 (en) 2015-06-10 2022-07-21 Qiagen Gmbh Method for isolating extracellular nucleic acids using anion exchange particles
JP6675167B2 (en) 2015-08-28 2020-04-01 シスメックス株式会社 Method for releasing and recovering peptide, peptide releasing agent and reagent kit
AU2016369603A1 (en) * 2015-12-18 2018-07-05 Clear Gene, Inc. Methods, compositions, kits and devices for rapid analysis of biological markers
WO2017151195A1 (en) * 2016-02-29 2017-09-08 The Penn State Research Foundation Nucleic acid molecular diagnosis
US20170298413A1 (en) * 2016-04-13 2017-10-19 New York Genome Center Methods for the isolation of biomolecules and uses thereof
US11161107B2 (en) 2016-05-25 2021-11-02 Integrated Micro-Chromatography Systems, Inc. Dispersive pipette extraction system for purification of large biomolecules
CN109863249A (en) 2016-08-10 2019-06-07 纽约基因组中心 Ultralow coverage gene order-checking and its application
EP3570974B1 (en) * 2017-01-20 2021-03-31 Dionex Corporation Multimodal chromatographic media for protein separation
WO2018187779A1 (en) 2017-04-07 2018-10-11 Sage Science, Inc. Systems and methods for detection of genetic structural variation using integrated electrophoretic dna purification
CN111094564A (en) 2017-07-12 2020-05-01 伊鲁米纳公司 Nucleic acid extraction materials, systems and methods
GB201819726D0 (en) 2018-12-03 2019-01-16 Diagnostics For The Real World Ltd HCV detection
GB2580385B (en) * 2019-01-08 2022-10-12 Quantumdx Group Ltd Oligonucleotide deposition onto polypropylene substrates
GB2580384B (en) * 2019-01-08 2021-01-27 Quantumdx Group Ltd Oligonucleotide deposition onto polypropylene substrates
JP2020124129A (en) * 2019-02-01 2020-08-20 積水化学工業株式会社 Nucleic acid isolating method and nucleic acid isolating device
EP4077659A1 (en) 2019-12-16 2022-10-26 QIAGEN GmbH Enrichment method
AU2020404874A1 (en) 2019-12-18 2022-06-23 Life Technologies Corporation Systems, methods, and devices for automated nucleic acid and protein isolation
AU2021240470A1 (en) 2020-03-23 2022-11-24 Diagnostics For The Real World, Ltd Coronavirus detection
JP2023530938A (en) * 2020-06-17 2023-07-20 インテグリス・インコーポレーテッド Ion exchange membrane, filter and method
US20230310453A1 (en) * 2020-07-20 2023-10-05 Insmed, Inc. Methods for extracting neutrophil serine proteases and treating dipeptidyl peptidase 1-mediated conditions
GB202017751D0 (en) 2020-11-10 2020-12-23 Life Tech As Sample protection
KR20220075978A (en) 2020-11-30 2022-06-08 (주)바이오니아 Method of Separating Nucleic Acid by Using Binding Buffer with Chemicals Having Low-Mid Dielectric Constant
WO2022263500A1 (en) 2021-06-17 2022-12-22 Qiagen Gmbh Method for isolating non-vesicular mirna
WO2023084369A1 (en) 2021-11-12 2023-05-19 3M Innovative Properties Company Method of processing polynucleic acids
EP4215613A1 (en) * 2022-01-24 2023-07-26 Sartorius BIA Separations d.o.o. Method of separating single stranded rna molecules from other nucleic acid species, use of solid phase therefor, and method of separating double stranded rna molecules by size
US20230382875A1 (en) * 2022-04-22 2023-11-30 Promega Corporation Compounds, compositions, and methods for isolation of nucleic acids

Citations (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4648975A (en) * 1983-08-17 1987-03-10 Pedro B. Macedo Process of using improved silica-based chromatographic supports containing additives
US4843012A (en) * 1986-09-17 1989-06-27 Genetics Institute, Inc. Novel composition for nucleic acid purification
US4908318A (en) * 1987-09-04 1990-03-13 Integrated Genetics, Inc. Nucleic acid extraction methods
US4921805A (en) * 1987-07-29 1990-05-01 Life Technologies, Inc. Nucleic acid capture method
US4923978A (en) * 1987-12-28 1990-05-08 E. I. Du Pont De Nemours & Company Process for purifying nucleic acids
US5057426A (en) * 1986-11-22 1991-10-15 Diagen Institut Fur Molekular-Biologische, Diagnostik Gmbh Method for separating long-chain nucleic acids
US5124444A (en) * 1989-07-24 1992-06-23 Microprobe Corporation Lactam-containing compositions and methods useful for the extraction of nucleic acids
US5128247A (en) * 1989-08-14 1992-07-07 Board Of Regents, The University Of Texas System Methods for isolation of nucleic acids from eukaryotic and prokaryotic sources
US5204246A (en) * 1990-12-26 1993-04-20 Pioneer Hi-Bred International, Inc. Dna isolation method
US5234809A (en) * 1989-03-23 1993-08-10 Akzo N.V. Process for isolating nucleic acid
US5334499A (en) * 1989-04-17 1994-08-02 Eastman Kodak Company Methods of extracting, amplifying and detecting a nucleic acid from whole blood or PBMC fraction
US5508164A (en) * 1990-10-29 1996-04-16 Dekalb Genetics Corporation Isolation of biological materials using magnetic particles
US5596092A (en) * 1990-02-14 1997-01-21 Talent S.R.L. Extraction of genomic DNA from blood using cationic detergents
US5599667A (en) * 1987-03-02 1997-02-04 Gen-Probe Incorporated Polycationic supports and nucleic acid purification separation and hybridization
US5612473A (en) * 1996-01-16 1997-03-18 Gull Laboratories Methods, kits and solutions for preparing sample material for nucleic acid amplification
US5622822A (en) * 1994-09-13 1997-04-22 Johnson & Johnson Clinical Diagnostics, Inc. Methods for capture and selective release of nucleic acids using polyethyleneimine and an anionic phosphate ester surfactant and amplification of same
US5631146A (en) * 1995-01-19 1997-05-20 The General Hospital Corporation DNA aptamers and catalysts that bind adenosine or adenosine-5'-phosphates and methods for isolation thereof
US5641628A (en) * 1989-11-13 1997-06-24 Children's Medical Center Corporation Non-invasive method for isolation and detection of fetal DNA
US5652348A (en) * 1994-09-23 1997-07-29 Massey University Chromatographic resins and methods for using same
US5654179A (en) * 1990-11-14 1997-08-05 Hri Research, Inc. Nucleic acid preparation methods
US5660984A (en) * 1994-12-09 1997-08-26 Davis; Thomas E. DNA isolating apparatus comprising a non-porous DNA binding, anion exchange resin and methods of use thereof
US5705628A (en) * 1994-09-20 1998-01-06 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles
US5770712A (en) * 1997-03-14 1998-06-23 Virginia Tech Intellectual Properties, Inc. Crosslinked hydrogel beads from chitosan
US5916746A (en) * 1996-05-09 1999-06-29 Kirkegaard & Perry Laboratories, Inc. Formazan-based immunoassay
US5981735A (en) * 1996-02-12 1999-11-09 Cobra Therapeutics Limited Method of plasmid DNA production and purification
US5981235A (en) * 1996-07-29 1999-11-09 Promega Corporation Methods for isolating nucleic acids using alkaline protease
US6051380A (en) * 1993-11-01 2000-04-18 Nanogen, Inc. Methods and procedures for molecular biological analysis and diagnostics
US6060246A (en) * 1996-11-15 2000-05-09 Avi Biopharma, Inc. Reagent and method for isolation and detection of selected nucleic acid sequences
US6090288A (en) * 1996-02-19 2000-07-18 Amersham Pharmacia Biotech Ab Process for chromatographic separation of peptides and nucleic acid, and new high affinity ion exchange matrix
US6194562B1 (en) * 1998-04-22 2001-02-27 Promega Corporation Endotoxin reduction in nucleic acid purification
US6270970B1 (en) * 1999-05-14 2001-08-07 Promega Corporation Mixed-bed solid phase and its use in the isolation of nucleic acids
US6284470B1 (en) * 1998-04-22 2001-09-04 Promega Corporation Kits for cell concentration and lysate clearance using paramagnetic particles
US6310199B1 (en) * 1999-05-14 2001-10-30 Promega Corporation pH dependent ion exchange matrix and method of use in the isolation of nucleic acids
US6342387B1 (en) * 1997-09-22 2002-01-29 Riken Method for isolating DNA
US6534262B1 (en) * 1998-05-14 2003-03-18 Whitehead Institute For Biomedical Research Solid phase technique for selectively isolating nucleic acids
US6562573B2 (en) * 1999-01-27 2003-05-13 Folim G. Halaka Materials and methods for the purification of polyelectrolytes, particularly nucleic acids

Family Cites Families (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4055469A (en) * 1976-12-10 1977-10-25 Eastman Kodak Company Purification of microbial enzyme extracts using synthetic polyelectrolytes
JPH01125395A (en) 1987-07-29 1989-05-17 Life Technol Inc Nuclic acid catching reagent
EP0338591A3 (en) 1988-04-21 1991-09-04 Microprobe Corporation Nucleic acid extraction method
IE883226L (en) 1988-10-25 1990-04-25 Dna Prep Galway Ltd S 22 Separation of nucleic acids from protein material using¹cation exchangers
US5512439A (en) * 1988-11-21 1996-04-30 Dynal As Oligonucleotide-linked magnetic particles and uses thereof
WO1990008159A1 (en) 1989-01-23 1990-07-26 Invitron Corporation Method for removing dna from protein preparations
NL8900725A (en) 1989-03-23 1990-10-16 Az Univ Amsterdam METHOD AND COMBINATION OF AGENTS FOR INSULATING NUCLEIC ACID.
GB9003253D0 (en) 1990-02-13 1990-04-11 Amersham Int Plc Precipitating polymers
CA2067711C (en) 1991-05-03 2000-08-08 Daniel Lee Woodard Solid phase extraction purification of dna
JPH07504801A (en) 1991-08-06 1995-06-01 ザ ワールド ヘルス オーガニゼイション DNA isolation method
AT397510B (en) * 1991-11-06 1994-04-25 Wimmer Theodor METHOD FOR PRODUCING FATTY ACID ESTERS OF SHORT-CHAIN ALCOHOLS
US5710028A (en) * 1992-07-02 1998-01-20 Eyal; Nurit Method of quick screening and identification of specific DNA sequences by single nucleotide primer extension and kits therefor
GB9223334D0 (en) * 1992-11-06 1992-12-23 Hybaid Ltd Magnetic solid phase supports
GB2282138B (en) 1993-09-28 1997-09-03 Tosoh Corp Method of extracting nucleic acids and method of detecting specified nucleic acid sequences
GB9323305D0 (en) 1993-11-11 1994-01-05 Medinnova Sf Isoaltion of nucleic acid
GB2284208A (en) * 1993-11-25 1995-05-31 Pna Diagnostics As Nucleic acid analogues with a chelating functionality for metal ions
DE4432654C2 (en) 1994-09-14 1998-03-26 Qiagen Gmbh Process for the isolation of nucleic acids from natural sources
WO1995027718A2 (en) 1994-04-08 1995-10-19 Hybridon, Inc. Purification of oligodeoxynucleotide phosphorothioates using anion exchange chromatography
US5582988A (en) 1994-09-15 1996-12-10 Johnson & Johnson Clinical Diagnostics, Inc. Methods for capture and selective release of nucleic acids using weakly basic polymer and amplification of same
WO1996018732A2 (en) 1994-12-15 1996-06-20 Board Of Trustees Of The University Of Illinois Sequence-specific inhibition of dna synthesis by triplex-forming oligonucleotides
AR003122A1 (en) 1995-05-19 1998-07-08 Merck & Co Inc A PROCESS FOR ISOLATION AND PURIFICATION OF PLASMIDS IN LARGE SCALE FERMENTATORS AND ISOLATED AND PURIFIED DNA OBTAINED THROUGH SUCH A PROCESS.
KR19990022612A (en) 1995-06-08 1999-03-25 퍼니스 챨스 엠 DNA Extraction Method and Extraction Apparatus
US5783686A (en) 1995-09-15 1998-07-21 Beckman Instruments, Inc. Method for purifying nucleic acids from heterogenous mixtures
DE29601618U1 (en) 1996-01-31 1996-07-04 Invitek Gmbh Multiple simultaneous isolation device
US5874221A (en) * 1996-08-28 1999-02-23 The United States Of America As Represented By The Secretary Of Agriculture Species specific method for the PCR detection of phythophthora
CA2214495C (en) 1996-09-25 2002-02-05 Daniel L. Woodard Hydrated zirconium silicate composition for purification of nucleic acids
US5795748A (en) 1996-09-26 1998-08-18 Becton Dickinson And Company DNA microwell device and method
EP0853123A1 (en) 1997-01-10 1998-07-15 Roche Diagnostics GmbH Purification of DNA by 'cross-flow-filtration'
EP0897978A3 (en) 1997-08-22 2001-10-17 Becton, Dickinson and Company Zirconium oxide and related compounds for purification of nucleic acids
DE19746874A1 (en) 1997-10-23 1999-04-29 Qiagen Gmbh Isolation of nucleic acids
ATE218140T1 (en) * 1997-12-06 2002-06-15 Dna Res Innovations Ltd ISOLATION OF NUCLEIC ACIDS
DE19907023A1 (en) 1999-02-19 2000-08-24 Bayer Ag Isolation of nucleic acids, useful for e.g. genetic diagnosis by amplification, by adsorption onto polymeric beads at neutral or acidic pH then release at basic pH
CA2270106C (en) 1999-04-23 2006-03-14 Yousef Haj-Ahmad Nucleic acid purification and process
JP4551568B2 (en) 1999-05-14 2010-09-29 プロメガ コーポレイション Cell collection and lysate clarification using paramagnetic particles
US7501284B2 (en) 2000-07-31 2009-03-10 Applera Corporation Apparatus and method for specific release of captured extension products
AU2001281000A1 (en) 2000-08-24 2002-03-04 Bio-Rad Laboratories, Inc. Flow-through system for lysate clarification and nucleic acid binding in a single step

Patent Citations (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4648975A (en) * 1983-08-17 1987-03-10 Pedro B. Macedo Process of using improved silica-based chromatographic supports containing additives
US4843012A (en) * 1986-09-17 1989-06-27 Genetics Institute, Inc. Novel composition for nucleic acid purification
US5057426A (en) * 1986-11-22 1991-10-15 Diagen Institut Fur Molekular-Biologische, Diagnostik Gmbh Method for separating long-chain nucleic acids
US5599667A (en) * 1987-03-02 1997-02-04 Gen-Probe Incorporated Polycationic supports and nucleic acid purification separation and hybridization
US4921805A (en) * 1987-07-29 1990-05-01 Life Technologies, Inc. Nucleic acid capture method
US4908318A (en) * 1987-09-04 1990-03-13 Integrated Genetics, Inc. Nucleic acid extraction methods
US4923978A (en) * 1987-12-28 1990-05-08 E. I. Du Pont De Nemours & Company Process for purifying nucleic acids
US5234809A (en) * 1989-03-23 1993-08-10 Akzo N.V. Process for isolating nucleic acid
US5334499A (en) * 1989-04-17 1994-08-02 Eastman Kodak Company Methods of extracting, amplifying and detecting a nucleic acid from whole blood or PBMC fraction
US5124444A (en) * 1989-07-24 1992-06-23 Microprobe Corporation Lactam-containing compositions and methods useful for the extraction of nucleic acids
US5128247A (en) * 1989-08-14 1992-07-07 Board Of Regents, The University Of Texas System Methods for isolation of nucleic acids from eukaryotic and prokaryotic sources
US5641628A (en) * 1989-11-13 1997-06-24 Children's Medical Center Corporation Non-invasive method for isolation and detection of fetal DNA
US5596092A (en) * 1990-02-14 1997-01-21 Talent S.R.L. Extraction of genomic DNA from blood using cationic detergents
US5508164A (en) * 1990-10-29 1996-04-16 Dekalb Genetics Corporation Isolation of biological materials using magnetic particles
US5654179A (en) * 1990-11-14 1997-08-05 Hri Research, Inc. Nucleic acid preparation methods
US5204246A (en) * 1990-12-26 1993-04-20 Pioneer Hi-Bred International, Inc. Dna isolation method
US6051380A (en) * 1993-11-01 2000-04-18 Nanogen, Inc. Methods and procedures for molecular biological analysis and diagnostics
US5622822A (en) * 1994-09-13 1997-04-22 Johnson & Johnson Clinical Diagnostics, Inc. Methods for capture and selective release of nucleic acids using polyethyleneimine and an anionic phosphate ester surfactant and amplification of same
US5705628A (en) * 1994-09-20 1998-01-06 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles
US5898071A (en) * 1994-09-20 1999-04-27 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles
US5652348A (en) * 1994-09-23 1997-07-29 Massey University Chromatographic resins and methods for using same
US5660984A (en) * 1994-12-09 1997-08-26 Davis; Thomas E. DNA isolating apparatus comprising a non-porous DNA binding, anion exchange resin and methods of use thereof
US5631146A (en) * 1995-01-19 1997-05-20 The General Hospital Corporation DNA aptamers and catalysts that bind adenosine or adenosine-5'-phosphates and methods for isolation thereof
US5612473A (en) * 1996-01-16 1997-03-18 Gull Laboratories Methods, kits and solutions for preparing sample material for nucleic acid amplification
US5981735A (en) * 1996-02-12 1999-11-09 Cobra Therapeutics Limited Method of plasmid DNA production and purification
US6090288A (en) * 1996-02-19 2000-07-18 Amersham Pharmacia Biotech Ab Process for chromatographic separation of peptides and nucleic acid, and new high affinity ion exchange matrix
US5916746A (en) * 1996-05-09 1999-06-29 Kirkegaard & Perry Laboratories, Inc. Formazan-based immunoassay
US5981235A (en) * 1996-07-29 1999-11-09 Promega Corporation Methods for isolating nucleic acids using alkaline protease
US6060246A (en) * 1996-11-15 2000-05-09 Avi Biopharma, Inc. Reagent and method for isolation and detection of selected nucleic acid sequences
US5770712A (en) * 1997-03-14 1998-06-23 Virginia Tech Intellectual Properties, Inc. Crosslinked hydrogel beads from chitosan
US6342387B1 (en) * 1997-09-22 2002-01-29 Riken Method for isolating DNA
US6194562B1 (en) * 1998-04-22 2001-02-27 Promega Corporation Endotoxin reduction in nucleic acid purification
US6284470B1 (en) * 1998-04-22 2001-09-04 Promega Corporation Kits for cell concentration and lysate clearance using paramagnetic particles
US6534262B1 (en) * 1998-05-14 2003-03-18 Whitehead Institute For Biomedical Research Solid phase technique for selectively isolating nucleic acids
US6562573B2 (en) * 1999-01-27 2003-05-13 Folim G. Halaka Materials and methods for the purification of polyelectrolytes, particularly nucleic acids
US6270970B1 (en) * 1999-05-14 2001-08-07 Promega Corporation Mixed-bed solid phase and its use in the isolation of nucleic acids
US6310199B1 (en) * 1999-05-14 2001-10-30 Promega Corporation pH dependent ion exchange matrix and method of use in the isolation of nucleic acids

Cited By (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060024701A1 (en) * 2001-01-09 2006-02-02 Whitehead Institute For Biomedical Research Methods and reagents for the isolation of nucleic acids
US20090130736A1 (en) * 2003-02-06 2009-05-21 Becton, Dickinson And Company Pretreatment method for extraction of nucleic acid from biological samples and kits therefor
US7727727B2 (en) 2003-02-06 2010-06-01 Becton Dickinson And Company Pretreatment method for extraction of nucleic acid from biological samples and kits therefor
US20070031880A1 (en) * 2003-02-06 2007-02-08 Becton, Dickinson And Company Chemical treatment of biological samples for nucleic acid extraction and kits therefor
US20040197780A1 (en) * 2003-04-02 2004-10-07 Agencourt Bioscience Corporation Method for isolating nucleic acids
US20070054285A1 (en) * 2003-04-02 2007-03-08 Mckernan Kevin Method for isolating nucleic acids
US11078523B2 (en) 2003-07-31 2021-08-03 Handylab, Inc. Processing particle-containing samples
US11441171B2 (en) 2004-05-03 2022-09-13 Handylab, Inc. Method for processing polynucleotide-containing samples
US20060177836A1 (en) * 2004-07-30 2006-08-10 Mckernan Kevin J Methods of isolating nucleic acids using multifunctional group-coated solid phase carriers
US7527929B2 (en) 2004-07-30 2009-05-05 Agencourt Bioscience Corporation Methods of isolating nucleic acids using multifunctional group-coated solid phase carriers
US20060084089A1 (en) * 2004-08-03 2006-04-20 Becton, Dickinson And Company Use of magnetic material to direct isolation of compounds and fractionation of multipart samples
US20060030056A1 (en) * 2004-08-03 2006-02-09 Becton, Dickinson And Company Use of magnetic material to fractionate samples
US20060127887A1 (en) * 2004-12-10 2006-06-15 Lee Young-Sun Isolation and purification method of biomolecules using hydrogel
US7579151B2 (en) * 2004-12-10 2009-08-25 Samsung Electronics Co., Ltd. Isolation and purification method of biomolecules using hydrogel
US20060199199A1 (en) * 2005-01-20 2006-09-07 Kim Kui-Hyun Method of removing nucleic acid amplification inhibitor from biological sample and PCR system
US11666903B2 (en) 2006-03-24 2023-06-06 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using same
US11806718B2 (en) 2006-03-24 2023-11-07 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system
US11142785B2 (en) 2006-03-24 2021-10-12 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
US11141734B2 (en) 2006-03-24 2021-10-12 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system
US11085069B2 (en) 2006-03-24 2021-08-10 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
US10900066B2 (en) 2006-03-24 2021-01-26 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
US10913061B2 (en) 2006-03-24 2021-02-09 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using the same
KR100785011B1 (en) 2006-04-07 2007-12-11 삼성전자주식회사 Method for increasing the specificity of nucleic acid hybridization using zwitterionic compounds
US20080026374A1 (en) * 2006-07-31 2008-01-31 Sigma Aldrich Co. Compositions and Methods for Isolation of Biological Molecules
US20080026375A1 (en) * 2006-07-31 2008-01-31 Sigma Aldrich Co. Compositions and Methods for Isolation of Biological Molecules
US20080023395A1 (en) * 2006-07-31 2008-01-31 Sigma Aldrich Co. Compositions and Methods for Isolation of Biological Molecules
WO2008043551A1 (en) * 2006-10-10 2008-04-17 Qiagen Gmbh Methods and kit for isolating nucleic acids
US8460941B2 (en) 2006-10-10 2013-06-11 Qiagen Gmbh Methods and kit for isolating nucleic acids
EP1911844A1 (en) * 2006-10-10 2008-04-16 Qiagen GmbH Methods and kit for isolating nucleic acids
US20100056769A1 (en) * 2006-10-10 2010-03-04 Qiagen Gmbh Methods and kit for isolating nucleic acids
US8206990B2 (en) 2006-10-10 2012-06-26 Qiagen Gmbh Methods and kit for isolating nucleic acids
US8999636B2 (en) 2007-01-08 2015-04-07 Toxic Report Llc Reaction chamber
US20100120101A1 (en) * 2007-01-08 2010-05-13 U.S. Genomics, Inc. Reaction chamber
US20090061497A1 (en) * 2007-06-29 2009-03-05 Becton, Dickinson And Company Methods for Extraction and Purification of Components of Biological Samples
US20100294665A1 (en) * 2007-07-12 2010-11-25 Richard Allen Method and system for transferring and/or concentrating a sample
US11060082B2 (en) 2007-07-13 2021-07-13 Handy Lab, Inc. Polynucleotide capture materials, and systems using same
US11549959B2 (en) 2007-07-13 2023-01-10 Handylab, Inc. Automated pipetting apparatus having a combined liquid pump and pipette head system
US11845081B2 (en) 2007-07-13 2023-12-19 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
US10875022B2 (en) 2007-07-13 2020-12-29 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
US11466263B2 (en) 2007-07-13 2022-10-11 Handylab, Inc. Diagnostic apparatus to extract nucleic acids including a magnetic assembly and a heater assembly
US11266987B2 (en) 2007-07-13 2022-03-08 Handylab, Inc. Microfluidic cartridge
US11254927B2 (en) 2007-07-13 2022-02-22 Handylab, Inc. Polynucleotide capture materials, and systems using same
US8361716B2 (en) 2008-10-03 2013-01-29 Pathogenetix, Inc. Focusing chamber
US20100112576A1 (en) * 2008-10-03 2010-05-06 U.S. Genomics, Inc. Focusing chamber
US9006419B2 (en) 2009-10-22 2015-04-14 Industrial Technology Research Institute Method for isolating nucleic acids
US11788127B2 (en) 2011-04-15 2023-10-17 Becton, Dickinson And Company Scanning real-time microfluidic thermocycler and methods for synchronized thermocycling and scanning optical detection
EP2771462A1 (en) * 2011-10-27 2014-09-03 GE Healthcare Bio-Sciences AB Purification of nucleic acid
US9340828B2 (en) 2011-10-27 2016-05-17 Ge Healthcare Bio-Sciences Ab Purification of nucleic acid
WO2013062476A1 (en) * 2011-10-27 2013-05-02 Ge Healthcare Bio-Sciences Ab Purification of nucleic acid
EP2771462A4 (en) * 2011-10-27 2015-04-08 Ge Healthcare Bio Sciences Ab Purification of nucleic acid
US11453906B2 (en) 2011-11-04 2022-09-27 Handylab, Inc. Multiplexed diagnostic detection apparatus and methods
US8685708B2 (en) 2012-04-18 2014-04-01 Pathogenetix, Inc. Device for preparing a sample
US9808798B2 (en) 2012-04-20 2017-11-07 California Institute Of Technology Fluidic devices for biospecimen preservation
WO2013159117A1 (en) 2012-04-20 2013-10-24 SlipChip, LLC Fluidic devices and systems for sample preparation or autonomous analysis
US9822356B2 (en) 2012-04-20 2017-11-21 California Institute Of Technology Fluidic devices and systems for sample preparation or autonomous analysis
US9803237B2 (en) 2012-04-24 2017-10-31 California Institute Of Technology Slip-induced compartmentalization

Also Published As

Publication number Publication date
US20070231892A1 (en) 2007-10-04
DE60107468D1 (en) 2004-12-30
US20130338245A1 (en) 2013-12-19
DK1345952T3 (en) 2005-03-21
US6914137B2 (en) 2005-07-05
WO2002048164A2 (en) 2002-06-20
CA2432075A1 (en) 2002-06-20
US20010018513A1 (en) 2001-08-30
ATE283275T1 (en) 2004-12-15
AU2002216203A1 (en) 2002-06-24
WO2002048164A3 (en) 2002-10-17
ES2233560T3 (en) 2005-06-16
EP1345952A2 (en) 2003-09-24
EP1473299A3 (en) 2006-08-16
US20120196944A1 (en) 2012-08-02
US20120197009A1 (en) 2012-08-02
EP1345952B1 (en) 2004-11-24
DE60107468T2 (en) 2005-12-29
JP4868697B2 (en) 2012-02-01
JP2004521881A (en) 2004-07-22
EP1473299A2 (en) 2004-11-03
US20030054395A1 (en) 2003-03-20
US20080305528A1 (en) 2008-12-11

Similar Documents

Publication Publication Date Title
US6914137B2 (en) Isolation of nucleic acids
EP1234832B1 (en) Isolation of nucleic acids
US20060024712A1 (en) Separation of nucleic acid
US20080261202A1 (en) Tagged Polyfunctional Reagents Capable of Reversibly Binding Target Substances in a pH-dependent Manner
US7560228B2 (en) Solid-phase nucleic acid isolation
CA2773320C (en) Compositions and methods for recovery of nucleic acids or proteins from tissue samples fixed in cytology media
JP2005532799A (en) Binding of target substance
WO2003095646A1 (en) Isolating nucleic acid
CA2279131C (en) Electrophoretic separation of nucleic acids from proteins at low ph
CN117751186A (en) Methods for reducing endotoxin levels for nucleic acid purification
MXPA00005474A (en) Isolation of nucleic acids

Legal Events

Date Code Title Description
AS Assignment

Owner name: INVITROGEN CORPORATION, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BAKER, MATTHEW JOHN;DNA RESEARCH INNOVATIONS LIMITED;REEL/FRAME:017198/0593

Effective date: 20051130

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: LIFE TECHNOLOGIES CORPORATION,CALIFORNIA

Free format text: MERGER;ASSIGNOR:INVITROGEN CORPORATION;REEL/FRAME:023882/0551

Effective date: 20081121

Owner name: LIFE TECHNOLOGIES CORPORATION, CALIFORNIA

Free format text: MERGER;ASSIGNOR:INVITROGEN CORPORATION;REEL/FRAME:023882/0551

Effective date: 20081121

AS Assignment

Owner name: LIFE TECHNOLOGIES CORPORATION, CALIFORNIA

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE APPLICATION NO 09452626 PREVIOUSLY RECORDED ON REEL 023882 FRAME 0551. ASSIGNOR(S) HEREBY CONFIRMS THE MERGER SHOULD NOT HAVE BEEN RECORDED AGAINST THIS PATENT APPLICATION NUMBER;ASSIGNOR:INVITROGEN CORPORATION;REEL/FRAME:034217/0490

Effective date: 20081121