WO1994011103A1 - Magnetic solid phase supports - Google Patents
Magnetic solid phase supports Download PDFInfo
- Publication number
- WO1994011103A1 WO1994011103A1 PCT/GB1993/002289 GB9302289W WO9411103A1 WO 1994011103 A1 WO1994011103 A1 WO 1994011103A1 GB 9302289 W GB9302289 W GB 9302289W WO 9411103 A1 WO9411103 A1 WO 9411103A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- msps
- magnetisable
- particles according
- particles
- magnetisable particles
- Prior art date
Links
- 230000005291 magnetic effect Effects 0.000 title claims description 13
- 239000007790 solid phase Substances 0.000 title description 8
- 239000002245 particle Substances 0.000 claims abstract description 54
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- 102000039446 nucleic acids Human genes 0.000 claims abstract description 25
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 25
- 239000003446 ligand Substances 0.000 claims abstract description 20
- 239000013612 plasmid Substances 0.000 claims abstract description 17
- 229920000642 polymer Polymers 0.000 claims abstract description 13
- 229920001222 biopolymer Polymers 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 30
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 claims description 27
- 239000000725 suspension Substances 0.000 claims description 27
- 238000010828 elution Methods 0.000 claims description 23
- 229920000936 Agarose Polymers 0.000 claims description 22
- 239000000203 mixture Substances 0.000 claims description 20
- 239000000463 material Substances 0.000 claims description 12
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 claims description 11
- 238000001125 extrusion Methods 0.000 claims description 11
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 claims description 11
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
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- ZWVMLYRJXORSEP-LURJTMIESA-N (2s)-hexane-1,2,6-triol Chemical compound OCCCC[C@H](O)CO ZWVMLYRJXORSEP-LURJTMIESA-N 0.000 claims description 2
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 claims description 2
- 239000007983 Tris buffer Substances 0.000 claims description 2
- MZVQCMJNVPIDEA-UHFFFAOYSA-N [CH2]CN(CC)CC Chemical group [CH2]CN(CC)CC MZVQCMJNVPIDEA-UHFFFAOYSA-N 0.000 claims description 2
- 210000003850 cellular structure Anatomy 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 2
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
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- MBYLVOKEDDQJDY-UHFFFAOYSA-N tris(2-aminoethyl)amine Chemical compound NCCN(CCN)CCN MBYLVOKEDDQJDY-UHFFFAOYSA-N 0.000 description 4
- 0 *CC(COCCN(CCO)CO)O Chemical compound *CC(COCCN(CCO)CO)O 0.000 description 3
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 description 2
- 238000007399 DNA isolation Methods 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 2
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- 239000007900 aqueous suspension Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000007885 magnetic separation Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229920000768 polyamine Polymers 0.000 description 2
- 150000003141 primary amines Chemical class 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000007873 sieving Methods 0.000 description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 description 2
- 239000012279 sodium borohydride Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- XLVSIIHGQYACPK-UHFFFAOYSA-N 5-(oxiran-2-yl)-4-(oxiran-2-ylmethyl)pentane-1,4-diol Chemical compound C1OC1CC(O)(CCCO)CC1CO1 XLVSIIHGQYACPK-UHFFFAOYSA-N 0.000 description 1
- 108091027075 5S-rRNA precursor Proteins 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- WRKDZVUJABRFSB-UHFFFAOYSA-N NCCN(CCN)CCNCC(CO)O Chemical compound NCCN(CCN)CCNCC(CO)O WRKDZVUJABRFSB-UHFFFAOYSA-N 0.000 description 1
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- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
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- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 1
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- JECOSSASMXAXFV-UHFFFAOYSA-N chloroethane;hydrochloride Chemical compound Cl.CCCl JECOSSASMXAXFV-UHFFFAOYSA-N 0.000 description 1
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- 150000004676 glycans Chemical class 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
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- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
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Classifications
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
- B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
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- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
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- B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
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- B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
- B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
- B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
- G01N2400/32—Galactans, e.g. agar, agarose, agaropectin, carrageenan
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/20—Magnetic particle immunoreagent carriers the magnetic material being present in the particle core
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/30—Magnetic particle immunoreagent carriers the magnetic material being dispersed in the polymer composition before their conversion into particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
- G01N2446/84—Polymer coating, e.g. gelatin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
- G01N2446/90—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids characterised by small molecule linker used to couple immunoreagents to magnetic particles
Definitions
- This invention relates to the isolation and purification of molecules of biological interest eg proteins, nucleic acids, and other biopolymers.
- An affinity chromatography step is often the final or penultimate step. Ideally this step should be employed as early as possible in the process to maximise the yield of desired product and minimise the time required to generate it. Usually this is not feasible as the substance of interest (ligate) is present in a medium not readily amenable to conventional column affinity chromatography. Often the ligate may be present in a mixture of substances containing particulate and/or semi-solid or colloidal material such as cell debris and denatured cell components and/or in a mixture which is highly viscous.
- the present invention is concerned with the development of relatively simple techniques for use with complex mixtures of high viscosity and/or those containing particulate matter and which are based on the use of magnetisable solid phase support (MSPS) materials.
- MSPS magnetisable solid phase support
- the present invention comprises magnetisable polymer based particles derivatised with ligands possessing direct-binding affinities for nucleic acids and other types of biomolecule, and the use of these particles for nucleic acid (and other biomolecular) purification and speciation, including double or single stranded nucleic acids, genomic DNA, plasmid DNA, and cellular RNA.
- the direct binding of the ligand to the target molecule is important to achieve the benefits of this invention as contrasted with prior proposals based on indirect linking of the target molecules eg. through intercalating agents as described in EP 301,899 A.
- the present invention comprises particulate support material having magnetic properties and bearing a ligand which binds to molecules of a specific type. From another aspect the present invention comprises a method of separating biomolecules of various types by binding such molecules to ligands which are selective therefore and which are attached to magnetisable particulate support materials. The invention is further apparent from the appended claims.
- the magnetic susceptibility of MSPS materials in accordance with the present invention is a function of the weight and size of the particulate material and the quantity of paramagnetic material incorporated therein. This property may therefore be adjusted as desired in order to provide an appropriate selection parameter.
- a product range of MSPS materials may be manufactured in order to cater for the wide range of separations encountered in both analytical and preparative procedures.
- the MSPS materials in accordance with this invention are preferably gels and are conveniently in the form of beads. Such gels may be stabilised by crosslinking eg with epichlorohydrin, leading to greater particle stability especially at high temperatures.
- Many polymers may be used for the purposes of this invention including cellulose, dextran, polyacrylamide and the synthetic material trisacryl.
- One of the most preferred materials is agarose.
- the positive benefits of this polymer are gel strength and biological inertness.
- the abundance of hydroxyl groups possessed by the agarose matrix allows for easy covalent attachment of various ligands desirable for molecular biology applications and its non-covalent secondary structure also engenders structural stability, porosity and ease of shaping into spheres. Since many molecular biology methodologies are performed at temperatures above that at which agarose melts it would also be desirable to introduce thermal stability into the agarose solid phase support.
- MSPS materials consist of at least two separate components a non-magnetisable component which may be chemically derivatised with suitable ligands and a magnetisable component or core.
- Paramagnetic properties can be introduced into the solid phase support by addition of powdered paramagnetic iron oxide (Fe 3 0 4 ) to the agarose during the preparation of the MSPS, which leads to entrapment of the paramagnetic component within the solid phase matrix and gives the particles their magnetisability.
- MSPS particles should be within a size range which allows the greatest particle surface area exposure to ligate, but at the same time allows the best particle magnetic sedimentation rate.
- the magnetisable component should also have a high degree of magnetisable susceptibility to optimise magnetic sedimentation rates.
- the ideal magnetisable component can be selected on the basis of the above equation, but in practice relatively few paramagnetic molecules have been used in MSPS. Iron oxide, barium ferrite and nickel oxide may be used.
- MSPS construction since it possesses a high magnetic susceptibility, is readily available, inexpensive and non toxic.
- a highly preferred system for our purposes entails iron oxide as the magnetisable component for
- MSPS and agarose as the polymer matrix in which to embed it.
- other natural and synthetic polymers may be used as MSPS matrix material including cellulose, alginate, dextran and perfluorocarbon based supports.
- MSPS may be prepared either by encapsulation of the magnetisable component during the preparation of the particle, or by addition of the magnetisable component subsequent to particle formation.
- the magnetisable component can be mixed in an aqueous suspension of cellulose or agarose and allowed to cure, the water is removed and the dried material ground to the size range required.
- MSPS may be prepared by spraying or droplet formation and this leads to beaded particles.
- MSPS may be prepared by addition of a suspension of sodium alginate and iron oxide dropped into a solution of calcium chloride. Droplet formation can be brought about through emulsification techniques and thee have been employed for the formation of spherical magnetisable dextran, albumin, acrylates, acrolein and polyglutaraldehyde. In this system an aqueous emulsion of the matrix polymer and magnetisable component is prepared. This is added to mineral oil and the two phase system stirred. A precipitate is formed of beaded particles.
- the particle is coated with an aqueous solution of polyacrylamide containing ferric oxide.
- the resulting particles tend to be large >250 ⁇ and very porous. However, they are amenable to activation via either the polysaccharide or polyacrylamide component.
- agarose MSPS In one method we have used to prepare agarose MSPS an aqueous suspension is extruded into an immiscible organic phase.
- a suspension of paramagnetic iron oxide, Fe 3 0 4 , (4% w/v) in molten agarose (2% w/v) was extruded into vegetable oil stirred with an overhead paddle stirrer using a specially adapted plastic syringe. The end of the syringe was sealed, then drilled with a small hole to allow the molten agarose/iron oxide mixture to be extruded into the oil phase.
- the MSPS was collected, washed with water and sized by sieving, then checked under a light microscope set up for K ⁇ hler illumination for morphological homogeneity. The particles formed were highly bead like and uniformly spherical, a consequence of both the extrusion technique and the immiscibility of the oil and water.
- the highly spherical nature of the particles produced is particularly desirable because it prevents local concentration change effects that might occur with particles whose surface is more heterogeneous. It is particularly important to maintain uniform ionic concentrations around the surface of a particle in order that local absorption and/or desorption does not occur and alter the bulk properties of the support.
- MSPS When the MSPS was viewed under the light microscope, small yellow inclusions were observed incorporated within the agarose matrix which were believed to be vegetable oil retained in the matrix of the agarose during the preparation of the MSPS. To remove these inclusions the MSPS was washed following preparation in an acetone/water series.
- MSPS size, quantity and quality of the MSPS produced.
- a range of differently sized MSPS (>500 ⁇ m - ⁇ 150 ⁇ m) were obtained when stir rate, hole bore diameter and extrusion rate were altered. Results indicated that the faster the stir rate of the oil phase, the narrower the bore size of the pierced hole and the higher the extrusion rate, the greater the proportion of smaller diameter MSPS (less than 150 ⁇ m) was produced.
- Small particles are sought for affinity chromatography as they possess a greater surface area/volume ratio, significantly enhancing their ligand loading capacity and subsequent ability to take up and release macromolecules during chromatography.
- cross-linked MSPS In order to confer thermal and chemical stability on the MSPS following manufacture, it may be cross-linked with epichlorohydrin under basic conditions using known methods.
- the cross-linking reaction covalently binds together the polymeric agarose strands by a three-carbon link, instilling a physical rigidity in the particles that reduces the risk of shear damage.
- Once cross- linked MSPS can be autoclaved (120°C, 15psi, 15 min) without damage or alteration of its physical properties, and is resistant to a whole range of chemical reagents used in further derivatisations.
- Cross-linked MSPS can be stored quite satisfactorily at room temperature for several months as a suspension in 20% aqueous methanol (to act as an anti-bacterial agent) without any deterioration. No leaching of iron oxide from the agarose matrix was observed.
- DEAE-, ECTEOLA-, spermine-, TAEA-, EHEP, and HDA-MSPS can all be used for the general isolation of nucleic aids and are listed in decreasing order of affinity for nucleic acids.
- the degree of ligand loading may be expressed as the ability of a known weight of MSPS to adsorb a known amount of DNA, and the adsorption measured by ultra ⁇ violet spectroscopy. This serves as a method of quality control for the MSPS to check that surface derivatisation had proceeded satifactorily.
- a suspension of DEAE-MSPS 100 mg ml was incubated with a solution of salmon sperm DNA (50 ⁇ g ml -1 ) at room temperature in an Eppendorf tube. After 30 minutes the MSPS was immobilised with a magnetic concentrator, the supernatant removed and its adsorbance at 260nm and 280nm recorded, and compared with that of the original stock solution of DNA to calculate the level of nucleic acid uptake. Typically, for DEAE-MSPS a figure of at least 95% uptake of salmon sperm DNA was obtained. Similar results were obtained for ECTEOLA-, TAEA- and spermine-MSPS (Table 1).
- tertiary amines are more basic than secondary or primary amines, solid phase supports derivatised with tertiary amine-containing ligand have a higher positive charge density and therefore a greater affinity for nucleic acids.
- the adsorbed DNA could be eluted from all samples with at least 80% efficiency (based on A 250 readings) by incubation for up to 30 minutes with 1M NaCl/50mM arginine free base at 65°C.
- the derivatised MSPS can be stored at 4°C for several months in suspensions of 20% aqueous methanol without any decrease in performance or capacity for nucleic acids.
- MSPS magnetisable solid phase support
- the majority of the cleared oil phase is decanted and the aqueous phase containing the beaded MSPS re-washed with deionised water (100ml).
- the suspension of MSPS is initially sized by sieving through a series of Endecott sieves of mesh size 500 ⁇ m, 250 ⁇ m, 200 ⁇ m, 180 ⁇ m, and 150 ⁇ m using a Fritsch sieve shaker.
- the fraction containing particles of less than 150 ⁇ m diameter is further sieved with sieves of mesh size lOO ⁇ m, 50 ⁇ m, 32 ⁇ m and 20 ⁇ m.
- Cross-linked MSPS (0.7 g moist weight) is suspended in 40% sodium hydroxide (3ml) and cooled to 0°C in an ice- water bath. A solution of triethanolamine (0.4ml) in epichlorohydrin (0.7ml) is added drop-wise with occasional shaking, maintaining the temperature at 0°C. After 30 minutes the suspension is allowed to warm 23°C, shaken vigorously using a flask shaker and left to stand for 16 hours. The mixture is then poured into a large volume of 1M hydrochloric acid with stirring, filtered, then washed successively with 1M sodium hydroxide, sterile distilled water, 20% aqueous methanol then finally water. The ECTEOLA-MSPS is stored in 20% aqueous methanol to give a particle density of 100 mg/ml.
- Cross-linked MSPS (0.5 g moist weight) is suspended in 1M sodium hydroxide (0.4ml). Sodium borohydride (l g) is added followed by diglycidylbutane-1,4-diol (0.5ml). The suspension is shaken at 23°C for 6 hours, then filtered, washed thoroughly with deionised water and immediately resuspended in 5M tris(2-aminoethyl)amine (l l) and shaken at 30"c for 2 hours. The suspension is 0 washed successively with sterile distilled water, 1M sodium chloride, sterile distilled water and finally 0.05M phosphate buffer (pH7). The TAEA-MSPS is stored in 20% aqueous methanol to a final particle density of 100 mg/ml. 5
- MSPS (2g moist weight) is activated with oxirane groups as described for TAEA-MSPS, then suspended in 5M
- Cross-linked MSPS (0.9 g moist weight) is suspended in 3040% sodium hydroxide (4ml) and cooled to 0°C in an ice- water bath.
- the mixture is then poured into a large volume of 1M hydrochloric acid with stirring, filtered, then washed successively with 1M sodium hydroxide, sterile distilled water, 20% aqueous methanol and finally sterile distilled water.
- the EHEP-MSPS is stored in 20% aqueous methanol to give a final particle density of 100 mg/ml.
- nucleic acids to be adsorbed by the MSPS can be in a variety of forms, including aqueous solutions of one, or more than one, species of nucleic acid, or semi-crude cell lysate mixtures. Nucleic acids are adsorbed non- specifically by the MSPS at ambient temperature (23°C) from solution at pH7 or below. Nucleic acids can be eluted specifically in pure form from the MSPS by treatment with elution solutions of differing ionic strengths.
- RNA and DNA can be adsorbed simultaneously by the DEAE-derivatised beads, the RNA eluted using 0.lMNaCl/50mM arginine (free base) at 65°C for 30 minutes.
- Plasmid DNA can be adsorbed by the MSPS from a cell lysis mixture (boiling lysis) which has been treated with RNAse. Any remaining small fragments of oligoribonucleotide which co-adsorb with the plasmid DNA can be eluted using 0.1M/50mM arginine (free base), then the pure plasmid DNA can be eluted using 1.0M/50mM arginine (free base) at 65°C for 15 minutes.
- RNA could be adsorbed to and eluted from the DEAE- MSPS, but elution occurred at much lower ionic concentrations and temperatures and at a significantly faster rate.
- DEAE-MSPS was incubated with a solution of total RNA (50 ⁇ g ml "1 ) at 23°C for 30 minutes, magnetically immobilised and the supernatant removed. Comparison of the A 2 go values of the supernatant and original stock solution of RNA showed that, on average, 90 % of the RNA had been adsorbed by the MSPS.
- the RNA could be eluted with an efficiency of 80-85% at salt concentrations of 0.1M NaCl up to 1.0M NaCl (50mM arginine free base) at 23°C.
- DEAE-MSPS can also be used for differentiating plasmid DNA and total cellular RNA.
- a solution of plasmid pUC 18 was prepared by standard boiling lysis, which contained amounts of RNA carried over from the lysis step.
- DEAE-MSPS was incubated with this solution under the same conditions used for other protocols, magnetically immobilised, then treated with 0.1M NaCl, to remove adsorbed oligoribonucleotides, then the plasmid DNA eluted using 1.0M/50mM arginine free base. Eluted samples were analysed by agarose gel electrophoresi ⁇ , which showed firstly that the MSPS will adsorb and release plasmid DNA satisfactorily using the standard uptake and elution protocol, and secondly, that RNA was eluted at 0.05M-0.1 NaCl, whilst the plasmid DNA was eluted at a salt concentration of 1.0M NaCl. Therefore, DEAE-MSPS can be used to separate unwanted RNA resulting from cell lysis from plasmid DNA, and provide plasmid DNA of a purity and quality suitable for further applications.
- DEAE-MSPS appears to selectively release them according to size upon application of elution solutions.
- the DEAE-MSPS was non-specific in its uptake of a mixtures of nucleic acids from solution, but by application of different concentrations of an elution solution, DNA and RNA could be eluted specifically from the DEAE-MSPS, a procedure which can be scaled down to a microtitre plate format without loss of efficiency.
- plasmid DNA and RNA could be speciated on the DEAE-MSPS.
- Total RNA can be isolated from a mixture of RNA and DNA using a method similar to that described above. 250 ⁇ l of a suspension of DEAE-MSPS (100 mg ml -1 ) is aliquotted into an Eppendorf tube and washed twice with sterile distilled water. The nucleic acid solution containing RNA and DNA (up to 50 ⁇ g total) is added to the MSPS and the suspension mixed by end-over-end rotation for 30 minutes at 23°C. The MSPS is magnetically immobilised and the supernatant removed, then 1ml of an elution solution (0.1M NaCl, 50mM arginine free base) is added.
- an elution solution 0.1M NaCl, 50mM arginine free base
- 1.5 ml of cell culture is spun in a microcentrifuge for 20 seconds in an Eppendorf tube.
- the supernatant is aspirated and the cell pellet resuspended in 350 ⁇ l STET buffer (0.1M NaCl, lOmM Tris-HCl pH 8, l EDTA, 5% triton X-100) containing 2 ⁇ l of RNAse A solution (lOmg ml "1 ).
- 25 ⁇ l of a freshly prepared solution of lysozyme (lOmg ml "1 ) is added in lOmM tris-HCl, pH8.
- the solution is placed in a boiling water bath for 40 seconds, then spun in a centrifuge at 13,000 rpm for 10 minutes.
- the pelleted cell debris is removed using a disposable pipette tip, and the lysate solution added to 25mg of DEAE-MSPS (250 ⁇ l of 100 mg ml "1 suspension) in an Eppendorf tube.
- the suspension is mixed by end-over- end rotation for 30 minutes at 23°C, the MSPS magnetically immobilised and the supernatant removed.
- the DEAE-MSPS is washed twice with sterile distilled water, and then washed two times with O.lmM NaCl/50mM arginine free base to remove any oligoribonucleotides present in the lysis mixture which may become adsorbed to the MSPS.
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP93924712A EP0621802A1 (en) | 1992-11-06 | 1993-11-05 | Magnetic solid phase supports |
AU54273/94A AU5427394A (en) | 1992-11-06 | 1993-11-05 | Magnetic solid phase supports |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB929223334A GB9223334D0 (en) | 1992-11-06 | 1992-11-06 | Magnetic solid phase supports |
GB9223334.5 | 1992-11-06 |
Publications (1)
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WO1994011103A1 true WO1994011103A1 (en) | 1994-05-26 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/GB1993/002289 WO1994011103A1 (en) | 1992-11-06 | 1993-11-05 | Magnetic solid phase supports |
Country Status (4)
Country | Link |
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EP (1) | EP0621802A1 (en) |
AU (1) | AU5427394A (en) |
GB (1) | GB9223334D0 (en) |
WO (1) | WO1994011103A1 (en) |
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Also Published As
Publication number | Publication date |
---|---|
AU5427394A (en) | 1994-06-08 |
EP0621802A1 (en) | 1994-11-02 |
GB9223334D0 (en) | 1992-12-23 |
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