EP1025113A1 - Improved process for performing polynucleotide separations - Google Patents
Improved process for performing polynucleotide separationsInfo
- Publication number
- EP1025113A1 EP1025113A1 EP98923524A EP98923524A EP1025113A1 EP 1025113 A1 EP1025113 A1 EP 1025113A1 EP 98923524 A EP98923524 A EP 98923524A EP 98923524 A EP98923524 A EP 98923524A EP 1025113 A1 EP1025113 A1 EP 1025113A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- eluting solvent
- separation media
- polynucleotide
- solution
- separation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 238000000926 separation method Methods 0.000 title claims abstract description 192
- 102000040430 polynucleotide Human genes 0.000 title claims abstract description 116
- 108091033319 polynucleotide Proteins 0.000 title claims abstract description 116
- 239000002157 polynucleotide Substances 0.000 title claims abstract description 116
- 238000000034 method Methods 0.000 title claims abstract description 83
- 230000008569 process Effects 0.000 title description 23
- 239000002904 solvent Substances 0.000 claims abstract description 115
- 239000012634 fragment Substances 0.000 claims abstract description 94
- 150000001768 cations Chemical class 0.000 claims abstract description 93
- 239000011148 porous material Substances 0.000 claims abstract description 61
- 239000000203 mixture Substances 0.000 claims abstract description 53
- 239000000463 material Substances 0.000 claims abstract description 48
- 229910052751 metal Inorganic materials 0.000 claims abstract description 46
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- 239000000356 contaminant Substances 0.000 claims abstract description 20
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- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 claims description 24
- 239000010936 titanium Substances 0.000 claims description 24
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- 239000001301 oxygen Substances 0.000 claims description 9
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- FFUAGWLWBBFQJT-UHFFFAOYSA-N hexamethyldisilazane Chemical compound C[Si](C)(C)N[Si](C)(C)C FFUAGWLWBBFQJT-UHFFFAOYSA-N 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 7
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000011260 aqueous acid Substances 0.000 claims description 4
- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 claims description 4
- 150000002430 hydrocarbons Chemical group 0.000 claims 2
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical class Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 11
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 11
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 10
- 238000010828 elution Methods 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 229910021645 metal ion Inorganic materials 0.000 description 10
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- 229920000642 polymer Polymers 0.000 description 10
- 238000000527 sonication Methods 0.000 description 10
- VKIGAWAEXPTIOL-UHFFFAOYSA-N 2-hydroxyhexanenitrile Chemical compound CCCCC(O)C#N VKIGAWAEXPTIOL-UHFFFAOYSA-N 0.000 description 9
- 239000002253 acid Substances 0.000 description 9
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- 235000013980 iron oxide Nutrition 0.000 description 4
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- 229920000573 polyethylene Polymers 0.000 description 4
- MCJGNVYPOGVAJF-UHFFFAOYSA-N quinolin-8-ol Chemical compound C1=CN=C2C(O)=CC=CC2=C1 MCJGNVYPOGVAJF-UHFFFAOYSA-N 0.000 description 4
- 238000010561 standard procedure Methods 0.000 description 4
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- 239000005725 8-Hydroxyquinoline Substances 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical group CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 150000003983 crown ethers Chemical class 0.000 description 2
- NNBZCPXTIHJBJL-UHFFFAOYSA-N decalin Chemical compound C1CCCC2CCCCC21 NNBZCPXTIHJBJL-UHFFFAOYSA-N 0.000 description 2
- 238000007872 degassing Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
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- 238000001914 filtration Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000004190 ion pair chromatography Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 229910052755 nonmetal Inorganic materials 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229960003540 oxyquinoline Drugs 0.000 description 2
- 238000005191 phase separation Methods 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 229940005657 pyrophosphoric acid Drugs 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
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- 239000004094 surface-active agent Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000000825 ultraviolet detection Methods 0.000 description 2
- SNUSZUYTMHKCPM-UHFFFAOYSA-N 1-hydroxypyridin-2-one Chemical compound ON1C=CC=CC1=O SNUSZUYTMHKCPM-UHFFFAOYSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
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- ISWQCIVKKSOKNN-UHFFFAOYSA-L Tiron Chemical compound [Na+].[Na+].OC1=CC(S([O-])(=O)=O)=CC(S([O-])(=O)=O)=C1O ISWQCIVKKSOKNN-UHFFFAOYSA-L 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
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- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
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- 238000006731 degradation reaction Methods 0.000 description 1
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- 239000001307 helium Substances 0.000 description 1
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- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 208000016861 hereditary angioedema type 3 Diseases 0.000 description 1
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- 239000011261 inert gas Substances 0.000 description 1
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- VBMVTYDPPZVILR-UHFFFAOYSA-N iron(2+);oxygen(2-) Chemical class [O-2].[Fe+2] VBMVTYDPPZVILR-UHFFFAOYSA-N 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 229910052759 nickel Inorganic materials 0.000 description 1
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical group OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- GGOZGYRTNQBSSA-UHFFFAOYSA-N pyridine-2,3-diol Chemical group OC1=CC=CN=C1O GGOZGYRTNQBSSA-UHFFFAOYSA-N 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- QEMXHQIAXOOASZ-UHFFFAOYSA-N tetramethylammonium Chemical compound C[N+](C)(C)C QEMXHQIAXOOASZ-UHFFFAOYSA-N 0.000 description 1
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 1
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- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical group CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
- B01D15/166—Fluid composition conditioning, e.g. gradient
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/265—Adsorption chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
Definitions
- This invention is directed to the separation of polynucleotide fragments by liquid chromatography. More specifically, the invention is directed to a system and method, which enhances the chromatographic separation of polynucleotides on non-polar, wide pore separation media.
- Samples containing mixtures of polynucleotides can result from total synthesis of polynucleotides, cleavage of DNA with restriction endonucleases or RNA, as well as polynucleotide samples which have been multiplied or amplified using polymerase chain reaction (PCR) techniques or other amplifying techniques.
- PCR polymerase chain reaction
- MIPC Matched Ion Polynucleotide Chromatography
- the invention of parent application Serial No. 08/748,376 is based on the discovery that trace levels of multivalent metal ions, even when present below the limits of detection, interfere with the MIPC separation process. Special steps to prevent, remove or complex any trace multivalent ions result in enhanced separation of polynucleotides and lower the detection threshold.
- the invention provides an improved method for separating a mixture of polynucleotide fragments wherein multivalent cations are eliminated from the all aspects of the separation process.
- the method comprises applying a solution of said fragments and counterion agent to a column containing separation media having a non-polar surface, wherein said
- separation media have a pore size greater than 30 Angstroms and an
- the method further comprises contacting the solution of said fragments and the eluting solvent with a multivalent cation capture resin to remove any multivalent cations therein before entering the column.
- the separation media have
- An optimum embodiment of the invention comprises contacting the solution of said fragments and eluting solvent with a multivalent cation capture resin before entering the column, treating the separation media to remove residual traces of multivalent cations from the surfaces therefrom, and ensuring that surfaces which are contacted by the solution of the fragments and the eluting solvent are materials which do not release
- the polynucleotide fragments are double stranded, having more than 5 base pairs. Such fragments are separated by size or by polarity.
- the polynucleotide fragments are single stranded having 2 or more nucleotides. Such fragments are separated by size and by polarity.
- the separation media are organic polymer, or an inorganic substrate selected from the group consisting of inorganic substrates, silica, zirconia, and alumina.
- the inorganic substrates support a non-polar material on their surface. Said non-polar material may be organic polymer or long chain, C1 to C24 hydrocarbon groups bound to the inorganic substrate, wherein residual polar groups of the substrate are end capped with trimethylsilyl chloride or
- surfaces which are contacted by the solution of polynucleotide fragments and eluting solvent are titanium, coated stainless steel, organic polymer or combinations thereof. Removal of traces of residual multivalent metal cations from the separation process is further ensured by treating said surfaces with a solution comprising aqueous acid and chelating agent, by adding a chelating agent to the solution of polynucleotide mixture and eluting solvent, and by treating the eluting solvent to remove oxygen therefrom.
- the improved method for separating said mixture of polynucleotides comprises Matched Ion Polynucleotide Chromatography.
- the improved method of the invention may also be practiced as a
- the batch process method of the invention comprises applying
- the method further comprises contacting the separation media with a first eluting solvent and counterion agent, the first eluting solvent having a concentration of organic component sufficient to release from the separation media all polynucleotide fragments having a size smaller than the selected size and removing the first eluting solvent from the separation media.
- the selected size fragments are obtained by contacting the separation media with a second eluting solvent having a concentration of organic component sufficient to release from the separation media the polynucleotide fragments having the selected size and removing the second eluting solvent from the separation media.
- a second eluting solvent having a concentration of organic component sufficient to release from the separation media the polynucleotide fragments having the selected size and removing the second eluting solvent from the separation media.
- surfaces which are contacted by the solution of polynucleotide fragments and the eluting solvent are material which does not release multivalent metal cations therefrom.
- the separation media are rinsed with fresh first eluting solvent to remove residual released polynucleotide fragments therefrom.
- the separation media are rinsed with fresh second eluting solvent to remove residual released polynucleotide fragments of selected size therefrom.
- a preferred embodiment of the invention comprises contacting the solution of polynucleotide mixture and eluting solvent with a multivalent cation capture resin before contacting the separation media.
- the method comprises treating the separation media to remove residual traces of multivalent cations therefrom.
- the separation media comprises treating the separation media to remove residual traces of multivalent cations therefrom.
- separation media have been treated to remove residual traces of multivalent cations therefrom and the solution of polynucleotide mixture and eluting solvent have been contacted with a multivalent cation capture resin before contacting the separation media.
- Said separation media are contained in a column, a web, a membrane, or container.
- the batch process can be used to separate mixtures of double stranded polynucleotides or single stranded polynucleotides.
- the separation media are organic polymer, or an inorganic substrate selected from the group consisting of inorganic substrates, silica, zirconia, and alumina.
- the inorganic substrates support a non-polar material on their surface.
- Said non-polar material may be organic polymer or long chain, C1 to C24 hydrocarbon groups bound to the inorganic substrate, wherein residual polar groups of the substrate are end capped with trimethylsilyl chloride or hexamethyldisilazane.
- the surfaces contacted by the solution of polynucleotide fragments and eluting solvent are, preferably, comprised of material selected from the group consisting of titanium, coated stainless steel, and organic polymer, or combinations thereof. Removal of traces of residual multivalent metal cations from the separation process is further ensured by treating said surfaces with a solution comprising aqueous acid and chelating agent, by adding a chelating agent to the solution of polynucleotide mixture and eluting solvent, and by treating the eluting solvent to remove oxygen therefrom.
- FIG. 1 shows a guard disk having a one-piece annular ring.
- FIG. 2 is an exploded view of a guard disk having a two-piece annular
- guard disk material i.e., a layer or pad of multivalent cation capture resin which has been incorporated into a fabric or membrane.
- FIG. 3 shows an assembled view of the guard disk of FIG. 2.
- FIG. 4 shows placement of a chelating guard column and chelating guard disk in a liquid chromatographic system for polynucleotide separation.
- FIG. 5 shows placement of a chelating guard disk positioned between a chromatographic separation column and a column top, where the guard disk is in direct contact with a titanium frit at the top portion of the separation column.
- FIG 6. shows the chromatography of a 500 base pair DNA fragment on
- PRX-1 (Sarasep, San Jose, CA) non-polar, wide pore polymer separation
- FIG. 7 shows the effect on the chromatography result shown in FIG. 6 when Cr(lll) cations were added to the column before sample injection.
- FIG. 8 shows the effect on the chromatography result of FIG. 7 when a Cr(lll) contaminated column was treated with EDTA prior to sample injection.
- FIG 9. shows the chromatography of a 500 base pair DNA fragment on INERTSIL (MetaChem, Torrance, CA) C-18 non-polar, wide pore silica separation media which have been washed with EDTA to remove multivalent metal cations thereform.
- INERTSIL MetalChem, Torrance, CA
- FIG. 10 shows the effect on the chromatography result shown in FIG. 9
- FIG. 11 shows the effect on the chromatography result of FIG. 10 when a Cr(lll) contaminated column was treated with EDTA prior to sample injection.
- FIG .12 shows the chromatography of a 20 mer single stranded DNA fragment standard (Seq 2A, CTGen, Milpitas, CA) on INERTSIL (MetaChem, Torrance, CA) C-18 non-polar, wide pore silica separation media which have been washed with EDTA to remove multivalent metal cations thereform.
- FIG. 13 shows the effect on the chromatography result shown in FIG. 12 when Cr(lll) cations were added to the column before sample injection.
- FIG. 14 shows the chromatographic separation at 56 °C of a 209 base
- DNA standard 4 component heteroduplex and homoduplex mixture on a freshly packed, untreated DNASep column (Transgenomic, Inc., San Jose, CA).
- FIG. 15 shows the effect on the chromatography result shown in FIG 14 when the column was treated with EDTA prior sample injection.
- polynucleotide as used herein, is defined as a linear polymer containing an indefinite number of nucleotides, linked from one ribose (or deoxyribose) to another via phosphate residues.
- the present invention can be used in the separation of RNA or of double or single stranded DNA. For purposes of simplifying the description of the invention and not by way of limitation, the separation of double stranded DNA will be described hereinafter, it being understood that all polynucleotides are intended to be included within the scope of this invention.
- the present invention is an improved chromatographic and batch process method for separating mixtures of polynucleotide fragments on wide pore non-polar separation media.
- the improvement comprises ensuring the removal of all traces of residual multivalent metal cations from the sample mixture, the eluting solvent as well as components of the chromatographic or batch process equipment which contact said sample mixture and eluting solvent.
- the system used to implement the method of the invention comprises a liquid chromatographic system.
- the liquid chromatography system comprises a column containing a separation bed of non-polar, wide pore separation media held in the column between porous frits positioned at each end of the column.
- Other components of the liquid chromatography system are also components of the liquid chromatography system.
- Eluting solvent supply means is (are) connected to the injection valve, and the injection valve is connected to the inlet of the chromatographic separation column, by means of conduit (e.g., tubing), as illustrated in FIG. 5.
- conduit e.g., tubing
- the mixture of polynucleotide fragments is separated by Matched Ion Polynucleotide Chromatography (MIPC).
- MIPC Matched Ion Polynucleotide Chromatography
- MIPC Matched Ion Polynucleotide Chromatography
- the pores of the separation media may be contiguous, i.e., extend from one surface of the media to another surface of the media.
- the pores of the separation media may also be non-contiguous, i.e., extend into the media at one point on the surface but not through to another point on the surface.
- the non-polar, wide pore separation media can be an inorganic substrate, including silica, zirconia, alumina, or other material; or can be polymeric, including crosslinked resins of polystyrene, polyacrylates, polyethylene, or other organic polymeric material.
- the non-polar, wide pore separation media can also be a "rod column” or “monolith column”. Such columns contain silica or polymer separation media which have been formed inside the column as a continuous structure which has through pores or interstitial spaces which allow eluting solvent and analyte to pass through. The only requirement for the non-polar, wide pore separation media is that
- non-polar, wide pore separation medium is defined to denote any material which has surface pores having a diameter that is greater than 30 Angstroms or the approximate size and shape of the smallest polynucleotide fragment in the separation in the solvent medium used therein or greater, and is capable of separating polynucleotide fragments.
- the non-polar, wide pore separation medium may be any shape, those comprising alkylated wide pore polymer beads bead having an average diameter of 1-100 microns are preferred. Such particles are described in the references cited hereinbelow.
- the separation media are washed with acid followed by methanol to ensure removal of residual multivalent cation contaminants.
- the separation media can also be washed with EDTA or other chelating agent.
- Non-polar, wide pore separation media and their use for the separation of polynucleotide mixtures are well known in the art and are commercially
- Monolith or rod columns are commercially avialable form Merck & Co (Darmstadt, Germany) and described in the following references: U.S. Patent No. 5453185 to J. M. J. Frechet and F. Svec; M. Petro, et. al., Analytical Chemistry, 68, 315-321 (1996).
- the references cited above and the references contained therein are incorporated in their entirety herein.
- the components of the liquid chromatography system have surfaces (i.e., "process solution contacting surfaces") which contact process solutions held within the components (e.g., the eluting solvent supply means) or flowing through the components (e.g., the porous frits, chromatographic column, injection valve, and conduits).
- process solution as used herein
- process solution contacting surface refers to any surface of a liquid chromatography system which contacts said process solutions.
- ⁇ J3 release multivalent cations into solutions flowing through the column, or collect said cations from other sources.
- the material is preferably titanium, coated stainless steel, or organic polymer, or combinations thereof, but is most preferably acid treated titanium as described hereinbelow.
- coated stainless steel refers to stainless steel that has been coated so that it does not release, or is prevented from releasing, multivalent cations.
- a non-limiting example of a coating material is polytetrafluoroethylene (i.e., Teflon®).
- Teflon® polytetrafluoroethylene
- Coated stainless steel as used herein also refers to stainless steel that has been pre-treated with an agent such as EDTA or phosphoric acid which forms coordination complexes with multivalent metal ions.
- Passivated stainless steel refers to stainless steel that has been treated with an agent that removes oxidized metals and also metals that are easily oxidized such as iron.
- the most common passivating agent for stainless steel is nitric acid. Nitric acid will removed any oxidized metals, but will also remove iron that is located on the surface of the metal, leaving other metals such as chromium and nickel.
- Some chelating agents can coat and passivate. EDTA will first coat oxidized metals especially colloidal iron oxide particles. As treatment continues, the EDTA will bind and dissolve the iron oxide. However, as individual iron molecules leave the particle, other chelating molecules must coat the newly exposed surfaces for the surface to
- a chelating agent does not
- a chelating agent may, eventually, dissolve oxidized metals.
- the chelating agents used depend upon the type of ion contamination which is present. For example, Tiron chelating agent is selective for titanium and iron oxides. EDTA is selective for most metal oxides at pH 7.
- Other chelating agents include cupferron, 8 hydroxyquinoline, oxine, and various iminodiacetic acid derivatives.
- the metal ion chelate complex for example, EDTA-metal ion complex
- the metal ion chelate complex is soluble in the fluid.
- Chelating agents that form insoluble complexes for example 8- hydroxyquinoline, perform coating functions only.
- oxidized and positively charged metals such as oxides of iron on the surface of stainless steel can trap negatively charged molecules such as DNA leading to degradation of the chromatographic separation, and that the pre-treatment masks or shields these surface charges.
- EDTA can be added, for example, in an amount sufficient to shield any surface sites which would interfere with the chromatographic separation.
- a solution of a metal chelating agent such as EDTA can be applied in a batch process to coat the surface, for example by a single injection of EDTA solution into the HPLC system.
- EDTA is included as an additive in the eluting solvent in an amount sufficient to complex the metal ions present.
- components of the liquid chromatography system are preferably titanium, coated stainless steel, or organic polymer such as polyetherether
- the preferred eluting solvent inlet filters are composed of non-polar, porous,
- non-stainless steel material which can be PEEK, polyethylene, or other polymeric material.
- the preferred solvent pump is also made of a non- stainless steel material; the pump heads, check valves, and solvent filters are preferably titanium, PEEK, or other polymeric material.
- the preferred means for removing oxygen from the eluting solvent is an inline degasser placed prior to the pump inlet.
- the sample injection valve is also preferably titanium, PEEK, or other polymeric material.
- a standard detector and eluting solvent reservoirs can be used, with no modifications necessary.
- all of the process solution-contacting surfaces are subjected to a multivalent cation removal
- a non-limiting example of a multivalent cation removal treatment is an acid wash treatment.
- This wash treatment can include flushing or soaking and can include sonication.
- An example of an acid wash treatment is
- Other treatments include contacting the surfaces with chelating agents such as EDTA, pyrophosphoric acid, or phosphoric acid (e.g. 30% by weight phosphoric acid).
- PEEK and titanium can be treated with dilute acids including nitric and hydrochloric acids.
- PEEK is not compatible with concentrated sulfuric or concentrated nitric acids.
- Titanium is not compatible with concentrated hot hydrochloric acid.
- Treatment with a chelating agent can be performed before, but preferably after treatment with an acid. 20 mM tetrasodium EDTA is a preferred chelating agent treatment.
- the preferred treatment for titanium frits is sonication for 10 minutes with cold hydrochloric acid, sonication with water until neutral pH, 2 hour sonication with 0.5 M tetrasodium EDTA, storage several days in 0.5 M tetrasodium EDTA, sonication with water until neutral pH, and then washing with methanol, followed by drying.
- Preferred treatment for PEEK frits is sonication for 15-30 minutes each with THF, concentrated hydrochloric acid, 20% nitric acid, sonication with water until neutral pH, and then washing with
- the ionic contaminant is organic, then organic solvents or a combination of
- organic solvents and acids can be used. Also, organic ionic contaminants
- Nonionic contaminants such as greases and oils will also contaminate the separation column, generally leading to poor peak shape, but depending upon the size of the fragment.
- Nonionic organic contaminants such as oils will require detergents, soaps or surfactants to remove.
- Column tubing can be treated under sonication with Decalin (D5039, Sigma) to remove silicon greases and oils. Removal of colloidal metal oxides such as colloidal iron oxide can require repeated or continuous treatment as the surface of the particle is dissolved and new metal oxides are exposed.
- the preferred embodiment of the liquid chromatography system of the present invention utilizes methods to minimize the exposure of all process solution contacting surfaces to oxygen. Dissolved oxygen within the eluting
- the liquid chromatography system preferably employs a degassing method for essentially removing dissolved oxygen from the eluting solvent prior to contact with the rest of the chromatography system.
- degassing methods include sparging of the eluting solvent with an inert gas such as argon or helium, or filtering the eluting solvent under vacuum.
- a preferred method uses a vacuum type degasser which employs inline
- a stainless steel HPLC system can be used if a component for removing multivalent cations, herein referred to as a "multivalent cation capture resin," is also used.
- a multivalent cation capture resin is preferably a cation exchange resin or chelating resin. Any suitable cation exchange resin or chelating resin can be used. Preferred cation exchange and chelating resins are described hereinbelow.
- Cation exchange resins having an ion exchange moiety selected from the group consisting of iminodiacetate, nitriloacetate, acetylacetone, arsenazo, hydroxypyridinone, and 8-hydroxyquinoline groups are particularly preferred.
- Cation exchange resins having hydroxypyridinone groups are especially useful for removing iron from the system.
- Cation exchange resins having iminodiacetate groups are particularly preferred for use in the present invention because of their wide availability in resin format.
- a chelating (i.e., coordination binding) resin is an organic compound which is capable of forming more than one non-covalent bond with a metal.
- Chelating resins include iminodiacetate and crown ethers. Crown ethers are
- cyclic oligomers of ethylene oxide which are able to interact strongly with alkali or alkaline earth cations and certain transition metal cations.
- a cavity in the center of the molecule is lined with oxygen atoms which hold cations by electrostatic attraction.
- Each ether has a strong preference for cations whose ionic radius best fits the cavity.
- the multivalent cation capture resin is preferably contained in a guard
- guard cartridge or guard disk.
- Guard columns and cartridges are
- the guard column or cartridge typically contain packing material which is similar to the stationary phase of the separation column.
- the guard column or cartridge must contain a multivalent cation capture resin.
- the guard disc or guard column must contain particles which trap the metal ions.
- a preferred guard cartridge has a void volume of less than 5 mL, more
- the preferred cartridge has a 10 x 3.2 mm bed volume.
- guard disks are described in detail in U.S. Patent No. 5,338,448, which is incorporated herein by reference in its entirety.
- a guard disk comprises a layer or pad of a multivalent
- the guard disk is circular, having a rigid annular outer ring or collar for easy handling.
- the annular ring can be constructed of any suitable material which is inert to the chromatographic separation, such as inert conventional engineering plastic. The only requirement for the material is that it must be inert to the eluting solvent and sample and have
- guard disk material refers to a layer or pad of multivalent cation capture resin which has been incorporated
- one or more pads of guard disk material 2 are
- the fabric can be cut to a
- the rigid annular outer ring can comprise two flanged rings, as shown in FIG. 2 and 3, an outer flanged ring 6 and an inner flanged
- the inner flanged ring 8 is then inserted into the outer ring to form
- the guard disk pad(s) is (are) frictionally held within the press-fit ring or collar.
- the inner diameter (b) of the outer flanged ring and the inner diameter (a) of the inner flanged ring are substantially the same.
- the rigid annular outer ring can be incorporated into the guard disk holder or chromatographic column cap.
- the annular ring is a flange that is part of one or both sides of the disk holder or the column cap.
- the guard disk does not have an outer ring.
- a circle of the guard disk sheet material is placed into the holder or column cap.
- the flange in the holder column cap is annular so that, when the holder or column cap is tightened, the flange pinches or seals the outer annular portion of the guard disk.
- the center portion of the guard disk not pinched is in a chamber or depression in the holder or cap.
- a multivalent cation capture resin contained in a guard column, guard cartridge, or guard disk is placed upstream of the separation column.
- the guard column, cartridge, or disk containing the resin is placed upstream of the
- a guard disk a guard cartridge or column can be used as long as the dead volume of the cartridge or column is not excessive and an effective eluting solvent gradient can be produced.
- a guard disk, column, or cartridge can be placed before the injection valve and a second guard disk, column, or cartridge also placed between the sample injection valve and the separation column.
- the second guard disk (or cartridge or column) can be avoided if the contaminants are sufficiently cleaned by a guard column placed upstream of the injection valve, or if the contaminants are avoided through the use of non- metal or titanium components throughout the HPLC system.
- the eluting solvent reservoirs 12 contain eluting solvent inlet filters 14
- solvent pump 16 is connected to a chelating column 20 by system tubing 18.
- the chelating column 20 is connected to the sample injection valve 22 by
- the sample injection valve has means for injecting a
- the sample injection valve 22 is connected to a
- the chelating guard disk 24 by system tubing 18.
- the chelating guard disk 24 is
- Detector 28 is connected to the separation column 26.
- the system tubing, eluting solvent inlet filters, solvent pump, sample injection valve, and separation column are preferably made of titanium, coated stainless steel, or organic polymer.
- the material is preferably treated so that it does not release multivalent cations.
- the treatment can include treatment with nitric acid, phosphoric acid,
- Detector 28 is located downstream from
- FIG. 5 illustrates a specific embodiment of the invention in which a chelating guard disk is placed in direct contact with a titanium frit at the top
- the column top 30 is adapted to receive the
- solvent pump 46 pumps elution solvent to sample
- transducer 50 which is electrically connected to a display device 52.
- a chelating guard column, cartridge, or disk can be used in conjunction with a conventional, stainless steel liquid chromatography system, or with a system containing non-metal or titanium
- washing solvents comprise tetrahydrofuran, hydrochloric acid, and water.
- An example of a preferred washing procedure is described in Examplel .
- the methods of the invention comprise using the improved systems described above to separate mixtures of polynucleotide fragments, particularly double-stranded polynucleotide fragments.
- the methods of the present invention can be used to separate polynucleotide fragments having up to about 1500 base pairs using non-polar, wide pore separation media under the chromatography conditions described herein.
- the most preferred method of the invention comprises contacting a
- a guard cartridge or guard column containing multivalent cation capture resin is placed at the front of the column or in line between the eluting
- the polynucleotide fragments are separated by releasing said fragments from the separation media using an eluting solvent comprising an organic component, water, and a counterion agent.
- the separation of the polynucleotide components is based on the size or polarity of the fragments.
- the fragments are released from the separation media in order of size by increasing the concentration of organic component in the eluting solvent.
- the concentration of the organic component can be increased in stepwise fashion by means of a step gradient, or continuously, by means of a continuous gradient.
- non-polar, wide pore silica and polymer separation media is presented in Examples 2 - 7 and FIGs. 6 - 15.
- Example 2 describes a polynucleotide fragment separation on non- polar, wide pore organic polymer separation media using the optimized
- Example 3 is identical to Example 2, except that non-polar, wide pore silica separation media were used in the chromatography.
- Example 4 a series of three separations of a 500 base pair DNA fragment was performed using non-polar, wide pore polymer separation media are described. In the first separation, the separation media was
- Example 5 describes an sequence similar to Example 4, except that non-polar, wide pore silica separation media was used and washed with EDTA solution prior to sample injection. Once again, a sharp sample peak
- Example 6 chromatographic separation of a 20 mer single stranded DNA standard is described in Example 6 and shown by the complete loss of resolution as seen in FIG. 13 (after deliberate Cr (III) contamination) compared to FIG. 12 (column cleaned with EDTA).
- a standard 4 component mixture of double stranded DNA consisting of two 209 base pair homoduplex fragments and two 209 base pair
- heteroduplex fragments were chromatographed at 56 °C as described in
- Example 6 on a freshly packed column DNASep column (Transgenomic, Inc., San Jose, CA).
- FIG. 14 shows only partial resolution of the 4 component mixture.
- EDTA solution when the column was treated with EDTA solution followed by re-injection and elution of the 4 component homoduplex/heteroduplex DNA mixture as described in Example 6, a clean separation of all 4 components was achieved, as seen in FIG. 15.
- This result clearly indicates that trace levels of multivalent cations were present in a freshly packed column, and that said cations interfered with the separation of double stranded DNA fragments.
- the method of the invention can also be used to separate
- polynucleotide mixtures in a batch process useful for production and isolation of pure polynucleotide fragments of a plurality of selected sizes, on a small or
- the method of the invention comprises contacting a solution of a
- the separation media are held in a container.
- the container may be a column, a membrane, a container, or a
- the polynucleotide mixture is held on the separation media since the concentration of the organic component of the solvent in which the mixture is dissolved is not sufficient to release the polynucleotide fragments therefrom.
- the separation media are then contacted with a first eluting solvent and a counterion agent, said first eluting solvent having a concentration of the organic component sufficient to remove all polynucleotide fragments from the separation media which are smaller than the selected size.
- the eluting solvent is then separated from the separation media.
- the separation media are rinsed with the first eluting solvent to remove any remaining released polynucleotides.
- the separation media are then contacted with a second eluting solvent and counterion agent, said second eluting solvent having a concentration of the organic component sufficient to release the polynucleotide fragment having the selected size from the separation media.
- the second eluting solvent is separated from the separation media and the particles are rinsed with the second eluting solvent. This process can be repeated to release polynucleotide of any selected size which are present in the mixture.
- fragments of any specific base pair length can be determined experimentally. For example, isolation of a 102 base pair fragment from the polynucleotide mixture may be desired, and said fragment may be eluted with 15.9%
- the separation media holding the polynucleotide mixture may be contacted with
- Non-polar, wide pore separation media and their use for the separation of polynucleotide mixtures are well known in the art and are commercially available, e.g., Hamilton HPLC Application Handbook, (1993), Hamilton Company, Inc., 4970 Energy Way, Reno, Nevada 89502. This, and references contained therein, are incorporated in their entirety herein.
- Another reference, which is incorporated in its entirety herein, describing polynucleotide separations on non-polar, wide pore reverse phase particles is Chromatography, 5 th edition, Part B, edited by E. Heftmann, Elsevier (1992). Separation of tRNA and DNA fragment mixtures on non-polar, wide pore silica particles is described by R. Bischoff and L.W.
- the non-polar, wide pore reverse phase separation media were washed three times with tetrahydrofuran, then two times with methanol.
- the non-polar, wide pore separation media were then stirred for 12 hours with a mixture containing 100 mL of tetrahydrofuran and 100 mL of concentrated hydrochloric acid.
- the non-polar, wide pore separation media were washed with tetrahydrofuran/water (1 :1) until neutral
- Non-polar, Wide pore Polymer Separation Media Non-polar, wide pore PRX-1 separation media (Sarasep, Inc. San Jose, CA.) of polystyrene/divinylbenzene polymer having a pore size of 50 - 200 Angstroms (average pore size is 80 Angstroms) and a bead diameter of 5 microns are washed as described in Example 1 and packed in a 4.6 x 50 mm
- the flow rate is 0.75 mL/min, UV detection at 260 nm, column temp.
- colloidal iron suspension is injected onto the column and allowed to stand for
- INERTSIL MetalChem, Torrance, CA
- Example 4 Standard Procedure for Demonstrating the Effects of Chromium(lll) On The Separation of Double Stranded DNA Using Non-polar, Wide Pore
- Non-polar, wide pore PRX-1 separation media (Sarasep, Inc., San
- Example 2 a pore size of 50 - 200Angstroms (80 Angstrom average pore size) was washed as described in Example 1 and packed in 4.6 x 50 mm HPLC column.
- the flow rate was 0.60 mL/min, UV detection at 260 nm, column
- a 520 ppm aqueous solution of Cr(lll) was prepared from Cr 3 (S0 4 ) 2 .
- Example 4 showed a peak (FIG. 11)corresponding to the 500 base pair DNA standard
- Example 5 Using Non-polar, Wide Pore Silica Separation Media The column described in Example 5 was cleaned with Na 4 EDTA as decribed in Example 5 and equilibrated with 60%A eluting solvent to a constant bse line. A 20 mer, single stranded DNA standard (Seq2A purchased from CTGen, Milpitas, CA) was injected onto the column and eluted with the gradient protocol of Example 4.
- FIG 12 shows a major peak corresponding to the 20 mer standard and some well resolved impurity peaks.
- Example 4 A single 5 mL injection of the Cr(lll) solution described in Example 4 was followed by re-injection of the 20 mer standard and elution using the gradient protocol of Example 4. The results seen in FIG. 13 show a greatly diminished peak corresponding to the 20 mer, and essentially no resolution of the impurities.
- a freshly packed DNASep column (Transgenomic, Inc., San Jose, CA) was equilibrated with eluting solvent 50%A.
- a mixture of DNA 209 base pair standard fragments (Transgenomic, Inc., San Jose, CA, Cat. No. 560012) consisting of 2 heteroduplex DNA fragmetns and 2 homoduplex DNA fragments was injected (5 ⁇ L) onto the column. The mixture was eluted at a
- FIG. 14 shows the results of a chromatography run at 56 °C, on a
- cation contamination is more critical at higher column temperatures, especially as related to the difficult separation of homoduplex and heteroduplex fragments of identical base pair length.
Abstract
Description
Claims
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US6576133B2 (en) | 1996-11-13 | 2003-06-10 | Transgenomic, Inc | Method and system for RNA analysis by matched ion polynucleotide chromatography |
US6475388B1 (en) | 1996-11-13 | 2002-11-05 | Transgenomic, Inc. | Method and system for RNA analysis by matched ion polynucleotide chromatography |
US7138518B1 (en) | 1996-11-13 | 2006-11-21 | Transgenomic, Inc. | Liquid chromatographic separation of polynucleotides |
US6258264B1 (en) | 1998-04-10 | 2001-07-10 | Transgenomic, Inc. | Non-polar media for polynucleotide separations |
WO2001066216A1 (en) * | 2000-03-09 | 2001-09-13 | Transgenomic, Inc. | Method and system for rna analysis by matched ion polynucleotide chromatography |
WO2001081566A2 (en) * | 2000-04-21 | 2001-11-01 | Transgenomic, Inc. | Apparatus and method for separating and purifying polynucleotides |
US6521411B2 (en) * | 2000-09-28 | 2003-02-18 | Transgenomic, Inc. | Method and system for the preparation of cDNA |
WO2002040130A1 (en) * | 2000-11-16 | 2002-05-23 | Transgenomic, Inc. | Liquid chromatographic separation of polynucleotides |
US20020086291A1 (en) * | 2000-11-29 | 2002-07-04 | Hornby David P. | Methods and reagents for analysis of rna structure and function |
WO2003031580A2 (en) | 2001-10-05 | 2003-04-17 | Transgenomic, Inc. | Methods and compositions for mutation analysis by liquid chromatography |
WO2004047981A1 (en) * | 2002-11-25 | 2004-06-10 | Varian, Inc. | Irregularly-shaped macroporous copolymer particles and methods of using same |
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US5482628A (en) * | 1994-04-15 | 1996-01-09 | Upchurch Scientific, Inc. | Column for liquid chromatography |
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AT398973B (en) * | 1992-11-18 | 1995-02-27 | Bonn Guenther Dr | METHOD FOR SEPARATING NUCLEIC ACIDS |
-
1998
- 1998-05-19 WO PCT/US1998/010228 patent/WO1998056798A1/en not_active Application Discontinuation
- 1998-05-19 JP JP50248799A patent/JP2001524133A/en active Pending
- 1998-05-19 CA CA002288813A patent/CA2288813A1/en not_active Abandoned
- 1998-05-19 AU AU75796/98A patent/AU743789B2/en not_active Ceased
- 1998-05-19 EP EP98923524A patent/EP1025113A4/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5338448A (en) * | 1992-10-16 | 1994-08-16 | Sarasep, Inc. | Method of preventing contamination of a chromatography column |
US5482628A (en) * | 1994-04-15 | 1996-01-09 | Upchurch Scientific, Inc. | Column for liquid chromatography |
Non-Patent Citations (1)
Title |
---|
See also references of WO9856798A1 * |
Also Published As
Publication number | Publication date |
---|---|
CA2288813A1 (en) | 1998-12-17 |
WO1998056798A1 (en) | 1998-12-17 |
JP2001524133A (en) | 2001-11-27 |
AU743789B2 (en) | 2002-02-07 |
EP1025113A4 (en) | 2002-01-09 |
AU7579698A (en) | 1998-12-30 |
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