EP1231993A4 - Colonne amelioree pour la separation de l'adn en chromatographie de nucleotides a ions apparies (mipc) - Google Patents

Colonne amelioree pour la separation de l'adn en chromatographie de nucleotides a ions apparies (mipc)

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Publication number
EP1231993A4
EP1231993A4 EP00961961A EP00961961A EP1231993A4 EP 1231993 A4 EP1231993 A4 EP 1231993A4 EP 00961961 A EP00961961 A EP 00961961A EP 00961961 A EP00961961 A EP 00961961A EP 1231993 A4 EP1231993 A4 EP 1231993A4
Authority
EP
European Patent Office
Prior art keywords
column
beads
separation
dna
fragments
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00961961A
Other languages
German (de)
English (en)
Other versions
EP1231993A1 (fr
Inventor
Douglas T Gjerde
Robert M Haefele
Karl H Hecker
Raquel Roque
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.)
Precipio Inc
Original Assignee
Transgenomic Inc
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 US09/501,775 external-priority patent/US6372142B1/en
Application filed by Transgenomic Inc filed Critical Transgenomic Inc
Publication of EP1231993A1 publication Critical patent/EP1231993A1/fr
Publication of EP1231993A4 publication Critical patent/EP1231993A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/366—Ion-pair, e.g. ion-pair reversed phase
    • 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
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
    • 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
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • 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
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • 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
    • B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G01N2030/3084—Control of physical parameters of the fluid carrier of temperature ovens

Definitions

  • This invention relates to DNA separation systems and methods suitable for effecting a size-based (base pair length) separation of DNA.
  • the invention concerns an improved separation column for increasing the range of base pair length of the DNA fragments that can be separated by Matched Ion Polynucleotide Chromatography (MIPC) and for improving the separation of heteroduplex and homoduplex DNA using MIPC under partially denaturing conditions.
  • MIPC Matched Ion Polynucleotide Chromatography
  • DNA molecules are polymers comprising sub-units called deoxynucleotides.
  • the four deoxynucleotides found in DNA comprise a common cyclic sugar, deoxyribose, which is covalently bonded to any of the four bases, adenine (a purine), guanine (a purine), cytosine (a pyrimidine), and thymine (a pyrimidine), referred to herein as A, G, C, and T respectively.
  • a phosphate group links a 3'-hydroxyl of one deoxynucleotide with the 5'- hydroxyl of another deoxynucleotide to form a polymeric chain.
  • double stranded DNA two strands are held together in a helical structure by hydrogen bonds between what are called complimentary bases.
  • the complimentarity of bases is determined by their chemical structures.
  • double stranded DNA each A pairs with a T and each G pairs with a C, i.e., a purine pairs with a pyrimidine.
  • DNA is replicated in exact copies by DNA polymerases during cell division in the human body or in other living organisms.
  • DNA strands can also be replicated in vitro by means of the Polymerase Chain Reaction (PCR). Sometimes, exact replication fails and an incorrect base pairing occurs. Further replication of the new strand produces double stranded DNA offspring containing a heritable difference in the base sequence from that of the parent. Such heritable changes in base pair sequence are called mutations.
  • double stranded DNA is referred to as a duplex.
  • a duplex When a base sequence of one strand is entirely complimentary to a base sequence of the other strand, the duplex is called a homoduplex.
  • a duplex contains at least one base pair which is not complimentary, the duplex is called a heteroduplex.
  • a heteroduplex is formed during DNA replication when an error is made by a DNA polymerase enzyme and a non-complimentary base is added to a polynucleotide chain being replicated. Further replications of a heteroduplex will, ideally, produce homoduplexes which are heterozygous, i.e., these homoduplexes will have an altered sequence compared to the original parent DNA strand.
  • the parent DNA has a sequence which predominates in a naturally occurring population, the sequence is generally referred to as a "wild type".
  • DNA mutations include, but are not limited to, "point mutation” or “single base pair mutations” in which an incorrect base pairing occurs.
  • the most common point mutations comprise “transitions” in which one purine or pyrimidine base is replaced for another and “transversions” wherein a purine is substituted for a pyrimidine (and visa versa).
  • Point mutations also comprise mutations in which a base is added or deleted from a DNA chain.
  • Such "insertions” or “deletions” are also known as “frameshift mutations”. Although they occur with less frequency than point mutations, larger mutations affecting multiple base pairs can also occur and may be important.
  • frameshift mutations are also known as “frameshift mutations”.
  • the sequence of base pairs in DNA is a code for the production of proteins.
  • a DNA sequence in the exon portion of a DNA chain codes for a corresponding amino acid sequence in a protein. Therefore, a mutation in a DNA sequence may result in an alteration in the amino acid sequence of a protein. Such an alteration in the amino acid sequence may be
  • Detection of mutations is therefore of great importance in diagnosing diseases, understanding the origins of disease, and the development of potential treatments. Detection of mutations and identification of similarities or differences in DNA samples is also of critical importance in increasing the world food supply by developing diseases resistant and/or higher yielding crop strains, in forensic science, in the study of evolution and populations, and in scientific research in general (Guyer, et al., Proc. Natl. Acad. Sci. USA 92:10841 (1995); Cotton, TIG 13:43 (1997)). Alterations in a DNA sequence which are benign or have no negative consequences are sometimes called "polymorphisms". For the purposes of this application, all alterations in the DNA sequence, whether they have negative consequences or not, are defined herein as "mutations”.
  • mutation is used herein to mean an alteration in the base sequence of a DNA strand compared to a reference strand (generally, but not necessarily, a wild type).
  • mutation includes the term “polymorphism” or any other similar or equivalent term of art.
  • dsDNA double-stranded deoxyribonucleic acids
  • the stationary phase is provided by the surface of solid materials which can comprise many different materials in the form of particles or passageway surfaces of cellulose, silica gel, coated silica gel, polymer beads, polysaccharides, and the like. These materials can be supported on solid surfaces such as on glass plates or packed in a column.
  • the mobile phase can be a liquid or a gas in gas chromatography. This invention relates to liquid mobile phases.
  • the separation principles are generally the same regardless of the materials used, the form of the materials, or the apparatus used.
  • the different components of a mixture have different respective degrees of solubility in the stationary phase and in the mobile phase. Therefore, as the mobile phase flows over the stationary phase, there is an equilibrium in which the sample components are partitioned between the stationary phase and the mobile phase. As the mobile phase passes through the column, the equilibrium is constantly shifted in favor of the mobile phase. This occurs because the equilibrium mixture, at any time, sees fresh mobile phase and partitions into the fresh mobile phase. As the mobile phase is carried down the column, the mobile phase sees fresh stationary phase and partitions into the stationary phase. Eventually, at the end of the column, there is no more stationary phase and the sample simply leaves the column in the mobile phase.
  • a separation of mixture components occurs because the mixture components have slightly different affinities for the stationary phase and/or solubilities in the mobile phase, and therefore have different partition equilibrium values. Therefore, the mixture components pass down the column at different rates.
  • HPLC has enabled the use of a far greater variety of types of particles used to separate a greater variety of chemical structures than was possible with large particle gravity columns.
  • the separation principle is still the same.
  • MIPC Magnetic Ion Polynucleotide Chromatography
  • Matched Ion Polynucleotide Chromatography is defined as a process for separating single and double stranded polynudeotides using non-polar separation media, wherein the process uses a counter-ion agent, and an organic solvent to release the polynudeotides from the separation media.
  • MIPC separations can be complete in less than 10 minutes, and frequently in less than 5 minutes.
  • the MIPC separation process differs from the traditional HPLC separation processes in that the separation is not achieved by a series of equilibrium separations between the mobile phase and the stationary phase s as the liquids pass through the column. Instead, the sample is fed into the column using a solvent strength which permits the sample dsDNA to bind to the separation media surface. Strands of a specific base pair length are removed from the stationary phase surface and are carried down the column by a specific solvent concentration. By passing an increasing gradient of solvent through the sample, successively larger base pair lengths are removed in succession and passed through the column.
  • MIPC Magnetic Ink Characterization
  • a reliable way to detect mutations is by hybridization of the putative mutant strand in a sample with the wild type strand (Lerman, et al., Meth. Enzymol., 155:482 (1987)). If a mutant strand is present, then two homoduplexes and two heteroduplexes will be formed as a result of the hybridization process. Hence separation of heteroduplexes from homoduplexes provides a direct method of confirming the presence or absence of mutant DNA segments in a sample.
  • DMIPC Matched Ion Polynucleotide Chromatography
  • the hybridization process creates two homoduplexes and two heteroduplexes.
  • DMIPC can separate heteroduplexes that differ by as little as one base pair.
  • separations of homoduplexes and heteroduplexes are poorly resolved (e.g., as described by Liu et al. Nucleic Acids Res. 26: 1396-1400 (1998)).
  • the presence of mutations may even be missed entirely.
  • only two peaks or partially resolved peak(s) are observed in DMIPC analysis.
  • the two homoduplex peaks may appear as one peak or a partially resolved peak and the two heteroduplex peaks may appear as one peak or a partially resolved peak. In some cases, only a broadening of the initial peak is observed under partially denaturing conditions.
  • the invention concerns an improved separation column for separating a mixture of double stranded DNA fragments by Matched Ion Polynucleotide Chromatography (MIPC).
  • the mixture contains fragments having lengths exceeding about 1000 base pairs.
  • the column includes a cylinder having an ID greater than about 5 mm and containing polymer beads.
  • the beads have an average diameter of 1 to 100 microns and are unsubstituted polymer beads or are polymer beads substituted with a hydrocarbon moiety having from 1 to 1 ,000,000 carbons.
  • the preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA.
  • the column ID is greater than about 7 mm.
  • the column ID is greater than about 10 mm.
  • the column ID is greater than about 50 mm.
  • the column ID is in the range of about 5 mm to about 1 m.
  • the invention concerns an improved method for separating a mixture of double stranded DNA fragments by MIPC in which the mixture includes fragments having lengths exceeding about 1000 base pairs.
  • the method includes a first step (a) of applying a solution of the DNA fragments fragments and counterion reagent to separation beads.
  • the beads are retained within a separation column.
  • the column has an ID greater than about 5 mm.
  • the beads have an average diameter of 1 to 100 microns, and are composed of unsubstituted polymer beads or polymer beads substituted with a hydrocarbon moiety having from 1 to 1 ,000,000 carbons.
  • the preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA.
  • Step (b) involves eluting the fragments with a gradient of eluting solvent of increasing organic component concentration containing a counterion agent.
  • surfaces which are contacted by the solution of the fragments and the eluting solvent are materials which do not trap or release multivalent metal cations therefrom.
  • the eluting is carried out under conditions effective to at least partially denature the heteroduplexes and where the eluting results in the separation of the heteroduplexes from the homoduplexes.
  • the column ID is greater than about 7 mm. In another embodiment, the column ID is greater than about 10 mm. In yet another embodiment, the column ID is greater than about 50 mm. In still another embodiment the column ID is in the range of about 5 mm to about 1 m.
  • the invention concerns an improved method for separating heteroduplex and homoduplex DNA molecules in a mixture.
  • the t method includes a first step (a) of applying a solution of the fragments and counterion reagent to separation beads, said beads retained within a separation column having an ID greater than about 5 mm.
  • the beads have an average diameter of 1 to 100 microns.
  • the beads are unsubstituted polymer beads or polymer beads substituted with a hydrocarbon moiety having from 1 to 1 ,000,000 carbons.
  • the preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA, said column having an ID greater than about 5 mm.
  • step (b) fragments are eluted with a gradient eluting solvent of increasing organic component concentration containing a counterion agent.
  • the surfaces which are contacted by the solution of the fragments and the eluting solvent are materials which do not trap or release multivalent metal cations therefrom.
  • the eluting is carried out under conditions effective to at least partially denature the heteroduplexes and where the eluting results in the separation of the heteroduplexes from the homoduplexes.
  • the column ID is greater than about 7 mm. In another embodiment, the column ID is greater than about 10 mm. In yet another embodiment, the column ID is greater than about 50 mm. In still another embodiment the column ID is in the range of about 5 mm to about 1 m.
  • the invention concerns an improved separation column for separating heteroduplex and homoduplex DNA molecules in a mixture, by Denaturing Matched Ion Polynucleotide Chromatography (DMIPC).
  • the DNA molecules in the mixture consist of fragments having equal lengths.
  • the column includes a cylinder having an ID greater than about 5 mm and containing polymer beads.
  • the beads have an average diameter of 1 to 100 microns and are unsubstituted polymer beads or are polymer beads substituted with a hydrocarbon moiety having from 1 to 1 ,000,000 carbons.
  • the preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA.
  • the column ID is greater than about 7 mm.
  • the column ID is greater than about 10 mm.
  • the column ID is greater than about 50 mm.
  • the column ID is in the range of about 5 mm to about 1 m.
  • FIG. 1 is a schematic representation of a single column MIPC system using valves and valve controls to establish elution solvent gradients.
  • FIG. 2 is a partial schematic representation of a pump system for establishing elution solvent gradients.
  • FIG. 3 is a schematic representation of an autosampler subsystem.
  • FIG. 4 is a schematic representation of an injection valve used in the MIPC system.
  • FIG. 5 is a schematic representation of an injection valve in the filled loop load position.
  • FIG. 6 is a schematic representation of an injection valve in the filled loop injection position.
  • FIG. 7 is a schematic representation of an injection valve in the partial loop load position.
  • FIG. 8 is a schematic representation of the injection valve in the partial loop injection position.
  • FIG. 9 is a front view of the separation compartment of an improved HPLC DNA analyzer column oven according to this invention.
  • FIG. 10 is a top view of the HPLC DNA analyzer column oven shown in FIG. 9.
  • FIG. 11 is an end view of the compact column heater embodiment of this invention.
  • FIG. 12 is a cross-sectional view taken along the line A — A in FIG. 11.
  • FIG. 13 is a schematic view of a Peltier heater/cooler embodiment of this invention.
  • FIG. 14 is a representation of the physical structure of a representative separation column.
  • FIG. 15 is a MIPC separation of pGEM DNA markers on a 50mm x 4.6mm ID column. Peaks are labeled with the number of base pairs of the eluted fragment.
  • FIG. 16 is a MIPC separation of pGEM DNA markers on a 50mm x 7.8mm ID column. Peaks are labeled with the number of base pairs of the eluted fragment.
  • FIG. 17 shows a schematic representation of a hybridization to form homoduplex and heteroduplex.
  • FIG. 18 is a mutation separation profile of a 209 bp homoduplex/heteroduplex mixture using a 50mm x 4.6mm ID column.
  • FIG. 19 is a mutation separation profile of a 209 bp homoduplex/heteroduplex mixture using a 50mm x 7.8mm ID column.
  • FIG. 20 is a MIPC separation of a ⁇ DNA Hind III digest standard on a 50mm x 4.6mm ID column.
  • FIG. 21 is a MIPC separation of a ⁇ DNA Hind III digest standard on a 50mm x 7.8mm ID column.
  • This invention concerns an improved separation column.
  • the column can be used in an MIPC as described herein.
  • the MIPC system provides automated options for sample selection, mobile phase gradient selection and control, column and mobile phase temperature control, and fragment collection for a wide variety of MIPC separation processes.
  • U.S. Patents or Patent Applications U.S. Patent Nos. 5,772,889; 5,997,742; 5,972,222; 5,986,085; U.S.
  • MIPC separation processes can be applied to effect size-based separation of DNA fragments, mutation detection, DNA fragment purification, PCR process monitoring, and other novel processes.
  • polynucleotide is defined as a linear polymer containing an indefinite number of nucleotides, linked from one ribose (or deoxyribose) to another via phosphoric residues.
  • the present invention can be used in the separation of double- or single-stranded DNA or RNA. For purposes of simplifying the description of the invention, and not by way of limitation, the separation of double-stranded DNA will be described in the examples herein, it being understood that all polynudeotides are intended to be included within the scope of this invention.
  • FIG. 1 is a schematic layout of the system in accordance with one embodiment of the present invention.
  • a plurality of containers can be used as reservoirs for solutions, such as solvents, counter-ions, and other solutions, which make up the mobile phase.
  • container 2 can contain an aqueous component of a mobile phase such as an aqueous solution of counter ion agent (e.g., triethylammonium acetate (TEAA)), and container 4 can contain an aqueous solution of counterion agent plus organic (driving) solvent (e.g., TEAA plus acetonitrile).
  • An auxiliary liquid e.g., a co-solvent
  • These solutions are mixed to achieve a selected concentration of organic solvent in the mobile phase during a separation.
  • the containers have respective transport tubing such as counter-ion solution transport tubing 8, solvent solution transport tubing 10, and auxiliary liquid transport tubing 12 communicating therewith, and leading to degasser 14.
  • the degasser 14 removes dissolved gases from the liquids.
  • An example of a suitable degasser is the Degassit Model 6324. Removal of dissolved oxygen is particularly important because its presence increases the risk of oxidizing ferrous or other oxidizable metals in the system components and thus introducing the corresponding cations into the mobile phase liquid.
  • Column cleaning solution is contained in cleaning solution container 16 which likewise has a cleaning solution transport conduit 18 communicating therewith leading to the degasser 14.
  • the cleaning solution can flow by gravity pressure if the container 16 is elevated above the degasser and injection valve 54.
  • a pump 110 as shown in FIG. 2 can be provided to achieve cleaning solution flow.
  • the system of the invention incorporates conventional mobile phase flow control means which controls flow of solvent solution and aqueous components of a mobile phase.
  • the mobile phase flow control means comprises a set of flow control valves, each with automatic opening controls under computer control as described hereinbelow.
  • the mobile phase flow control means comprises a set of pumps, the flow setting of which are responsive to computer control as described hereinbelow.
  • the system illustrated in FIG. 1 utilizes one embodiment of a mobile phase flow control means which includes a set of flow control valves.
  • Degassed counterion solution conduit 20, degassed solvent solution conduit 22, and degassed auxiliary liquid conduit 24 leading from the degasser 14 communicate with respective aqueous component proportioning valve 26, solvent solution proportioning valve 28, and auxiliary liquid proportioning valve 30.
  • the settings for these proportioning valves are set and changed by valve operators such as stepper motors associated therewith, and these valve operators respond to establish a desired set of settings in response to commands from the mobile phase flow control software module described in greater detail hereinbelow.
  • the flow control valves 26, 28, and 30 comprise an embodiment of a mobile phase flow control means which controls the flow of solvent solution and other components of the mobile phase.
  • the settings for these valves control the ratio of liquids (co-solvents, solvent solution, etc.) through the injector valve and the separation column.
  • Conduits 32, 34, and 36 lead from respective proportioning valves 26, 28 and 30 to the intake of pump 38.
  • the cleaning solution transport conduit 31 leads to a cleaning solution valve 40.
  • An optional cleaning solution conduit 42 leads from the valve 40 and communicates with the inlet of pump 38.
  • Valve 33 controls flow through conduit 42.
  • valves 26, 28 and 30 accurately set the relative ratios of the organic solvent, and other components, within the mobile phase, a most important part of this system because the size-based DNA separation by MIPC is a function of solvent concentration.
  • the slope of the organic solvent gradient as a function of time is changed during the separation process, and the most critical phase may require a very precise gradient, or for some processes, a highly precise isocratic (constant solvent concentration) composition.
  • the settings of the valves 26, 28 and 30 are established by conventional valve actuators which can be remotely set by signals to a conventional valve control device.
  • the separation system is under computer control as represented at 35.
  • the computer includes Instrument Control Software, such as described in U.S. Patent Application No. 09/469,551 , which provides computer controlled instructions for establishing the settings of valves 26, 28 and 30 to precise flow values at appropriate times during the operation of the system.
  • the Instrument Control Software of the instant invention provides computer controlled instructions to establish the operational parameters of the pump 38, such as the off/on status of the pump and the pressure or flow rate settings of the pump.
  • Pump outflow conduit 44 communicates with the in-line mixer 46, directing the liquid flow through the mixer 46 for thorough mixing of the components.
  • Mixed liquid outflow conduit 48 communicates with optional guard column 50 to treat the mixed liquid to remove multivalent metal cations and other contaminants which would interfere with the separation of DNA fragments.
  • Guard column 50 can contain a cation exchange resin in sodium or hydrogen form for removal of multivalent metal cations by conventional ion exchange.
  • Conduit 52 communicates with the outlet of the guard column and an inlet port of a cleaning solution injector valve 54.
  • Cleaning solution supply conduit 56 connects valve 40 with the cleaning solution injector valve 54, and waste outlet conduit 58 leads to waste.
  • Conduit 60 leads from valve 54 to the sample injection valve 62.
  • Sample aliquot selector 64 communicates with injector valve 62 through sample conduit 66. Waste conduit 68 leads from the injector valve and removes waste liquids.
  • sample conduit 70 communicates with an outlet port of injector valve 62 and with the column prefilter 74 in the air bath oven 72.
  • the capillary tubing coil 76 communicates with the prefilter 74 and the inlet of separation column 78.
  • the extended length of the capillary coil 76 allows ample heat to pass from the heated oven air into the liquid passing through the coil, bringing the liquid within ⁇ 0.05 °C of a selected temperature.
  • the oven 72 establishes this temperature uniformity in the prefilter 74, coil 76, and separation column 78.
  • the separation column 78 is packed with beads having a unique separation surface which effects a size-based separation of DNA fragments in the presence of a counter-ion by the MICP process.
  • the separation process and details about the column and beads are described in detail hereinbelow.
  • a stream of mobile phase containing base pair length size-separated DNA fragments passes from the separation column 78 through conduit 80.
  • Conduit 80 communicates with a detector 84.
  • the detector can be a conventional UV absorbance device which measures the UV absorbance of the DNA fragment structures in the liquid mobile phase. The absorbance is a function of the concentration of the DNA fragments in the liquid being tested.
  • the detector can be a fluorescence detector which can continuously measure the level of the fluorescent marker in the liquid by detecting the emission level at the frequency most appropriate for the marker.
  • any detecting system capable of continuously measuring a characteristic of the liquid which is a function of the concentration of the DNA fragments therein is suitable and intended to be within the scope of this invention.
  • suitable detectors include the L-7420 UV-Vis detector, and the L- 7480 Fluorescence detector available from Hitachi.
  • the electrical output from the detector preferably is converted to a digital form by an A/D converter and recorded in standard digital format to a digital storage device such as a disk drive in computer 35. . Conduit 86 removes the tested liquid.
  • the mobile phase passes to the fragment collector 88 where selected portions of the mobile phase containing a separated DNA fraction are collected in vials for later processing or analysis. Uncollected fractions are removed through waste conduit 90.
  • the DNA separation process is impaired by the presence of multivalent cations.
  • the liquid flow system is described as a series of conduits.
  • the conduits are capillary tubing selected to avoid introduction of multivalent cations into the liquids.
  • the preferred capillary tubing materials are titanium and PEEK.
  • the other components of the system are preferably made of titanium or PEEK or have the surfaces exposed to the liquid coated with PEEK to protect them from oxidation and prevent the introduction of multivalent cations into the liquid.
  • Stainless steel can also be used provided it has been treated to remove all oxidized surface materials and the solutions contacting the stainless steel surfaces are free of dissolved oxygen.
  • FIG. 2 is a partial schematic representation of a pump system for establishing mobile phase composition.
  • This system relies on proportioning pumps to control the ratio of aqueous component and solvent solution, such as solutions A and B described hereinabove.
  • the inlets of proportioning pumps 92, 94 and 96 by way of their respective supply conduits 98, 100, and 102 communicate with the degasser 14, and by way of their respective outlet conduits 104, 106 and 108 communicate with the inline mixer 46.
  • the operational speed for these proportioning pumps are calibrated to flow rates therethrough and are controlled by a flow control software module described in greater detail hereinbelow.
  • the settings for these proportioning valves control the liquid flow speed and the ratio of liquids (co-solvents, driving solvents, etc.) through the injector valve and the separation column.
  • a pump 110 can supply cleaning solution to the system through optional conduit 112.
  • An optional conduit 107 leads from conduit 112 and communicates with the in-line mixer 46.
  • Valve 111 controls flow through conduit 107.
  • FIG. 3 is a schematic representation of an autosampler subsystem used in the MIPC system. This autosampler removes an aliquot having a predetermined volume from a selected well or vial (e.g., micro-centrifuge tube) supported in a multi-well 113. Microwell plates can have any predetermined number of wells 114 having a precise dimensional position for each well, such as the standard 96 well multiwell plate.
  • the sampling needle 115 is supported on a sampling carriage 116.
  • the sampling carriage 116 has a needle support
  • the flexible tubing 123 is mounted in sealed engagement with the needle 115 at one end and with the syringe needle 124 at the other end.
  • the syringe needle 124 communicates with the inner volume of the syringe cylinder 125.
  • the piston 126 is mounted on the syringe actuator rod 128 and forms a sealed engagement with the inner wall of the cylinder 125. In operation, vertical upward movement of the syringe actuator rod 128 displaces liquid in the cylinder 125, and vertical downward movement of the syringe actuator rod 128 pulls liquid into the syringe.
  • Rod 128 is attached to clamp 130 which is supported for movement along guide element 132.
  • valve 122 When valve 122 is positioned to provide communication between the needle 124 and the tubing 123, the downward movement of the piston 126 pulls sample into the needle 115 from a well 114. When needle 115 is positioned above injector valve port 119, upward movement of the piston 126 discharges sample from needle 115 into port 119.
  • Conduit 131 extends from valve 122 to the cleaning solution reservoir 121.
  • valve 122 When valve 122 is in the position providing communication between the needle 124 and the conduit 131 , the downward movement of the piston 126 draws cleaning solution into the needle.
  • valve 122 When the needle 115 is positioned above the injector port 119 and valve 122 is positioned to provide communication between the needle 124 and the conduit 123, upward movement of the piston 126 discharges cleaning solution into the injector port 119.
  • suitable autosamplers include the HITACHI Model L-7250 Programmable Autosampler and the HTS PAL High Throughput Autosampler (Shimadzu, Columbia, MD).
  • FIG. 4 is schematic representation showing the structure of the sample injection valve and cleaning solution injection valve for use in the MIPC system.
  • the same valve structure can be used for both the sample injection and cleaning solution injection.
  • the injection valve 150 is a six-port, rotary valve operated by a conventional valve motor such as a stepper motor (not shown). Exemplary valves include the LabPRO valves available from RHEODYNE (Cotati, CA).
  • the valve has six external ports permanently connected to inlet and outlet conduits.
  • External port 152 is connected with an injection line 154 for receiving a sample to be analyzed.
  • External port 156 is connected with a column supply conduit 158 communicating with the separation column 78 (FIG. 1 ).
  • External port 160 is connected with an inlet
  • FIGs. 5 and 6 describe the use of the valve for filled loop injection, the mode used when a larger volume of sample (or cleaning solution) is to be injected.
  • FIG. 5 is a schematic representation of an injection valve in the sample load position
  • FIG. 6 is a schematic representation of the injection valve in the injection position. In the load position shown in FIG.
  • a first internal passageway 174 of the valve connects the first end 176 of loop 172 with the sample injection line 154, and a second internal passageway 178 connects the second end 180 of loop 172 with the waste conduit 166.
  • a third internal passageway 182 connects the pump outlet conduit 162 with the conduit 158 to the separation column 78. While sample from the injection port 154 is introduced into the sample loop 172 through passageway 174, any surplus or liquid in the loop 172 is expelled to the waste conduit 166 through passageway 178. Simultaneously, mobile phase solutions flow from the pump conduit 162 to the separation column 78 through third conduit 182.
  • Passageway 179 connects one end 180 of the loop 172 with the conduit 158 leading to the separation column, and passageway 175 connects the other end 176 of the loop 172 with the inlet conduit 162 leading to the pump.
  • passageway 175 It from the pump enters passageway 175 and passes through the loop 172, expelling sample solution into the conduit 158 leading to the column and continues to rinse the loop, carrying any residue into the column conduit 158. Meanwhile, passageway 183 connects the sample injection conduit 154 to waste, permitting passage of cleaning solution, if desired, through passageway 183.
  • This procedure provides a reliable injection of a measured volume of sample solution into the conduit leading to the separation column 78 (FIG. 1 ), the liquid passing through the prefilter 74 and temperature regulating coil 76 before it reaches the separation column.
  • the system of the invention incorporates oven temperature control means for controlling the temperature of the separation column and the mobile phase entering the column.
  • FIGs. 9 and 10 illustrate one embodiment of a temperature control means.
  • FIG. 9 is a front view of the process compartment of an HPLC DNA analyzer column oven
  • FIG. 10 is a top view of the HPLC DNA analyzer column oven shown in FIG. 9.
  • the process compartment in the embodiment shown in FIGs. 9 and 10 is divided from the heating compartment by backwall 200 in which air exhaust port 202 is positioned.
  • a metal bar 204 enclosing a temperature sensor such as a thermocouple or thermister is positioned in the port 202 to measure the temperature of the air passing through the port.
  • Capillary tubing 206 leads from the sample injector (not shown) to a prefilter 208.
  • Prefilter 208 is an inline filter or guard cartridge, such as described in U.S. Patent No. 5,772,889, which removes contaminants from the incoming liquid.
  • An elongated coil 210 of capillary tubing has an inlet end in communication with prefilter 208 for receiving mobile phase liquid therefrom.
  • the elongated coil 210 has an outlet end communicating with the inlet end 212 of a separation column 214.
  • Separation column 214 preferably contains MIPC separation media.
  • Outlet tubing 216 leads from the outlet end 218 of the separation column 214 to detector 84 (FIG. 1 ).
  • Coil 210 is a liquid heating coil made of a DNA compatible, multivalent cation free tubing such as titanium or PEEK.
  • the length and diameter of tubing used is any length which is sufficient to enable liquid mobile phase passing therethrough to reach the equilibrium temperature of air in the processing compartment.
  • a tubing length of from 6 to 400 cm and a tubing ID of from 0.15 to 0.4 mm is usually sufficient. Since the length of tubing 210 does not degrade the separation of components achieved by the system, the length can be selected based on the length required to achieve effective heating of the process liquids.
  • air from the processing compartment 220 passes through the opening 202 in wall 200, through a heater/fan system 222 for temperature adjustment.
  • the adjusted air received by the heating compartment 224 recycles back to the processing compartment 220 along the passageways 226 defined by the spacing between the sidewalls 227 and the outer oven wall 228.
  • the heating coil in the embodiment shown in FIGs. 9 and 10 provides a temperature accuracy to within the range of ⁇ 0.2 °C and reduces the temperature equilibrium time between temperature settings to below 5 minutes for temperature changes of 5°C and below 2 minutes for temperature changes of up to 1°C.
  • FIGs. 11 and 12 illustrate another embodiment of a temperature control means.
  • FIG. 11 is an end view of a compact column heater
  • FIG. 12 is a cross-sectional view taken along the line A-A in FIG. 11.
  • This embodiment relies on direct metal-to-metal conduction of heat to and from the system components and does not depend upon an air bath to achieve temperature changes and accuracy.
  • This embodiment is shown for a two column system, although it could be used for a single column, if desired. It comprises heat conducting blocks (230,232) having receptacles sized and shaped to receive the system components.
  • Filter cavity or prefilter receptacles (234,236) have inner surfaces which are sized to receive prefilters (238,240) and establish heat transfer contact with the outer surfaces thereof.
  • Separation column receptacles (242,244) have inner surfaces sized to receive respective separation columns (246,248) and separation column couplers (250) (one is shown in FIG. 12) which connect capillary tubing to the respective separation columns.
  • Receptacles (242,244) are sized and shaped to establish heat transfer contacts between the inner heat transfer surfaces of blocks (230,232) and the separation column components received therein.
  • Capillary coil receptacles 252 (one is shown in FIG. 12) have an inner surface which is shaped to receive a coils of capillary tubing 254 (one is shown in FIG. 12) and to establish heat transfer contact with the outer surface thereof.
  • receptacles (234,236) and (242,244) can be cylindrical holes with approximately parallel central axes lying in a common plane. It would be readily apparent to a person skilled in the art that other configurations are equally suitable and all configurations are considered to be within the scope of this invention.
  • Temperature sensor receptacles (256,258) are provided in heat conducting blocks (230,232).
  • Capillary receptacle passageways 260 for receiving connecting tubing 262 in a heat-conducting relationship are also provided in the heating-conducting block (230,232).
  • the capillary coil receptacles 252 are shown in this figure to be cylindrical cavities with their axes perpendicular to the axes of receptacles (234,236) and (242,244).
  • a conductive metal cylinder (not shown) can be positioned within the capillary coils in heat conducting contact with the inner surfaces thereof to increase heat transfer area between the metal block heating assembly and the liquid in the coils.
  • a KAPTON resistance heater or other type of heating unit 264 is positioned between and in heat-conducting contact with surfaces 266 and 268 of heating blocks (230,232) to transfer heat to the heat- conducting blocks.
  • Heat sinks (270,272) are positioned in heat-conducting relationship with opposed cooling surfaces (274,276) of the heat conduct blocks (230,232) to remove heat therefrom.
  • Cooling fans 278 and 280 are in a heat removal relationship with the heat sinks 270 and 272 and are activated to accelerate heat removal therefrom.
  • the heat conducting blocks 230 and 232, and the heat sinks 270 and 272 are made of a material having high heat conductivity such as aluminum or copper, although they can be made of other heat-conducting solids such as ferrous metals or any other solid material having the requisite heat conductivity. Heat pipes can also be used as heat sinks.
  • the capillary tubing can be made of PEEK or titanium, although titanium is preferred for maximum heat transfer efficiency. With this improved heat transfer, the capillary coil can have a fully extended length as short as 5 cm although a minimum coil length of 10 cm is preferred. A longer coil of PEEK tubing would be required to achieve the same heat transfer as titanium capillary tubing.
  • FIGs. 11 and 12 The system shown in FIGs. 11 and 12 comprises two systems in mirror image. It will be readily apparent that for a single column, half the system would be sufficient and is intended to be included within the scope of this invention.
  • the position, alignment and spacing of the receptacles are not a critical feature of this invention. Any alignment and configuration which provides a compact and heat-transfer efficient result is intended to be included within the scope of this invention.
  • FIGs. 11 and 12 provide a compact heater which is more responsive to heater controls, provides rapid changes from one temperature platform to another, and maintains a temperature accuracy within ⁇ 0.5 °C of a set temperature.
  • the heat transfer rate obtained with the metal- to-metal contact between the heating block and the elements being heated is far greater than can be obtained in an air bath system, providing the more rapid response to a changed temperature and greater temperature accuracy. It also allows process liquid temperature adjustment with a shorter capillary tubing coil.
  • FIG. 13 shows a schematic view of a preferred Peltier heater/cooler embodiment.
  • Heating block 282 is in conductive contact with a Peltier heating element (not shown) for heating or cooling required to reach and maintain a desired temperature.
  • Channel 284 is a prefilter receptor having an inner surface 286 in heat conductive relationship with prefilter 288.
  • Channel 290 is a column and column guard receptor having an inner surface 292 in heat conductive relationship with coupler 294 and end nut elements 296 of separation column 298.
  • Capillary tubing 300 communicates with the prefilter 288 and the sample and solution sources (not shown).
  • Capillary tubing 302 from the outlet of the separation column 288 communicates with an analyzer 84 (FIG. 1 ).
  • Capillary tubing 304 connects the outlet end of the prefilter 288 with the coupler 294, which in turn communicates with the separation column 298.
  • Capillary tubing 304 is received in a labyrinth-like configuration of channels in the heating block 282 to provide increased capillary length and surface contact between the capillary tubing 304 and the heating block 282.
  • the configuration of the labyrinth and tubing can be any configuration which provides an adequate
  • the capillary tubing 304 can be PEEK or titanium, titanium being preferred because of its high heat conductivity.
  • the heating block 282 can be any heat conductive metal. Aluminum or copper are preferred because of their higher heat conductivity, although ferrous metals such as steel can be used.
  • the Peltier heater is controlled with a conventional temperature and control system (not shown) such as the systems used in Peltier thermocyclers. As with the embodiment shown in FIGs. 11 and 12, the temperature accuracy achieved by the Peltier heated block is ⁇ 0.5 °C.
  • Fig. 4 is a partially exploded representation of the physical structure of a representative separation column.
  • the column comprises a cylinder or tube 334 with external ferrule 335 on both ends.
  • the tube has internal diameter (ID) as shown at 333 and is filled with separation media 336.
  • ID internal diameter
  • a porous frit 338 is held against the upper surface of the separation media by the end fitting 340.
  • the end fitting 340 receives frit 338 and holds the frit against the end of the tube 334.
  • the internally threaded nut 342 receives the externally threaded fitting 340 in a threaded engagement.
  • the fitting 340 has an internally threaded end receptor 346 for receiving a capillary tubing end coupler (not shown).
  • the material comprising the cylinder can be polymer or metal.
  • Stainless steel tubes suitable for use in the present invention are available commercially, for example from Isolation Technologies Inc. (Hopedale, MA). Examples include stainless steel tubing having ID sizes such as 4.6, 6.5, 7.8, 10.0, 21.2, 30 and 50mm. Some separations are carried out using columns having diameters as large as 500mm. Columns as large as 1 m are used for large-scale commercial manufacture.
  • the column preferably includes porous
  • the separation media 336 comprises organic polymer materials or inorganic materials having the requisite structure and non-polar surfaces. Suitable materials are described hereinbelow and in copending, commonly assigned patent applications Serial No. 09/058,580 filed April 10,- 1998 and Serial No. 09/183,123 filed October 20, 1998. In a preferred embodiment of the present invention, all of the process solution-contacting surfaces are subjected to a multivalent cation removal treatment to remove any potential source of multivalent cation contamination.
  • 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 sonication of a titanium frit in the presence of aqueous nitric acid solution, followed by sonication in water until a neutral pH is achieved.
  • Other treatments include contacting the surfaces with chelating agents such as EDTA, pyrophosphoric acid, or phosphoric acid (e.g. 30% by weight phosphoric acid).
  • Example 1 A representative example of a size-based DNA separation is described in Example 1 (FIG. 15) in which a standard mixture of DNA fragments, pGEM® DNA Markers (Promega Corp. Madison, WI), was eluted using a 50 mm x 4.6 mm ID column.
  • the mixture included fragments of double stranded DNA having the base pair lengths as indicated in FIG. 15.
  • Improved resolution during the separation of DNA by MPIC is obtained using a column having a ID of greater than 5 mm, preferably greater than about 7 mm, more preferably greater than about 10 mm. In other embodiments, improved separation is obtained with a column can having an ID within the range of about 5 mm to about 1 m.
  • Another aspect of the present invention concerns an improved separation column and method for use in conducting DNA mutation detection by DMIPC.
  • the instant invention can be used to detect mutations in double stranded DNA.
  • the following definitions will be used herein:
  • a “homoduplex” is defined herein to mean, a double stranded DNA fragment wherein the bases in each strand are complimentary relative to their counterpart bases in the other strand.
  • a “heteroduplex” is defined herein to mean a double stranded DNA fragment wherein at least one base in each strand is not complimentary to at least one counterpart base in the other strand. This can be due to a mismatched base or a deletion. Since at least one base pair in a heteroduplex is not complimentary, it takes less energy to separate the bases at that site compared to its fully complimentary base pair analog in a homoduplex. This results in the lower melting temperature at the site of a mismatched base of a hetroduplex compared to a homoduplex.
  • hybridization refers to a process of heating and cooling a dsDNA sample, e.g., heating to 95°C followed by slow cooling.
  • the heating process causes the DNA strands to denature. Upon cooling, the strands re- combine into duplexes in a largely statistical fashion. If the sample contains a mixture of wild type and mutant DNA, then hybridization will form a mixture of hetero- and homoduplexes.
  • heteromutant site separation temperature T(hsst) is defined herein to mean the temperature which preferentially denatures the heteroduplex DNA at a site of mutation and which gives the greatest difference in the degree of denatu ration between the heteroduplexes and homoduplexes. This is a temperature which is optimal to effect a chromatographic separation of heteroduplexes and homoduplexes by DMIPC and hence, detect mutations.
  • heteromutant is defined herein to mean a DNA fragment containing a polymorphism or non-complimentary base pair.
  • mutant separation profile is defined herein to mean a DMIPC separation chromatogram which shows the separation of heteroduplexes from homoduplexes. Such separation profiles are characteristic of samples which contain mutations or polymorphisms and have been hybridized prior to being separated by DMIPC.
  • a reliable way to detect mutations is by hybridization of the putative mutant strand in a sample with the wild type strand (Lerman, et al., Meth. Enzymol., 155:482 (1987)). If a mutant strand is present, then two homoduplexes and two heteroduplexes will be formed as a result of the hybridization process, as shown in FIG. 17. Hence separation of heteroduplexes from homoduplexes provides a direct method of confirming the presence or absence of mutant DNA segments in a sample.
  • a standard containing 209 base pair homoduplexes and heteroduplexes was subjected to DMIPC as shown in FIG. 18. as a series of separation chromatograms and the separation process is described in Example 4.
  • the hybridization process created two homoduplexes and two heteroduplexes as shown schematically in FIG. 17.
  • the DMIPC analysis was performed using a separation column having an ID of 4.6 mm. There was partial overlap between the two heteroduplexes 350,352 and also between the two homoduplexes 354,356. Applicants surprisingly observed dramatic and unexpected improvement in the separation of the peaks when the separation of the 209 base pair mutation standard was conducted using a column having an ID of 7.8 mm (FIG. 19) as described in Example 4. The results demonstrated improved separation of two homoduplex fragments 350,352 from each other, the two heteroduplex fragments 354,356 from each other,. and increased separation between the homoduplex and heteroduplex pairs as indicated by distance "d".
  • the method is preferably performed using a column having a ID of greater than about 5 mm, more preferably greater than about 7 mm, most preferably greater than about 10 mm.
  • the column can have an ID within the range of about 5 mm to about 1 m.
  • the separation process used in the chromatography system of the invention concerns separation of polynudeotides, e.g. DNA, utilizing a stationary separation medium having non-polar surfaces. The preferred surfaces are essentially free from multivalent cation contamination which can trap polynudeotides.
  • the separation is performed on the stationary surface.
  • the surface can be porous, but preferably any surface pores are of a size which excludes the smallest polynucleotide being analyzed.
  • the only requirement for the separation beads of the present invention is that they must have a surface that is either intrinsically non-polar or be bonded with a material that forms a surface having sufficient non- polarity to interact with a counterion agent.
  • the non-porous polymeric beads can have an average diameter of about 0.5 -100 microns; preferably, 1 - 10 microns; more preferably, 1 - 5 microns. Beads having an average diameter of 1.0 - 3.0 microns are most preferred.
  • MIPC Polynucleotide Chromatography
  • nonporous is defined to denote a bead which has surface pores having a diameter that is less than the size and shape of the smallest DNA fragment in the separation in the solvent medium used therein. Included in this definition are polymer beads having these specified maximum size restrictions in their natural state or which have been treated to reduce their pore size to meet the maximum effective pore size required.
  • the surface conformations of nonporous beads of the present invention can include depressions and shallow pit-like structures which do not interfere with the separation process.
  • a pretreatment of a porous bead to render it nonporous can be effected with any material which will fill the pores in the bead structure and which does not significantly interfere with the MIPC process.
  • Pores are open structures through which mobile phase and other materials can enter the bead structure. Pores are often interconnected so that fluid entering one pore can exit from another pore. Applicants believe that pores having dimensions that allow movement of the polynucleotide into the interconnected pore structure and into the bead impair the resolution of separations or result in separations that have very long retention times. In MIPC, however, the beads are "nonporous" and the polynudeotides do not enter the bead structure.
  • the nonporous polymeric beads of the present invention are prepared by a two-step process in which small seed beads are initially produced by emulsion polymerization of suitable polymerizable monomers.
  • the emulsion polymerization procedure of the invention is a modification of the procedure of Goodwin, et al. (Colloid & Polymer Sci., 252:464-471 (1974)).
  • Monomers which can be used in the emulsion polymerization process to produce the seed beads include styrene, alkyl substituted styrenes, alpha-methyl styrene, and alkyl substituted alpha-methyl styrene.
  • the seed beads are then enlarged and, optionally, modified by substitution with various groups to produce the nonporous polymeric beads of the present invention.
  • the seed beads produced by emulsion polymerization can be enlarged by any known process for increasing the size of the polymer beads.
  • polymer beads can be enlarged by the activated swelling process disclosed in U.S. Patent No. 4,563,510.
  • the enlarged or swollen polymer beads are further swollen with a crosslinking polymerizable monomer and a polymerization initiator.
  • Polymerization increases the crosslinking density of the enlarged polymeric bead and reduces the surface porosity of the bead.
  • Suitable crosslinking monomers contain at least two carbon-carbon double bonds capable of polymerization in the presence of an initiator.
  • Preferred crosslinking monomers are divinyl monomers, preferably alkyl and aryl (phenyl, naphthyl, etc.) divinyl monomers and include divinyl benzene, butadiene, etc.
  • Activated swelling of the polymeric seed beads is useful to produce polymer beads having an average diameter ranging from 1 up to about 100 microns.
  • the polymer seed beads can be enlarged simply by heating the seed latex resulting from emulsion polymerization. This alternative eliminates the need for activated swelling of the seed beads with an activating solvent.
  • the seed latex is mixed with the crosslinking monomer and polymerization initiator described above, together with or without a water-miscible solvent for the crosslinking monomer.
  • Suitable solvents include acetone, tetrahydrofuran (THF), methanol, and dioxane.
  • THF tetrahydrofuran
  • methanol methanol
  • dioxane a solvent that is heated for about 1 - 12 hours, preferably about 4 - 8 hours, at a temperature below the initiation temperature of the polymerization initiator, generally, about 10°C - 80°C, preferably 30°C - 60°C.
  • the initiation temperature of the polymerization initiator generally, about 10°C - 80°C, preferably 30°C - 60°C.
  • ⁇ 7 temperature of the mixture can be increased by 10 - 20% and the mixture heated for an additional 1 to 4 hours.
  • the ratio of monomer to polymerization initiator is at least 100:1, preferably about 100:1 to about 500:1 , more preferably about 200:1 in order to ensure a degree of polymerization of at least 200.
  • Beads having this degree of polymerization are sufficiently pressure-stable to be used in high pressure liquid chromatography (HPLC) applications.
  • HPLC high pressure liquid chromatography
  • This thermal swelling process allows one to increase the size of the bead by about 110 - 160% to obtain polymer beads having an average diameter up to about 5 microns, preferably about 2 - 3 microns.
  • the thermal swelling procedure can, therefore, be used to produce smaller particle sizes previously accessible only by the activated swelling procedure.
  • Polymerization can be conducted, for example, by heating of the enlarged particles to the activation temperature of the polymerization initiator and continuing polymerization until the desired degree of polymerization has been achieved. Continued heating and polymerization allows one to obtain beads having a degree of polymerization greater than 500.
  • the packing material disclosed by Bonn et al. or U.S. Patent No. 4,563,510 can be modified through substitution of the polymeric beads with alkyl groups or can be used in its unmodified state.
  • the polymer beads can be alkylated with 1 or 2 carbon atoms by contacting the beads with an alkylating agent, such as methyl iodide or ethyl iodide. Alkylation is achieved by mixing the polymer beads with the alkyl halide in the presence of a Friedel-Crafts catalyst to effect electrophilic aromatic substitution on the aromatic rings at the surface of the polymer blend.
  • Suitable Friedel-Crafts catalysts are well-known in the art and include Lewis acids such as aluminum chloride, boron trifiuoride, tin tetrachloride, etc.
  • the beads can be hydrocarbon substituted by substituting the corresponding hydrocarbon halide for methyl iodide in the above procedure, for example.
  • alkyl as used herein in reference to the beads of the present invention is defined to include alkyl and alkyl substituted aryl groups, having from 1 to 1 ,000,000 carbons, the alkyl groups including straight chained, branch chained, cyclic, saturated, unsaturated nonionic functional groups of various types including aldehyde, ketone, ester, ether, alkyl groups, and the like, and the aryl groups including as monocyclic, bicyclic, and tricyclic aromatic hydrocarbon groups including phenyl, naphthyl, and the like.
  • Methods for alkyl substitution are conventional and well-known in the art and are not an aspect of this invention.
  • the substitution can also contain hydroxy, cyano, nitro groups, or the like which are considered to be non-polar, reverse phase functional groups.
  • the chromatographic material reported in the Bonn patent was limited to nonporous beads substituted with alkyl groups having at least 3 carbons because Bonn et al. were unsuccessful in obtaining separations using polymer beads lacking this substitution. Additionally, the polymer beads were limited to a small group of vinyl aromatic monomers, and Bonn et al. were unable to effect double stranded DNA separations with other materials.
  • successful separation of double stranded DNA can be achieved using underivatized nonporous beads as well as using beads derivatized with alkyl groups having 1 to 1 ,000,000 carbons.
  • the base polymer of the invention can also be other polymers, non- limiting examples of which include mono- and di-vinyl substituted aromatics such as styrene, substituted styrenes, alpha-substituted styrenes and divinylbenzene; acrylates and methacrylates; polyolefins such as polypropylene and polyethylene; polyesters; polyurethanes; polyamides; polycarbonates; and substituted polymers including fluorosubstituted ethylenes commonly known under the trademark TEFLON.
  • mono- and di-vinyl substituted aromatics such as styrene, substituted styrenes, alpha-substituted styrenes and divinylbenzene
  • acrylates and methacrylates polyolefins such as polypropylene and polyethylene
  • polyesters polyurethanes
  • polyamides polyamides
  • polycarbonates and substituted polymers including fluorosubsti
  • the base polymer can also be mixtures of polymers, non-limiting examples of which include poly(styrene-divinylbenzene) and poly(ethylvinylbenzene- divinylbenzene).
  • Methods for making beads from these polymers are conventional and well known in the art (for example, see U.S. Patent 4,906,378).
  • the physical properties of the surface and near-surface areas of the beads are the predominant influence on chromatographic efficiency.
  • the polymer, whether derivatized or not, must provide a nonporous, non-reactive, and non-polar surface for the MIPC separation.
  • the beads and other media of the invention are characterized by being substantially free from multivalent cations which are free to bind with DNA.
  • the preferred beads of the present invention are characterized by having been subjected to precautions during production, including a decontamination treatment, such as an acid wash treatment, designed to substantially eliminate any multivalent cation contaminants (e.g. Fe(lll), Cr(lll), or colloidal metal contaminants). Only very pure, non-metal containing materials should be used in the production of the beads in order that the resulting beads will have minimum metal content.
  • the separation column and all process solutions held within the column or flowing through the column are preferably substantially free of multivalent cation contaminants.
  • this can be achieved by supplying and feeding solutions that enter the separation column with components which have process solution-contacting surfaces made of material which does not release multivalent cations into the process solutions held within or flowing through the column, in order to protect the column from multivalent cation contamination.
  • the process solution-contacting surfaces of the system components are preferably material selected from the group consisting of titanium, coated stainless steel, passivated stainless steel, and organic polymer.
  • multivalent cation binding agents e.g., chelators
  • these binding agents can be incorporated into a solid through which the mobile phase passes. Contaminants are trapped before they reach places within the system that can harm the separation.
  • the functional group is attached to a solid matrix or resin (e.g., a flow-through cartridge, usually an organic polymer, but sometimes silica or other material).
  • the capacity of the matrix is preferably about 2 mequiv./g.
  • An example of a suitable chelating resin is available under the trademark CHELEX 100 (Dow Chemical Co.) containing an iminodiacetate functional group.
  • the multivalent cation binding agent can be added to the mobile phase.
  • the binding functional group is incorporated into an organic chemical structure.
  • the preferred multivalent cation binding agent fulfills three requirements. First, it is soluble in the mobile phase. Second, the complex with the metal is soluble in the mobile phase. Multivalent cation binding agents such as EDTA fulfill this requirement because both the chelator and the multivalent cation binding agent-metal complex contain charges which make them both water-soluble. Also, neither precipitate when acetonitrile, for example, is added.
  • the solubility in aqueous mobile phase can be enhanced by attaching covalently bound ionic functionality, such as, sulfate, carboxylate, or hydroxy.
  • a preferred multivalent cation binding agent can be easily removed from the column by washing with water, organic solvent or mobile phase. Third, the binding agent must not interfere with the chromatographic process.
  • the multivalent cation binding agent can be a coordination compound.
  • preferred coordination compounds include water soluble chelating agents and crown ethers.
  • Non-limiting examples of multivalent cation binding agents which can be used in the present invention include acetylacetone, alizarin, aluminon, chloranilic acid, kojic acid, morin, rhodizonic acid, thionalide, thiourea, ⁇ -furildioxime, nioxime, salicylaldoxime, dimethylglyoxime, ⁇ -furildioxime, cupferron, ⁇ -nitroso- ⁇ -naphthol, nitroso-R- salt, diphenylthiocarbazone, diphenylcarbazone, eriochrome black T, PAN, SPADNS, glyoxal-bis(2-hydroxyanil), murexide, ⁇ -benzoinoxime, mandelic acid, anthranilic acid, ethylenedi
  • ⁇ To achieve high resolution chromatographic separations of polynudeotides, it is generally necessary to tightly pack the chromatographic column with the solid phase polymer beads. Any known method of packing the column with a column packing material can be used in the present invention to obtain adequate high resolution separations. Typically, a slurry of the polymer beads is prepared using a solvent having a density equal to or less than the density of the polymer beads. The column is then filled with the polymer bead slurry and vibrated or agitated to improve the packing density of the polymer beads in the column. Mechanical vibration or sonication are typically used to improve packing density.
  • the separation method of the invention is generally applicable to the chromatographic separation of single stranded and double stranded polynudeotides of DNA and RNA.
  • Samples containing mixtures of polynudeotides can result from total or enzymatic synthesis of polynudeotides, cleavage of DNA or RNA with endonucleases or with other enzymes or chemicals, as well as nucleic acid samples which have been multiplied and amplified using polymerase chain reaction techniques.
  • the improved method of the present invention can be used to separate double stranded polynudeotides having up to about 10,000 base pairs.
  • the method can be used to separate polynudeotides having between about 5 to about 15,000-20,000 nucleotides.
  • the separation is by Matched Ion Polynucleotide Chromatography (MIPC).
  • MIPC Matched Ion Polynucleotide Chromatography
  • the nonporous beads of the invention are used as a reverse phase material that will function with counterion agents and a solvent gradient to effect the DNA separations.
  • the polynudeotides are paired with a counterion and then subjected to reverse phase chromatography using the nonporous beads of the present invention.
  • counterions suitable for use with MIPC include a mono-, di-, or trialkylamine that can be protonated to form a positive counter charge or a quaternary alkyl substituted amine that already contains a positive counter charge.
  • the alkyl substitutions may be uniform (for example, triethylammonium acetate or tetrapropylammonium acetate) or mixed (for example, propyldiethylammonium acetate).
  • the size of the alkyl group may be small (methyl) or large (up to 30 carbons) especially if only one of the substituted alkyl groups is large and the others are small.
  • octyldimethylammonium acetate is a suitable counterion agent.
  • Preferred counterion agents are those containing alkyl groups from the ethyl, propyl or butyl size range.
  • the purpose of the alkyl group is to impart a nonpolar character to the polynucleic acid through a matched ion process so that the polynucleic acid can interact with the nonpolar surface of the separation media.
  • the requirements for the extent of nonpolarity of the counterion-DNA pair depends on the polarity of the separation media, the solvent conditions required for separation, the particular size and type of fragment being separated.
  • the polarity of the counterion agent may have to change to match the polarity of the surface and increase interaction of the counterion-DNA pair.
  • Triethylammonium acetate is preferred although quaternary ammonium reagents such as tetrapropyl or tetrabutyl ammonium salts can be used when extra nonpolar character is needed or desired.
  • quaternary ammonium reagents such as the polarity of the alkyl group is increased, size specific separations, sequence independent separations become more possible. Quaternary counterion reagents are not volatile, making removal of the reagent more difficult. In some cases, it may be desired to increase the range of concentration of organic solvent used to perform the separation.
  • increasing the alkyl length on the counterion agent will increase the nonpolarity of the counterion-DNA pair resulting in the need to either increase the concentration of the mobile phase organic solvent, or increase the strength of the organic solvent type, e.g. acetonitrile is about two times more effective than methanol for eluting polynucleic acids.
  • concentration of the organic solvent required to elute a fragment from the column and the length of the fragment.
  • the polynucleotide could precipitate.
  • a strong organic solvent or a smaller counterion alkyl group can be used.
  • the alkyl group on the counterion reagent can also be substituted with halides, nitro groups, or the like to moderate polarity.
  • the mobile phase preferably contains a counterion agent.
  • Typical counterion agents include trialkylammonium salts of organic or inorganic acids, such as lower alkyl primary, secondary, and lower tertiary amines, lower trialkyammonium salts and lower quaternary alkyalmmonium salts.
  • Lower alkyl refers to an alkyl radical of one to six carbon atoms, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl, isoamyl, n-pentyl, and isopentyl.
  • counterion agents examples include octylammonium acetate, octadimethylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium a
  • anion in the above examples is acetate
  • other anions may also be used, including carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride, and bromide, or any combination of cation and anion.
  • These and other agents are described by Gjerde, et al. in Ion Chromatography, 2nd Ed., Dr. Alfred H ⁇ thig Verlag Heidelberg (1987).
  • Counterion agents that are volatile are preferred for use in the method of the invention, with triethylammonium acetate (TEAA) and triethylammonium hexafluoroisopropyl alcohol being most preferred.
  • TEAA triethylammonium acetate
  • TEAA triethylammonium hexafluoroisopropyl alcohol
  • EXAMPLE 1 DNA Fragment Sizing by MIPC using a 4.6 mm ID column MIPC analysis of pGEM DNA size markers (Part No. G174A, Promega Corp., Madison, Wl) was performed using octadecyl modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads packed in a 50 mm x 4.6 mm ID separation column (DNASEP® cartridge, Transgenomic, Inc., San Jose, CA) and using a WAVE® DNA Fragment Analysis System (Transgenomic).
  • Eluent A 0.1 M TEAA, pH 7.0
  • eluent B 0.1 TEAA, 25% (v/v) acetonitrile, pH 7.0.
  • the gradient conditions were as follows:
  • the flow rate was 0.9 mL/min and the column temperature was 50°C. UV detection was performed at 260nm. Injection volume was 10 ⁇ L. 80 ⁇ L of 0.1 M TEAA was mixed with 20 ⁇ L of pGEM® DNA Marker prior to injection. The amount of DNA injected was 2 ⁇ g. The chromatogram is shown in FIG. 15.
  • EXAMPLE 2 DNA Fragment Sizing by MIPC using a 7.8 mm ID column Analysis of the pGEM DNA Markers was performed using the same conditions as described in Example 1 except that the separation column was replaced by a 50 mm x 7.8 mm ID column packed with separation beads from the same lot. The chromatogram is shown in FIG. 16.
  • the standard mixture was hybridized as recommended by the vendor (Trangenomic). Injection volume was 4 ⁇ L.
  • the mutation separation profile is shown in FIG. 18.
  • the mutation standard (part no. 440582 from Transgenomic) contained a 209 base pair fragment from the human Y chromosome, locus DYS271 with an A to G mutation at position 168 as described by Seielstad et al., Hum. Mol. Genet. 3:2159 (1994).
  • the gas introduction tube was pulled out of the solution and positioned above the liquid surface.
  • the reaction mixture was subsequently stirred for 6.5 hours at 87°C.
  • the contents of the reactor were cooled down to ambient temperature and diluted to a volume yielding a concentration of 54.6 g of polymerized styrene in 1000 mL volume of suspension resulting from the first step.
  • the amount of polymerized styrene in 1000 mL was calculated to include the quantity of the polymer still sticking to the mechanical stirrer (approximately 5 - 10 g).
  • the diameter of the spherical beads in the suspension was determined by light microscopy to be about 1.0 micron.
  • Beads resulting from the first step are still generally too small and too soft (low pressure stability) for use as chromatographic packings.
  • the softness of these beads is caused by an insufficient degree of crosslinking.
  • the beads are enlarged and the degree of crosslinking is increased.
  • the protocol for the second step is based on the activated swelling method described by Ugelstad et al. (Adv. Colloid Interface Sci., 13:101-140 (1980)).
  • the aqueous suspension of polystyrene seeds (200 ml) from the first step was mixed first with 60 mL of acetone and then with 60 mL of a 1-chlorododecane emulsion.
  • the reaction mixture was transferred into a separation funnel.
  • the excess amount of the monomer separated from the layer containing the suspension of the polymeric beads and could thus be easily removed.
  • the remaining suspension of beads was returned to the reactor and subjected to a stepwise increase in temperature (63°C for about 7 hours, 73°C for about 2 hours, and 83°C for about 12 hours), leading to further increases in the degree of polymerization (> 500).
  • the pore size of beads prepared in this manner was below the detection limit of mercury porosimetry ( ⁇ 3 ⁇ A).
  • the dried beads (10 g) from step two were washed four times with 100 mL of n-heptane, and then two times with each of the following: 100 mL of diethylether, 100 mL of dioxane, and 100 mL of methanol. Finally, the beads were dried.
  • Example 5 The following procedures were carried out under nitrogen (Air Products, Ultra Pure grade, Allentown, PA) at a flow rate of 250-300 mL/min. 25 g of the beads prepared in Example 5 were suspended in 150-160 g of 1- chlorooctadecane (product no. 0235, TCI America, Portland, OR) using a bow shaped mixer (use a 250 mL wide neck Erienmeyer flask). The temperature was set to 50-60°C to prevent the 1-chlorooctadecane from solidifying. Larger pieces of polymer were broken up to facilitate suspending. The solution was mixed using a stirrer (Model RZRI, Caframo, ONT NOH2T0, Canada) with the speed set at 2.
  • 1- chlorooctadecane product no. 0235, TCI America, Portland, OR
  • the temperature was set to 50-60°C to prevent the 1-chlorooctadecane from solidifying. Larger pieces of polymer were broken up to facilitate suspending.
  • the polymer suspension was transferred into a three neck bottle (with reflux condenser, overhead stirrer and gas inlet). 52-62 g of 1- chlorooctadecane were used to rinse the Erienmeyer flask and were added to the three neck bottle. The bottle was heated in an ethylene glycol bath set at 80°C. The solution was mixed using a stirrer (Caframo) with the speed set at 0. After 20 minutes, the reaction was started by addition of 1.1 g AICI 3 powder (product no. 06218, Fluka, Milwaukee, Wl) and continued for 16-18 h.
  • AICI 3 powder product no. 06218, Fluka, Milwaukee, Wl
  • EXAMPLE 8 Column packing procedure After weighing out 3 grams of oven dried polymeric beads, form a slurry with 10 mL tetrahydrofuran (THF) and place in a sonicator under a fume hood for 15 min. The add 5 mL of THF and 5 mL of methanol (MeOH) and sonicate an additional 10 min. Pre-fill a packing assembly with 20 mL MeOH. Pour the slurry slowly into the packing assembly. Turn on a Haskel pump (Haskel International, Inc., Burbank, CA) and slowly increase packing pressure to 5000 psi for the initial packing phase. After 10 min, slowly increase packing pressure to 9000 psi and set the secondary packing phase for 20 min. After 20 min, change the packing eluent from MeOH to 0.05 M Na 4 EDTA. Then set the final packing phase for 40 min.
  • THF tetrahydrofuran
  • MeOH methanol
  • EXAMPLE 9 DNA Fragment Sizing by MIPC using a 4.6 mm ID column MIPC analysis of ⁇ DNA Hind III digest (containing fragments of sizes 125; 564, 2027, 2322, 4361 , 6557, 9416 and 23130 bp) (part no. 030204, Kramel Biotech, Transgenomic Ltd., Northumberland, UK) was performed using octadecyl modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads packed in a 50 mm x 4.6 mm ID separation column (DNASEP® cartridge) and using a WAVE® DNA Fragment Analysis System.
  • Eluent A 0.1 M TEAA, pH 7.0
  • eluent B 0.1 TEAA, 25% (v/v) acetonitrile, pH 7.0.
  • the gradient conditions were as follows:
  • the flow rate was 0.9 mL/min and the column temperature was 50°C.
  • UV detection was performed at 260nm.
  • 40 ⁇ L of 0.1 M TEAA was mixed with 100 ⁇ L of ⁇ DNA Hind III digest solution. Injection volume was 15 ⁇ L. The amount of DNA injected was 2 ⁇ g.
  • the chromatogram is shown in FIG. 20.
  • DNA Fragment Sizing by MIPC using a 7.8 mm ID column Analysis of the ⁇ DNA Hind III digest was performed using the same conditions as described in Example 9 except that the separation column was replaced by a 50 mm x 7.8 mm ID packed column. The chromatogram is shown in FIG. 21.

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Abstract

L'invention concerne une colonne de séparation améliorée et un procédé correspondant pour séparer un mélange de fragments d'ADN à double brin par chromatographie MIPC. La colonne cylindrique, qui a un diamètre interne supérieur à environ 5 mm, contient des perles de polymère dont le diamètre moyen est compris entre 1 et 100 microns. Il s'agit de perles non substituées ou substituées par une fraction hydrocarbure qui présente entre 1 atome de carbone et un million d'atomes de carbone. De préférence, ces perles sont sensiblement dépourvues de cations multivalents qui peuvent se lier librement avec l'ADN. La colonne améliorée assure une meilleure séparation des fragments d'ADN ayant une taille comprise entre environ 100 et 20 000 paires de base. En outre, cette colonne assure une séparation améliorée pour les molécules d'ADN hétéroduplex et homoduplex dans une procédure de détection de mutation où la chromatographie est accomplie selon des conditions de dénaturation partielle de l'ADN en un site de paires de base présentant un défaut d'appariement.
EP00961961A 1999-09-17 2000-09-18 Colonne amelioree pour la separation de l'adn en chromatographie de nucleotides a ions apparies (mipc) Withdrawn EP1231993A4 (fr)

Applications Claiming Priority (11)

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US15461499P 1999-09-17 1999-09-17
US154614P 1999-09-17
US15874399P 1999-10-12 1999-10-12
US158743P 1999-10-12
US16751599P 1999-11-24 1999-11-24
US167515P 1999-11-24
US17717700P 2000-01-20 2000-01-20
US177177P 2000-01-20
US501775 2000-02-10
US09/501,775 US6372142B1 (en) 1996-11-13 2000-02-10 Column for DNA separation by matched ion polynucleotide chromatography
PCT/US2000/025532 WO2001019485A1 (fr) 1999-09-17 2000-09-18 Colonne amelioree pour la separation de l'adn en chromatographie de nucleotides a ions apparies (mipc)

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DE60031891T2 (de) * 2000-04-21 2007-09-06 Transgenomic, Inc., San Jose Reinigungsverfahren für säulen zur passenden ionischen polynukleotidchromatographie
US7135289B2 (en) 2001-10-05 2006-11-14 Transgenomic, Inc. Methods and compositions for mutation analysis of polynucleotides by liquid chromatography
EP1565261A1 (fr) * 2002-11-25 2005-08-24 Varian, Inc. Particules copolymeres macroporeuses de forme irreguliere et leurs procedes d'utilisation
JP5670544B2 (ja) * 2013-11-27 2015-02-18 第一三共株式会社 オンライン不純物除去装置及び方法
RU2563277C1 (ru) * 2014-04-07 2015-09-20 Валерий Геннадьевич Брусов Способ автоматического управления узлом разгонки отходов брагоректификационной установки
GB201610792D0 (en) * 2016-06-21 2016-08-03 Ge Healthcare Bioprocess R&D Ab Method for packing chromatography columns

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WO1998056797A1 (fr) * 1997-06-10 1998-12-17 Transgenomic, Inc. Systeme et procede permettant de realiser des separations de polynucleotides grace a la chromatographie en phase liquide
WO1999007899A1 (fr) * 1997-08-05 1999-02-18 Transgenomic, Inc. Chromatographie par denaturation d'ions apparies de polynucleotide permettant de detecter des mutations

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US5772889A (en) * 1995-11-13 1998-06-30 Transgenomic, Inc. System and method for performing nucleic acid separations using liquid chromatography
US6066258A (en) * 1997-12-05 2000-05-23 Transgenomic, Inc. Polynucleotide separations on polymeric separation media

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WO1998056797A1 (fr) * 1997-06-10 1998-12-17 Transgenomic, Inc. Systeme et procede permettant de realiser des separations de polynucleotides grace a la chromatographie en phase liquide
WO1999007899A1 (fr) * 1997-08-05 1999-02-18 Transgenomic, Inc. Chromatographie par denaturation d'ions apparies de polynucleotide permettant de detecter des mutations

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JP2003509662A (ja) 2003-03-11

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