WO2000043534A1 - Procede et dispositif de production automatisee de ligands d'acide nucleique - Google Patents

Procede et dispositif de production automatisee de ligands d'acide nucleique Download PDF

Info

Publication number
WO2000043534A1
WO2000043534A1 PCT/US2000/001001 US0001001W WO0043534A1 WO 2000043534 A1 WO2000043534 A1 WO 2000043534A1 US 0001001 W US0001001 W US 0001001W WO 0043534 A1 WO0043534 A1 WO 0043534A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
target
nucleic acids
ligand
candidate mixture
Prior art date
Application number
PCT/US2000/001001
Other languages
English (en)
Inventor
Larry Gold
Dominic A. Zichi
Robert Jenison
Daniel J. Schneider
Original Assignee
Somalogic, 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 US09/232,946 external-priority patent/US6569620B1/en
Application filed by Somalogic, Inc. filed Critical Somalogic, Inc.
Priority to CA002360748A priority Critical patent/CA2360748A1/fr
Priority to EP00909909A priority patent/EP1144669A4/fr
Priority to KR1020017009027A priority patent/KR20010101573A/ko
Priority to MXPA01007352A priority patent/MXPA01007352A/es
Priority to AU32092/00A priority patent/AU777823B2/en
Priority to JP2000594942A priority patent/JP2002534985A/ja
Publication of WO2000043534A1 publication Critical patent/WO2000043534A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • 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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1048—SELEX
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00364—Pipettes
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00689—Automatic using computers
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00691—Automatic using robots
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • This invention is directed to a method for the generation of nucleic acid ligands having specific functions against target molecules using the SELEX process.
  • the methods described herein enable nucleic acid ligands to be generated in dramatically shorter times and with much less operator intervention than was previously possible using prior art techniques.
  • the invention includes a device capable of generating nucleic acid ligands with little or no operator intervention.
  • SELEX process Systematic Evolution of Ligands by Exponential enrichment
  • nucleic acids have three dimensional structural diversity not unlike proteins.
  • the SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in United States Patent Application Serial No. 07/536,428, filed June 11, 1990, entitled “Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, United States Patent Application Serial No. 07/714, 131 , filed June 10, 1991, entitled "Nucleic Acid Ligands," now United States Patent No. 5,475,096, United States Patent Application Serial No.
  • Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule.
  • the SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three- dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.
  • the SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity.
  • the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand- enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
  • nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
  • the present inventors have recognized that SELEX or SELEX-like processes could be used to identify nucleic acids which can facilitate any chosen reaction in a manner similar to that in which nucleic acid ligands can be identified for any given target.
  • SELEX or SELEX-like processes could be used to identify nucleic acids which can facilitate any chosen reaction in a manner similar to that in which nucleic acid ligands can be identified for any given target.
  • the present inventors postulate that at least one nucleic acid exists with the appropriate shape to facilitate each of a broad variety of physical and chemical interactions.
  • the basic SELEX method has been modified to achieve a number of specific objectives. For example, United States Patent Application Serial No. 07/960,093, filed October 14, 1992, entitled “Method for Selecting Nucleic Acids on the Basis of Structure," (See United States Patent No.
  • the SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in United States Patent Application Serial No. 08/284,063, filed August 2, 1994, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,” now United States
  • the SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in United States Patent Application Serial No. 08/434,465, filed May 4, 1995, entitled "Nucleic Acid Ligand Complexes.”
  • the automated SELEX process can be used to generate nucleic acid ligands against a single target using many different selection conditions in a single experiment.
  • the present invention greatly enhances the power of the SELEX process, and will make SELEX the routine method for the isolation of ligands.
  • the present invention includes methods and apparatus for the automated generation of nucleic acid ligands against virtually any target molecule.
  • This process is termed the automated SELEX process.
  • the method uses a robotic manipulator to move reagents to one or more work stations on a work surface where the individual steps of the SELEX process are performed.
  • the individual steps include: 1) contacting the candidate nucleic acid ligands with the target molecule(s) of interest immobilized on a solid support; 2) partitioning the nucleic acid ligands that have interacted in the desired way with the target molecule on the solid support away from those nucleic acids that have failed to do so; and 3) amplifying the nucleic acid ligands that have interacted with the target molecule.
  • Steps 1-3 are performed for the desired number of cycles by the automated SELEX process and apparatus; the resulting nucleic acid ligands are then isolated and purified.
  • Figure 1 demonstrates the effect of blocking reagents on background binding of RNA to microtiter plates.
  • the total number of RNA molecules remaining in wells of an Immulon 1 polystyrene plate, quantified with QPCR as described below are displayed for wells treated with various blocking reagents, (1) SHMCK alone, (2) SuperBlock, (3) SCHMK + Iblock, (4) SCHMK + SuperBlock, (5) SCHMK + Casein, (6) SCHMK + BSA.
  • Figure 2 demonstrates the effect of buffer reagents on background binding of RNA to microtiter plates.
  • the total number of RNA molecules remaining in unblocked wells of an Immulon 1 polystyrene plate, quantified with QPCR as described below are displayed for wells incubated and washed with solutions containing various buffer reagents, (1) SHMCK + 0.1% Iblock + 0.05% Tween 20 (SIT), (2) SHMCK + 0.01 % HAS (SA), (3) SCHMK + 0.05% Tween 20 (ST), (4) SCHMK + 0.01 % HSA+ 0.05% Tween 20 (SAT), (5) SCHMK.
  • SIT SHMCK + 0.1% Iblock + 0.05% Tween 20
  • SA SHMCK + 0.01 % HAS
  • SA SCHMK + 0.05% Tween 20
  • ST SCHMK + 0.01 % HSA+ 0.05% Tween 20
  • SAT SCHMK
  • Figure 3 depicts the binding and EDTA elution of aptamer 1901 from murine PS-Rg passively hydrophobically attached to an Immulon 1 polystyrene plate.
  • the amount of eluted aptamer for each of these concentrations is shown by filled triangles, and the amount of aptamer remaining in the protein coated wells after elution is shown by open squares. All samples were quantified by scintillation counting of 32 P.
  • Figure 4 depicts the quantification of passive adsorption of PS-Rg to Immulon 1 polystyrene plates.
  • the amount of PS-Rg capable of binding aptamer 1901 after protein immobilization through hydrophobic interactions (filled circles) is displayed as a function of input protein concentration.
  • the amount of active protein was obtained from the plateau values of aptamer binding curves.
  • Figure 5 depicts the progress of the automated in vitro selection process.
  • the number of RNA molecules eluted from plate wells for both manual (squares) and automated (circles) experiments are displayed for each of five rounds of SELEX performed.
  • the amount of RNA eluted from protein coated wells is denoted by the filled markers and background binding RNA is denoted by open markers, and the amount of coated protein used in each round is denoted by x markers.
  • Figure 6 depicts the solution phase binding curves of round 5 RNA pools to murine PS-Rg protein.
  • the binding curve measured for the enriched round five RNA pool generated with the automated SELEX process (+) is compared to the manual process (filled circles) as well as the starting random RNA pool (filled diamonds).
  • Figure 7 shows a perspective view of an embodiment of an apparatus for performing automated SELEX according to the present invention.
  • Figure 8 shows a plan elevation view of an embodiment of an apparatus for performing automated SELEX according to the present invention.
  • Figure 9 shows a front elevation view of an embodiment of an apparatus for performing automated SELEX according to the present invention.
  • Figure 10 shows a right side elevation view of an embodiment of an apparatus for performing automated SELEX according to the present invention.
  • nucleic acid ligand is a non-naturally occurring nucleic acid having a desirable action on a target.
  • Nucleic acid ligands are also sometimes referred to in this applications as “aptamers” or “clones.”
  • a desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way which modifies/alters the target or the functional activity of the target, covalently attaching to the target as in a suicide inhibitor, facilitating the reaction between the target and another molecule.
  • the action is specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.
  • Nucleic acid ligands include nucleic acids that are identified from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target, by the method comprising: a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the target molecule are identified.
  • candidate mixture is a mixture of nucleic acids of differing sequence from which to select a desired ligand.
  • the source of a candidate mixture can be from naturally-occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids, enzymatically synthesized nucleic acids or nucleic acids made by a combination of the foregoing techniques.
  • candidate mixture is also referred to as "40N8 RNA,” or as "RNA pool.”
  • each nucleic acid has fixed sequences surrounding a randomized region to facilitate the amplification process.
  • nucleic acid means either DNA, RNA, single-stranded or double- stranded, and any chemical modifications thereof.
  • Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.
  • modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like.
  • Modifications can also include 3' and 5' modifications such as capping.
  • SELEX methodology involves the combination of selection of nucleic acid ligands which interact with a target in a desirable manner, for example binding to a protein, with amplification of those selected nucleic acids. Optional iterative cycling of the selection/amplification steps allows selection of one or a small number of nucleic acids which interact most strongly with the target from a pool which contains a very large number of nucleic acids. Cycling of the selection amplification procedure is continued until a selected goal is achieved.
  • the SELEX methodology is described in the SELEX Patent Applications.
  • SELEX target or “target” means any compound or molecule of interest for which a ligand is desired.
  • a target can be a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. without limitation.
  • solid support is defined as any surface to which molecules may be attached through either covalent or non-covalent bonds. This includes, but is not limited to, membranes, plastics, paramagnetic beads, charged paper, nylon, Langmuir-Bodgett films, functionalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold and silver. Any other material known in the art that is capable of having functional groups such as amino, carboxyl, thiol or hydroxyl inco ⁇ orated on its surface, is also contemplated. This includes surfaces with any topology, including, but not limited to, spherical surfaces grooved surfaces, and cylindrical surfaces.
  • Partitioning means any process whereby ligands bound to target molecules can be separated from nucleic acids not bound to target molecules. More broadly stated, partitioning allows for the separation of all the nucleic acids in a candidate mixture into at least two pools based on their relative affinity to the target molecule. Partitioning can be accomplished by various methods known in the art. Nucleic acid-protein pairs can be bound to nitrocellulose filters while unbound nucleic acids are not. Columns which specifically retain nucleic acid- target complexes can be used for partitioning. For example, oligonucleotides able to associate with a target molecule bound on a column allow use of column chromatography for separating and isolating the highest affinity nucleic acid ligands.
  • Beads upon which target molecules are conjugated can also be used to partition nucleic acid ligands in a mixture.
  • Surface plasmon resonance technology can be used to partition nucleic acids in a mixture by immobilizing a target on a sensor chip and flowing the mixture over the chip, wherein those nucleic acids having affinity for the target can be bound to the target, and the remaining nucleic acids can be washed away.
  • Liquid-liquid partitioning can be used as well as filtration gel retardation, and density gradient centrifugation. In its most basic form, the SELEX process may be defined by the following series of steps:
  • a candidate mixture of nucleic acids of differing sequence is prepared.
  • the candidate mixture generally includes regions of fixed sequences (i.e., each of the members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences.
  • the fixed sequence regions are selected either: a) to assist in the amplification steps described below; b) to mimic a sequence known to bind to the target; or c) to enhance the concentration of a given structural arrangement of the nucleic acids in the candidate mixture.
  • the randomized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).
  • the candidate mixture is contacted with the selected target under conditions favorable for binding between the target and members of the candidate mixture. Under these circumstances, the interaction between the target and the nucleic acids of the candidate mixture can be considered as forming nucleic acid-target pairs between the target and those nucleic acids having the strongest affinity for the target.
  • nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a certain amount of the nucleic acids in the candidate mixture are retained during partitioning.
  • nucleic acids selected during partitioning as having relatively higher affinity to the target are then amplified to create a new candidate mixture that is enriched in nucleic acids having a relatively higher affinity for the target.
  • the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase.
  • the SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.
  • targets that can be used in the process; methods for the preparation of the initial candidate mixture; methods for partitioning nucleic acids within a candidate mixture; and methods for amplifying partitioned nucleic acids to generate enriched candidate mixtures.
  • the automated SELEX method uses one or more computer-controlled cartesian robotic manipulators to move solutions to and from a work station located on a work surface. The individual steps of the SELEX process are carried out at the work station.
  • each robotic manipulator is a movable arm that is capable of carrying tools in both horizontal and vertical planes.
  • One tool contemplated is a pipetting tool.
  • a robotic manipulator uses the pipetting tool to pick up liquid from a defined location on the work surface and then dispense the liquid at a different location.
  • the pipetting tool can also be used to mix liquids by repeatedly picking up and ejecting the liquid i.e. "sip and spit" mixing.
  • the robotic manipulator is also able to eject a disposable tip from the pipetting tool into a waste container, and then pick up a fresh tip from the appropriate station on the work surface.
  • the pipetting tool is connected to one or more fluid reservoirs that contain some of the various buffers and reagents needed in bulk for the SELEX process.
  • a computer controlled valve determines which solution is dispensed by the pipetting tool.
  • the pipetting tool is further able to eject liquid at desired locations on the work surface without the outside of the tip coming in contact with liquid already present at that location. This greatly reduces the possibility of the pipette tip becoming contaminated at each liquid dispensing step, and reduces the number of pipette tip changes that must be made during the automated SELEX process.
  • tips that are used at certain steps of the automated SELEX process can be reused.
  • a tip can be reused if it is used in each cycle of the SELEX process to dispense the same reagent.
  • the tip can be rinsed after each use at a rinse station, and then stored in a rack on the work surface until it is needed again. Reusing tips in this way can drastically reduce the number of tips used during the automated SELEX process.
  • a vacuum aspiration system is also attached to a separate robotic manipulator.
  • This system uses a fine needle connected to a vacuum source to withdraw liquid from desired locations on the work surface without immersing the needle in that liquid.
  • the pipetting tool and the vacuum aspirator are associated with separate robotic manipulators, the pipetting tool and the aspiration system can work simultaneously at different locations on the work surface.
  • a robotic manipulator is also capable of moving objects to and from defined locations on the work surface.
  • objects include lids for multi-well plates, and also the various pieces of apparatus used in the embodiments outlined below.
  • the robotic manipulator uses a "gripper" to mechanically grasp objects.
  • the vacuum aspiration system described above is also used to power a suction cup that can attach to the object to be moved.
  • the fine needle described above can pick up a suction cup, apply a vacuum to the cup, pick up an object using the suction cup, move the object to a new location, release the object at the new location by releasing the vacuum, then deposit the suction cup at a storage location on the work surface.
  • Suitable robotic systems contemplated in the invention include the MultiPROBETM system (Packard), the Biomek 200TM (Beckman Instruments) and the TecanTM (Cavro).
  • the system uses three robotic manipulators: one carries the pipetting tool, one carries a vacuum aspirator, and one carries the fluorometry cover (see below).
  • the automated SELEX process method involves:
  • Steps (a)-(g) are performed automatically by the computer-controlled robotic manipulator.
  • the computer also measures and stores information about the progress of the automated SELEX process procedure, including the amount of nucleic acid ligand eluted from the target molecule prior to each amplification step.
  • the computer also controls the various heating and cooling steps required for the automated SELEX process.
  • the work surface comprises a single work station where the individual SELEX reactions take place. This station comprises heating and cooling means controlled by the computer in order to incubate the reaction mixtures at the required temperatures.
  • One suitable heating and cooling means is a Peltier element.
  • the work station preferably also comprises a shaking mechanism to insure that SELEX reaction components are adequately mixed.
  • the work surface also comprises stations in which the enzymes necessary for SELEX are stored under refrigeration, stations where wash solutions and buffers are stored, stations where tools and apparatus are stored, stations where tools and apparatus may be rinsed, and stations where pipette tips and reagents are discarded.
  • the work surface may also comprise stations for archival storage of small aliquots of the SELEX reaction mixtures. These mixtures may be automatically removed from the work station by the pipetting tool at selected times for later analysis.
  • the work surface may also comprise reagent preparation stations where the robotic manipulator prepares batches of enzyme reagent solutions in preparation vials immediately prior to use.
  • the work surface comprises more than one work station.
  • the individual steps of the automated SELEX process are carried out at discrete work stations rather than at a single multi-functional work station.
  • the solutions of candidate nucleic acid mixtures are transferred from one work station to another by the robotic manipulator. Separate work stations may be provided for heating and cooling the reaction mixtures.
  • the individual steps of the automated SELEX process are carried out in a containment vessel that is arranged in an array format.
  • This allows many different SELEX reactions—using different targets or different reaction conditions—to take place simultaneously on a single work station.
  • the individual steps may be performed in the wells of microtitre plates, such as Immulon 1 plates.
  • an array of small plastic tubes is used. Typical tube arrays comprise 96 0.5 ml round-bottomed, thin- walled polypropylene tubes laid out in a 8 x 12 format. Arrays can be covered during the heating and cooling steps to prevent liquid loss through evaporation, and also to prevent contamination.
  • lids including heated lids
  • arrays allow the use of multipipettor devices, which can greatly reduce the number of pipetting steps required.
  • well will be used to refer to an individual containment vessel in any array format.
  • Solid supports suitable for attaching target molecules are well known in the art. Any solid support to which a target molecule can be attached, either covalently or non-covalently, is contemplated by the present invention. Covalent attachment of molecules to solid supports is well known in the art, and can be achieved using a wide variety of derivatization chemistries. Non-covalent attachment of targets can depend on hydrophobic interactions; alternatively, the solid support can be coated with streptavidin which will bind strongly to a target molecule that is conjugated to biotin.
  • the solid support is a paramagnetic bead.
  • complexes of target molecules and nucleic acid ligands can be rapidly partitioned from the candidate mixture by the application of a magnetic field to the wells.
  • the magnetic field is applied by an array of electromagnets adjacent to the walls of each well; when the electromagnets are activated by the computer, paramagnetic target beads are held to the sides of the wells.
  • the magnets can either be an integral part of the work station, or they can be attached to a cover that is lowered over the work station by the robotic manipulator.
  • the magnetic separator cover allows the magnets to be placed adjacent to the wells without blocking access to the wells themselves. In this way, the wells are accessible by the pipetting and aspirating units when the cover is in place. Following magnet activation, liquid can be aspirated from the wells, followed by the addition of wash solutions.
  • the magnetic separator cover can be stored on the work surface.
  • the magnets in the separator cover are permanent magnets. In this case, withdrawing the cover removes the influence of the magnets, and allows the beads to go into suspension.
  • the magnets used for bead separation are attached to a series of bars that can slide between adjacent rows of wells.
  • Each bar has magnets regularly spaced along its length, such that when the bar is fully inserted between the wells, each well is adjacent to at least one magnet.
  • an 8x12 array of wells would have 8 magnet bars, each bar with 12 magnets.
  • bead separation is achieved by inserting the bars between the wells; bead release is accomplished by withdrawing the bars from between the wells.
  • the array of bars can be moved by a computer-controlled stepper motor.
  • the paramagnetic target beads used in the above embodiments are preferably stored on the work surface in an array format that mirrors the layout of the array format on the work station.
  • the bead storage array is preferably cooled, and agitated to insure that the beads remain in suspension before use.
  • Beads can be completely removed from the wells of the work station using a second array of magnets.
  • this second array comprises an array of electromagnets mounted on a cover that can be placed by the robotic manipulator over the surface of the individual wells on the work station.
  • the electromagnets on this bead removal cover are shaped so that they project into the liquid in the wells.
  • the electromagnets are activated, the beads are attracted to them.
  • the beads can be efficiently removed from the work station.
  • the beads can either be discarded, or can be deposited back in the bead storage array for use in the next cycle of automated SELEX.
  • the bead removal cover can then be washed at a wash station on the work surface prior to the next bead removal step.
  • the automated SELEX process begins when the pipetting tool dispenses aliquots of the beads— with their bound target— to the individual wells of a microtitre plate located on the work station.
  • Each well already contains an aliquot of a candidate mixture of nucleic acid ligands previously dispensed by the robotic manipulator.
  • the robot optionally "sips and spits" the contents of each well up and down several times to facilitate thorough mixing.
  • the microtitre plate is then incubated at a preselected temperature on the work station in order to allow nucleic acid ligands in the candidate mixture to bind to the bead-bound target molecule. Agitation of the plate insures that the beads remain in suspension.
  • the magnetic separator cover is placed over the microtitre plate by the robotic manipulator.
  • the beads are then held to the sides of the wells, and the aspirator tool removes the solution containing unbound candidate nucleic acids from the wells.
  • a washing solution such as a low salt solution, can then be dispensed into each well by the pipetting tool.
  • the beads are released from the side of the wells by withdrawing the magnetic separator cover or deactivating the electromagnets, then resuspended in the wash solution by agitation and "sip and spit" mixing.
  • the magnetic separator cover is placed over the plate again, and the wash solution is aspirated. This wash loop can be repeated for a pre-selected number of cycles. At the end of the wash loop, the beads are held by the magnets to the sides of the empty wells.
  • the beads can then be resuspended in a solution designed to elute the nucleic acid ligands from the target molecule, such as dH 2 0.
  • the dissociation of nucleic acid ligand from target can also be achieved by heating the beads to a high temperature on the work station.
  • the pipetting tool can dispense into the wells the enzyme and buffer components necessary to perform amplification of the candidate nucleic acid ligands. After amplification, purification and quantification (see below), a predetermined amount of the amplified candidate mixture can then used in the next cycle of the automated SELEX process.
  • the pipetting tool can remove an aliquot of the candidate mixture and store it in an archive plate for later characterization. Furthermore, during incubation periods, the pipetting tool can prepare reaction mixtures for other steps in the SELEX process.
  • the preferred embodiments of the automated SELEX process method and apparatus use microtitre plates and magnetic beads to achieve selection.
  • the target molecule is coupled directly to the surface of the microtitre plate. Suitable methods for coupling in this manner are well known in the art.
  • the target molecule is coupled to affinity separation columns known in the art. The robotic device would dispense the candidate mixture into such a column, and the bound nucleic acid ligands could be eluted into the wells of a microtitre plate after suitable washing steps.
  • the solid support used in the automated SELEX process method is a surface plasmon resonance (SPR) sensor chip.
  • SPR surface plasmon resonance
  • the use of SPR sensor chips in the isolation of nucleic acid ligands is described in WO 98/33941, entitled “Flow Cell SELEX,” incorporated herein by reference in its entirety.
  • a target molecule is coupled to the surface of a surface plasmon resonance sensor chip.
  • the refractive index at the junction of the surface of the chip and the surrounding medium is extremely sensitive to material bound to the surface of the chip.
  • a candidate mixture of nucleic acid ligands is passed over the chip by the robotic device, and the kinetics of the binding interaction between the chip-bound target and nucleic acid ligands is monitored by taking readings of the resonance signal from the chip. Such readings can be made using a device such as the BIACore 2000TM (BIACore, Inc.). Bound nucleic acid ligands can then be eluted from the chip; the kinetics of dissociation can be followed by measuring the resonance signal. In this way it is possible to program the computer that controls the automated SELEX process to automatically collect nucleic acid ligands which have a very fast association rate with the target of interest and a slow off rate.
  • the collected nucleic acid ligands can then be amplified and the automated SELEX process cycle can begin again.
  • the solid support is a non-paramagnetic bead. Solutions can be removed from the wells containing such beads by aspirating the liquid through a hole in the well that is small enough to exclude the passage of the beads. For example, a vacuum manifold with a 0.2 ⁇ M filter could be used to partition 100 ⁇ M beads.
  • the resulting nucleic acid ligands can be isolated from the automated SELEX process apparatus for sequence analysis and cloning.
  • the candidate nucleic acid ligands must be amplified.
  • the amplification is achieved using the Polymerase Chain Reaction (PCR).
  • PCR Polymerase Chain Reaction
  • primers that bind to these regions are used to facilitate PCR.
  • the beads are removed from the wells before beginning the amplification procedure.
  • this can be done using the magnetic removal system described above.
  • Candidate nucleic acid ligands can be single-stranded DNA molecules, double- stranded DNA molecules, single-stranded RNA molecules, or double-stranded RNA molecules.
  • RNA nucleic acid ligands can be single-stranded DNA molecules, double- stranded DNA molecules, single-stranded RNA molecules, or double-stranded RNA molecules.
  • RT-PCR can be carried out using the automated SELEX process method by dispensing the necessary enzymes, primers and buffers to the wells on the work station containing the eluted ligand. The resulting reaction mixtures are then first incubated on the work station at a temperature that promotes reverse transcription.
  • the work station thermally-cycles the reaction mixtures to amplify the cDNA products.
  • the amount of amplified product is then measured to give a value for the amount of candidate nucleic acid ligand eluted from the target (see below).
  • the amplified DNA molecules must be transcribed to regenerate the pool of candidate RNA ligands for the next cycle of automated SELEX.
  • This can be achieved by using primers in the amplification step that contain sites that promote transcription, such as the T7 polymerase site. These primers become incorporated into the amplification product during the PCR step. Transcription from these sites can be achieved simply by dispensing the appropriate enzymes and buffer components into the amplified mixtures and then incubating at the appropriate temperature. A predetermined amount of the amplified mixture is then used in the next cycle of the automated SELEX process. Purification of RNA Ligands from Amplification Mixtures
  • amplified RNA ligands are purified from their DNA templates before beginning the next cycle of automated SELEX. This can be done using a second set of paramagnetic beads to which primers complementary to the 3' constant region of the RNA ligands are attached. When these primer beads are added to the transcribed amplification mixture, the newly transcribed full length RNA ligands hybridize to the bead-bound primer, whereas the amplified double-stranded DNA molecules remain in solution. The beads can be separated from the reaction mixture by applying a magnetic field to the wells and aspirating the liquid in the wells, as described above.
  • the beads can then be washed in the appropriate buffer at a preselected temperature, and then the RNA ligands may be eluted from the beads by heating in an elution buffer (typically dH 2 0). Finally, the beads may be removed from the wells on the work station, as described above to leave only a solution of candidate RNA ligands remaining in the wells. This point marks the completion of one cycle of the automated SELEX procedure.
  • an elution buffer typically dH 2 0
  • the amount of primer bead added determines the amount of RNA ligand that is retained in the wells. Therefore, the amount of RNA ligand that is used in the next cycle of the automated SELEX procedure can be controlled by varying the amount of primer bead that is added to the amplification mixture.
  • the amount of RNA ligand that is to be used can be determined through quantitation of the amount of PCR product (see below). Calculation of the Amount of Eluted Nucleic Acid Ligand in Each Amplification Mixture In certain embodiments, it may be important to measure the amount of candidate nucleic acid ligand eluted from the target before beginning the next cycle of the automated SELEX process. Such measurements yield information about the efficiency and progress of the selection process.
  • the automated SELEX process method uses a novel system for the automated real-time quantitation of PCR products during amplification. This, in turn, permits the progress of the selection experiment to be monitored in real time during the automated SELEX process.
  • the automated SELEX process method uses a fluorophore/quencher pair primer system. This system is used to calculate automatically the amount of eluted nucleic acid ligand introduced into the reaction mixture by measuring the fluorescence emission of the amplified mixture.
  • the PCR reaction is carried out using primers that have a short hairpin region attached to their 5' ends.
  • the stem of the hairpin has a fluorophore attached to one side and a quencher attached on the other side opposite the fluorophore.
  • the quencher and the fluorophore are located close enough to one another in the stem that efficient energy transfer occurs, and so very little fluorescent signal is generated upon excitation of the fluorophore. Examples of such primers are described in Example 2.
  • polymerase extension of the 3' end of DNA molecules that anneal to the primer disrupts the stem of the hairpin.
  • PCR reaction kinetics can be monitored in real time. In this way, the amount of candidate nucleic acid ligand eluted from target in each reaction can be quantitated. This information in turn is used to follow the progress of the selection process.
  • the candidate nucleic acid ligand templates are quantitated using the TaqManTM probe PCR system available from Roche Molecular Systems.
  • a TaqManTM probe is an oligonucleotide with a sequence complementary to the template being detected, a fluorophore on the 5' end, and a quencher on the 3' end.
  • the probe is added to a standard PCR reaction and anneals to the template between the primer binding sites during the annealing phase of each PCR cycle.
  • the probe is degraded by the 5'- 3' exonuclease activity of Taq Polymerase, separating the fluorophore from the quencher and generating a signal.
  • the probe Before PCR begins, the probe is intact and the excitation energy of the fluorophore is non-radioactively transferred to the quencher. During PCR, as template is amplified, the probe is degraded and the amount of fluorescent signal generated is directly proportional to the amount of PCR product formed.
  • the current invention contemplates the use of fluorometry instruments that can monitor the fluorescence emission profile of the reaction mixture(s) on the work station during thermal-cycling.
  • Suitable instruments contemplated comprise a source for excitation of the fluorophore, such as a laser, and means for measuring the fluorescence emission from the reaction mixture, such as a Charge Coupled Device (CCD) camera.
  • CCD Charge Coupled Device
  • Appropriate filters are used to select the correct excitation and emission wavelengths.
  • a fluorometry instrument mounted on an optically-transparent cover that can be placed over the wells on the work station by the robotic manipulator. When placed over the wells and then covered with a light shield, this fluorometry cover can capture an image of the entire array at pre-selected intervals. The computer interprets this image to calculate values for the amount of amplified product in each well at that time.
  • the robotic manipulator removes the light shield and fluorometry cover and returns them to a storage station on the work surface.
  • measurements of PCR product quantity are used to determine a value for the amount of eluted nucleic acid ligand introduced as template into the amplification reaction mixture. This can be done by comparing the amount of amplified product with values stored in the computer that were previously obtained from known concentrations of template amplified under the same conditions.
  • the automated SELEX process apparatus automatically performs control PCR experiments with known quantities of template in parallel with the candidate nucleic acid amplification reactions. This allows the computer to re-calibrate the fluorescence detection means internally after each amplification step of the automated SELEX process.
  • the value for the amount of candidate nucleic acid ligand eluted from the target is used by the computer to make optimizing adjustments to any of the steps of the automated SELEX process method that follow.
  • the computer can change the selection conditions in order to increase or decrease the stringency of the interaction between the candidate nucleic acid ligands and the target.
  • the computer can also calculate how much of the nucleic acid ligand mixture and/or target bead should be used in the next SELEX cycle. In embodiments using primer beads (above), the computer uses this information to determine the amount of primer bead suspension to be added to each well on the work station.
  • the computer can change the conditions under which the candidate nucleic acid ligands are amplified. All of this can be optimized automatically without the need for operator intervention.
  • Figures 7-10 show various views of an embodiment of an apparatus for performing automated SELEX according to the present invention. This embodiment is based on the
  • TecanTM (Cavro) robot system Each view shows the apparatus during the PCR amplification stage of the automated SELEX process.
  • FIGURE 7 a perspective view of this apparatus is shown.
  • the system illustrated comprises a work surface 71 upon which the work station 72 is located (work station is partially obscured in this perspective view but can be seen in FIGURES 8, 9 and 10 as feature 72).
  • the pipetting tool 74 and the aspirator 75 are attached to a central guide rail 73 by separate guide rails 77 and 78 respectively.
  • the pipetting tool 74 can thus move along the long axis of guide rail 77; guide rail 77 can then move orthogonally to this axis along the long axis of central guide rail 73.
  • the pipetting tool 74 can move throughout the horizontal plane; the pipetting tool can also be raised away from and lowered towards the work surface 71.
  • aspirator 75 is attached to guide rail 78, and guide rail 78 is attached to central guide rail 73 in such a way that aspirator 75 can move in the horizontal plane; aspirator 75 can also move in the vertical plane.
  • the fluorometry cover 76 is attached to guide rail 79 via bracket 710. Bracket 710 can move along the vertical axis of guide rail 79, thereby raising fluorometry cover 76 above the work station 72. When fluorometry cover 76 is positioned at the top of guide rail 79, then guide rails 77 and 78 can move underneath it to allow the pipetting tool 74 and the aspirator 75 to have access to work station 72. In this illustration, the fluorometry cover 76 is shown lowered into its working position on top of the work station 72.
  • Fluorometry cover 76 is attached to a CCD camera 71 la and associated optics 71 lb. A source of fluorescent excitation light is associated with the cover 76 also (not shown). When positioned on top of the work station 72, the cover 76 allows the CCD camera 71 la to measure fluorescence emission from the samples contained on the work station 72 during PCR amplification. For clarity, the light shield—which prevents ambient light from entering the fluorometry cover— is omitted from the drawing. When PCR amplification is finished, fluorometry cover 76, with attached CCD camera 711a and optics 71 lb, is simply raised up guide rail 79. Also not visible in this view, but visible in FIGURES 9 and 10, is the heated lid 91 , which is resting on top of the work station 72 underneath the fluorometry cover 76.
  • the work surface 71 also comprises a number of other stations, including: 4°C reagent storage stations 712, a -20°C enzyme storage station 713, ambient temperature reagent storage station 714, solution discard stations 715, pipette tip storage stations 716 and archive storage stations 717.
  • Pipetting tool 74 is also associated with a gripper tool 718 that can move objects around the work surface 71 to these various storage locations. Lid park
  • FIGURE 719 (shown unoccupied here) is for storage of the heated lid (see FIGURES 9 and 10).
  • FIGURE 8 shows the instrument of FIGURE 7 in a plan elevation view. Each element of the instrument is labelled with the same nomenclature as in FIGURE 7.
  • FIGURE 9 is a front elevation view of the instrument in FIGURE 7. Note that each element of the instrument is labelled with the same nomenclature as in FIGURE 7 and
  • FIGURE 8 Note also that in this view, it can be seen that work station 72, and chilled enzyme and reagent storage stations 712 are each associated with shaking motors 92.
  • the motors 92 are each under computer control, and can be momentarily stopped to allow reagent addition or removal, as appropriate, to the receptacle that is being agitated.
  • heated lid 91 which is resting on top of work station 72 to insure uniform heating of the samples.
  • FIGURE 10 is a right side elevation view of the instrument shown in FIGURES 7, 8 and 9. Every element of the instrument is labelled with the same nomenclature as in
  • Example 1 is illustrative embodiments of the invention. They are not to be taken as limiting the scope of the invention.
  • Example 1
  • the basis of the robotic workstation is a Packard MULTIProbe 204DTTM, a four probe pipetting station that utilizes disposable pipette tips to minimize nucleic acid contamination.
  • the workspace contains a 37°C constant temperature heat block used for equilibration of the binding reaction and in vitro transcription, a computer controlled thermal cycler for both RT and PCR reactions, a freezer unit for cold enzyme storage, various vessels for reagent storage, e.g., buffers, primers and mineral oil, and disposable pipette tip racks.
  • the tip racks utilize the greatest area on the work surface and vary depending on the number of samples processed in parallel. All steps for in vitro selection take place either on the heat block or in the thermal cycler, liquids are transferred primarily between these two stations, although some enzyme buffers are premixed in an adjacent reagent block prior to transfer to the plate or thermal cycler.
  • HAM High level Access Method
  • DOS based C programming language interpreter augmented with liquid handling functions for the Packard MULTIProbe.
  • HAM supports window based screen io, file handling, and RS-232 serial communications.
  • the software automatically adjusts the process to run any number of samples between one and 96, preparing only those enzyme solutions necessary during the current run.
  • Two way communication with the thermal cycler established with an RS-232 connection, allows the main computer to perform lid opening/closing operations, initiate programs stored on thermal cycler and monitor thermal cycler programs for completion.
  • the overall software design enables complete computer control of the process, from binding reaction incubation through transcription, to occur with no user intervention.
  • the process begins by placing a microtiter plate coated with protein on the 37°C block. All subsequent liquid handling up to gel purification of the enriched RNA pool is controlled by the software. During the initial two hour incubation of RNA with immobilized protein target, dH 2 0 is periodically added to the samples (to control evaporative loss) and each solution is mixed by repeated aspiration and dispensing, so-called sip-and-spit. After the binding reaction has equilibrated, partitioning bound from free RNA is easily accomplished in this format by simply removing the RNA solution from each well; bound nucleic acid remains on the immobilized target and unbound molecules are disposed.
  • Partitioning is followed by a series of wash steps, each wash comprised of pipetting a wash buffer solution into each well with subsequent repeated sip-and-spit mixing and finally disposal of the wash solution.
  • the elution process begins by addition of EDTA followed by a 30 minute incubation with periodic sip-and-spit mixing. After incubation, the solution is transferred to the thermal cycler and the wells washed as described above, with the exception that each wash solution here is added to the eluted material in the cycler. The sample is then ready for enzymatic amplification.
  • the first step for each of the three enzyme reactions requires the preparation of a fresh enzyme solution. This is done by pipetting an aliquot of enzyme from the freezer to the appropriate buffer located in the reagent block. The viscous enzyme solution is mixed carefully and thoroughly using slow sip-and-spit mixing to avoid foaming of detergents in the enzyme solution. An aliquot of the freshly prepared RT reaction mixture is added to the dry wells of the eluted plate for a wash to remove possible eluted RNA remaining in the well. The RT reaction mixture wash is then added to the appropriate well in the thermal cycler and capped with silicone oil to prevent evaporative loss during reaction incubation at 48°C. The thermal cycler lid is closed and a program initiated for the RT reaction.
  • the main computer monitors the reaction progress and upon detecting program completion, the lid is opened, a Taq polymerase reaction mixture is prepared and added to each completed RT reaction. This is followed by lid closure, PCR program initiation, monitoring and lid opening upon completion of PCR.
  • An aliquot of the amplified DNA is moved from the thermal cycler to appropriate wells in the 37°C plate for in vitro transcription of the DNA template.
  • a freshly prepared T7 RNA polymerase solution is added to each well thoroughly mixed.
  • a layer of silicone oil caps the reaction mixture that then incubates for 4 hours. This completes the automated process; the resulting transcribed RNA is gel purified offline and added to a microtiter plate with freshly coated protein wells for the next round of SELEX.
  • Typical Automated SELEX Process Run A typical automated SELEX process run using a multiwell plate begins with loading the various reagents and materials needed to the appropriate locations on the work surface.
  • Separate primer beads by placing magnetic separation cover on plate; aspirate each well; remove separation cover; pipette wash buffer to each well; incubate plate at 48°C for 5 minutes with shaking.
  • step 20 Repeat step 20) for the desired number of wash cycles.
  • Murine PS-Rg a recombinant murine selectin/IgG fusion (purchased from D. Vestweber) was manually coated in concentrations stated in results in 75 ⁇ l SHMCK buffer (10 mM HEPES pH 7.3, 120 mM NaCl, 5 mM KC1, 1 mM MgCl 2 , 1 mM CaCl 2 ) for two hours at room temperature (23°C) onto a round bottom Immulon 1 polystyrene 96 well microtiter plate. Control wells were prepared by coating SHMCK alone.
  • RNA pool was added in 75 ⁇ l SAT buffer.
  • the plate was placed on a 37°C heat block (USA Scientific) mounted on a MultiPROBE 204DT pipetting workstation (Packard) and samples were incubated uncovered at 37°C for two hours. All subsequent steps were performed by the robotic workstation except where noted. Every twenty minutes during the incubation of the RNA with the plate, 5 ⁇ l of dH 2 O was added to compensate for evaporative loss (rate of loss measured at 14.5 + 0.4 ⁇ l/hour) and to mix the reactions. Plates were then washed six times with 150 ⁇ l SAT buffer.
  • the RT mix was then moved into the thermocycler, added to the eluted RNA, and thoroughly mixed. To this 25 ⁇ l of silicone oil (Aldrich) was added to prevent evaporation. The thermocycler was then remotely turned on by the computer. The lid was closed and the reaction incubated at 48°C for 30 minutes followed by 60°C for 5 minutes. Upon completion of the RT reaction the lid was triggered to open and 10 ⁇ l of the reaction was manually removed to be measured manually by quantitative PCR
  • Taq polymerase (Perkin Elmer) stored in the Styrofoam cooler, was added to a prepared PCR buffer (Perkin Elmer Buffer 2 (50 mM KCL, 10 mM Tris-HCl pH 8.3), 7.5 mM MgCl 2 , 400 pmoles 5P8) and thoroughly mixed. 100 ⁇ l of the Taq mix was then added to each well, the lid closed, and PCR was initiated. PCR was run under the following conditions: 93°C for 3 minutes followed by a loop consisting of 93°C for 1 minute, 53°C for 1 minute, and 72°C for 1 minute for n cycles where n was determined by the input amount of RNA to the RT reaction (see qPCR description). Upon completion of PCR the lid was opened and 50 ⁇ l was removed and added to an empty plate well on the fixed 37°C heat block.
  • Perkin Elmer Buffer 2 50 mM KCL, 10 mM Tris-HCl pH 8.3
  • T7 RNA polymerase stored in the Styrofoam cooler, was added to a prepared Transcription buffer (40 mM Tris-HCl pH 8, 4% (w/v) PEG-8000,12 mM MgCl 2 , 5 mM DTT, 1 mM Spermidine, 0.002% Triton X-100, 100 units/ml pyrophosphatase (Sigma)) and thoroughly mixed. 200 ⁇ l of the Transcription buffer was then added to the PCR product well and mixed. To this reaction a 25 ⁇ l layer of silicone oil was added and the reaction was incubated for 4 hours at 37°C. The completed reaction was then removed and purified manually by PAGE. Plate Characterization 1. Test of various blocking agents
  • PS-Rg loading is indicated in ⁇ g/ml concentrations. (Binding of PS-Rg to the plate surface has been measured by loading fixed amounts of PS-Rg, washing as described, and then performing a binding curve by titrating high affinity aptamer #1901. This is done with several protein concentrations.
  • primers (5P7-FD2 and 5P8-FD2) were designed wherein the underlined portions are complementary to the N7 and N8 templates.
  • the hairpin in each primer has a Tm of ⁇ 85°C, and contains a fluorophore (6-FAM) on its 5' terminus and a quencher (DABCYL) opposite the fluorophore on its stem.
  • 6-FAM fluorophore
  • DABCYL quencher
  • the efficiency of energy transfer is dependent on the sixth power of the distance between the fluorophore and quencher. Because the fluorophore and quencher are in very close proximity in the closed hairpin conformation, little signal is generated by unincorporated primer. However, as primer is inco ⁇ orated into product during PCR, the fluorophore and quencher are further separated by a distance of 10 base pairs, and signal is increased. The increase in signal is directly proportional to the amount of product formed.
  • the equation for the standard curve can then be used to calculate template copy numbers in unknowns based on the Ct values.
  • This quantitative PCR technique was used to measure signal to noise ratios and absolute template copy number in a SELEX targeting PDGF adsorbed to polystyrene plates. Because very low protein loadings were used ( ⁇ 100 amol/reaction), quantitation by radiation was not possible.
  • An amplification plot illustrates quantitation of 10 amol RNA bound to the background well and 600 amol RNA bound to the target well, for a signal-to-noise ratio of 60.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

La présente invention comprend un procédé et un dispositif permettant de réaliser un SELEX (évolution systématique de ligand par enrichissement exponentiel) automatisé. Les étapes du procédé SELEX se déroulent sur un ou plusieurs postes de travail sur une surface de travail au moyen d'un manipulateur robotisé à coordonnées cartésiennes commandé par un ordinateur.
PCT/US2000/001001 1999-01-19 2000-01-14 Procede et dispositif de production automatisee de ligands d'acide nucleique WO2000043534A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA002360748A CA2360748A1 (fr) 1999-01-19 2000-01-14 Procede et dispositif de production automatisee de ligands d'acide nucleique
EP00909909A EP1144669A4 (fr) 1999-01-19 2000-01-14 Procede et dispositif de production automatisee de ligands d'acide nucleique
KR1020017009027A KR20010101573A (ko) 1999-01-19 2000-01-14 핵산 리간드의 자동 발생을 위한 방법 및 장치
MXPA01007352A MXPA01007352A (es) 1999-01-19 2000-01-14 Metodo y aparato para la generacion automatizada de ligandos de acido nucleico.
AU32092/00A AU777823B2 (en) 1999-01-19 2000-01-14 Method and apparatus for the automated generation of nucleic acid ligands
JP2000594942A JP2002534985A (ja) 1999-01-19 2000-01-14 核酸リガンドの自動化生成のための方法および装置

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US09/232,946 US6569620B1 (en) 1990-06-11 1999-01-19 Method for the automated generation of nucleic acid ligands
US35623399A 1999-07-16 1999-07-16
US09/356,233 1999-07-16
US09/232,946 1999-07-16

Publications (1)

Publication Number Publication Date
WO2000043534A1 true WO2000043534A1 (fr) 2000-07-27

Family

ID=26926487

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/001001 WO2000043534A1 (fr) 1999-01-19 2000-01-14 Procede et dispositif de production automatisee de ligands d'acide nucleique

Country Status (7)

Country Link
EP (1) EP1144669A4 (fr)
JP (1) JP2002534985A (fr)
KR (1) KR20010101573A (fr)
AU (1) AU777823B2 (fr)
CA (1) CA2360748A1 (fr)
MX (1) MXPA01007352A (fr)
WO (1) WO2000043534A1 (fr)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001096559A2 (fr) * 2000-06-15 2001-12-20 Board Of Regents The University Of Texas System Acides nucleiques pouvant etre regules et actifs du point de vue catalytique
WO2002016903A2 (fr) * 2000-08-23 2002-02-28 Imego Ab Systeme et procede de prelevement d'echantillons
EP1386138A1 (fr) * 2001-04-02 2004-02-04 Prolinx Incorporated Systemes et appareils d'analyse d'interactions moleculaires
WO2004024950A1 (fr) * 2002-09-10 2004-03-25 Noxxon Pharma Ag Procede de selection d'acides nucleiques ligands
US7179659B2 (en) 2001-04-02 2007-02-20 Agilent Technologies, Inc. Sensor surfaces for detecting analytes and methods of use
US7431887B2 (en) 2000-08-24 2008-10-07 David Storek Microfluidic device and method with trapping of sample in cavities having lids that can be opened or closed
US7960102B2 (en) 2002-07-25 2011-06-14 Archemix Corp. Regulated aptamer therapeutics
US7998746B2 (en) 2000-08-24 2011-08-16 Robert Otillar Systems and methods for localizing and analyzing samples on a bio-sensor chip
US20120064511A1 (en) * 2010-09-07 2012-03-15 Roche Molecular Systems, Inc. Generic Buffer For Amplification
CN103026238A (zh) * 2010-07-28 2013-04-03 株式会社日立高新技术 核酸分析装置
CN103399162A (zh) * 2002-05-17 2013-11-20 贝克顿·迪金森公司 用于分离、放大和检测目标核酸序列的自动化系统
CN103614291A (zh) * 2013-11-13 2014-03-05 戴小波 全自动荧光定量基因扩增仪
US9175332B2 (en) 2010-07-29 2015-11-03 Roche Molecular Systems, Inc. Generic PCR
US9315804B2 (en) 2007-10-22 2016-04-19 Caris Life Sciences Switzerland Holdings, GmbH Method of selecting aptamers
EP2755033B1 (fr) 2013-01-09 2019-05-22 Hamilton Bonaduz AG Système de traitement d'échantillons avec dispositif de dosage et thermocycleur
CN114923839A (zh) * 2022-07-18 2022-08-19 高分(北京)生物科技有限公司 全自动超高通量细胞成像计数仪及样品检测方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5156905B2 (ja) * 2007-04-20 2013-03-06 独立行政法人科学技術振興機構 アプタマー生成装置、アプタマー生成方法
WO2012013732A1 (fr) * 2010-07-29 2012-02-02 F. Hoffmann - La Roche Ag Pcr générique
CN114276896B (zh) * 2021-12-22 2023-12-29 成都瀚辰光翼科技有限责任公司 一种自动化核酸提取方法及存储介质

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5443791A (en) * 1990-04-06 1995-08-22 Perkin Elmer - Applied Biosystems Division Automated molecular biology laboratory
US5475096A (en) * 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
US5866336A (en) * 1996-07-16 1999-02-02 Oncor, Inc. Nucleic acid amplification oligonucleotides with molecular energy transfer labels and methods based thereon
US5985548A (en) * 1993-02-04 1999-11-16 E. I. Du Pont De Nemours And Company Amplification of assay reporters by nucleic acid replication

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1339653C (fr) * 1986-02-25 1998-02-03 Larry J. Johnson Appareil servant a l'amplification automatisee de sequences nucleotidiques et d'essais au moyen de cycle de chauffage et de refroidissement
US5861254A (en) * 1997-01-31 1999-01-19 Nexstar Pharmaceuticals, Inc. Flow cell SELEX
EP0736105B1 (fr) * 1993-09-17 2007-11-21 SomaLogic, Inc. Evolution systematique de ligands par enrichissement exponentiel : photoselection de ligands d'acide nucleique
EP0824540B1 (fr) * 1995-05-03 2004-09-15 Gilead Sciences, Inc. Ligands a acide nucleique pour cibles tissulaires
US5737498A (en) * 1995-07-11 1998-04-07 Beckman Instruments, Inc. Process automation method and apparatus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5443791A (en) * 1990-04-06 1995-08-22 Perkin Elmer - Applied Biosystems Division Automated molecular biology laboratory
US5475096A (en) * 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
US5985548A (en) * 1993-02-04 1999-11-16 E. I. Du Pont De Nemours And Company Amplification of assay reporters by nucleic acid replication
US5866336A (en) * 1996-07-16 1999-02-02 Oncor, Inc. Nucleic acid amplification oligonucleotides with molecular energy transfer labels and methods based thereon

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
COX ET AL.: "Automated RNA Selection", BIOTECHNOL.,, vol. 14, 1998, pages 845 - 850, XP002927340 *
See also references of EP1144669A4 *

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001096559A3 (fr) * 2000-06-15 2003-07-10 Univ Texas Acides nucleiques pouvant etre regules et actifs du point de vue catalytique
WO2001096559A2 (fr) * 2000-06-15 2001-12-20 Board Of Regents The University Of Texas System Acides nucleiques pouvant etre regules et actifs du point de vue catalytique
US7351592B2 (en) 2000-08-23 2008-04-01 David Storek Microfluidic device and method with trapping of sample in cavities having lids that can be opened or closed
WO2002016904A3 (fr) * 2000-08-23 2002-05-23 Imego Ab Procede et systeme de preparation d'echantillons
WO2002016903A3 (fr) * 2000-08-23 2002-05-30 Imego Ab Systeme et procede de prelevement d'echantillons
WO2002016904A2 (fr) * 2000-08-23 2002-02-28 Imego Ab Procede et systeme de preparation d'echantillons
WO2002016903A2 (fr) * 2000-08-23 2002-02-28 Imego Ab Systeme et procede de prelevement d'echantillons
US7998746B2 (en) 2000-08-24 2011-08-16 Robert Otillar Systems and methods for localizing and analyzing samples on a bio-sensor chip
US7431887B2 (en) 2000-08-24 2008-10-07 David Storek Microfluidic device and method with trapping of sample in cavities having lids that can be opened or closed
EP1386138A4 (fr) * 2001-04-02 2005-05-18 Agilent Technologies Inc Systemes et appareils d'analyse d'interactions moleculaires
US7179659B2 (en) 2001-04-02 2007-02-20 Agilent Technologies, Inc. Sensor surfaces for detecting analytes and methods of use
US7208322B2 (en) 2001-04-02 2007-04-24 Agilent Technologies, Inc. Sensor surfaces for detecting analytes
EP1386138A1 (fr) * 2001-04-02 2004-02-04 Prolinx Incorporated Systemes et appareils d'analyse d'interactions moleculaires
CN103399162A (zh) * 2002-05-17 2013-11-20 贝克顿·迪金森公司 用于分离、放大和检测目标核酸序列的自动化系统
US9696328B2 (en) 2002-05-17 2017-07-04 Becton, Dickinson And Company Automated system for isolating, amplifying and detecting a target nucleic acid sequence
CN103399162B (zh) * 2002-05-17 2016-04-27 贝克顿·迪金森公司 用于分离、放大和检测目标核酸序列的自动化系统
US7960102B2 (en) 2002-07-25 2011-06-14 Archemix Corp. Regulated aptamer therapeutics
WO2004024950A1 (fr) * 2002-09-10 2004-03-25 Noxxon Pharma Ag Procede de selection d'acides nucleiques ligands
DE10241938A1 (de) * 2002-09-10 2004-03-25 Noxxon Pharma Ag Verfahren zur Selektion von Nukleinsäureliganden
US9315804B2 (en) 2007-10-22 2016-04-19 Caris Life Sciences Switzerland Holdings, GmbH Method of selecting aptamers
CN103026238A (zh) * 2010-07-28 2013-04-03 株式会社日立高新技术 核酸分析装置
US9175332B2 (en) 2010-07-29 2015-11-03 Roche Molecular Systems, Inc. Generic PCR
US20120064511A1 (en) * 2010-09-07 2012-03-15 Roche Molecular Systems, Inc. Generic Buffer For Amplification
US9416398B2 (en) * 2010-09-07 2016-08-16 Roche Molecular Systems, Inc. Generic buffer for amplification
EP2755033B1 (fr) 2013-01-09 2019-05-22 Hamilton Bonaduz AG Système de traitement d'échantillons avec dispositif de dosage et thermocycleur
CN103614291A (zh) * 2013-11-13 2014-03-05 戴小波 全自动荧光定量基因扩增仪
CN114923839A (zh) * 2022-07-18 2022-08-19 高分(北京)生物科技有限公司 全自动超高通量细胞成像计数仪及样品检测方法
CN114923839B (zh) * 2022-07-18 2022-10-28 高分(北京)生物科技有限公司 全自动超高通量细胞成像计数仪及样品检测方法

Also Published As

Publication number Publication date
KR20010101573A (ko) 2001-11-14
CA2360748A1 (fr) 2000-07-27
AU3209200A (en) 2000-08-07
EP1144669A1 (fr) 2001-10-17
AU777823B2 (en) 2004-11-04
EP1144669A4 (fr) 2005-01-05
JP2002534985A (ja) 2002-10-22
MXPA01007352A (es) 2003-06-06

Similar Documents

Publication Publication Date Title
US6569620B1 (en) Method for the automated generation of nucleic acid ligands
US6716580B2 (en) Method for the automated generation of nucleic acid ligands
AU777823B2 (en) Method and apparatus for the automated generation of nucleic acid ligands
US20030054360A1 (en) Method and apparatus for the automated generation of nucleic acid ligands
Cox et al. Automated selection of anti-protein aptamers
US7258980B2 (en) Determining non-nucleic acid molecule binding to target by competition with nucleic acid ligands
JP4768598B2 (ja) ビーズエマルジョン核酸増幅
AU783689B2 (en) Sensitive, multiplexed diagnostic assays for protein analysis
EP0885958B1 (fr) Procede de traitement de biopolymeres, de micro-organismes ou de matieres a l'aide de plusieurs types de particules magnetiques
AU777180B2 (en) Device and method for mixing magnetic particles with a fluid
US7022479B2 (en) Sensitive, multiplexed diagnostic assays for protein analysis
US20200277653A1 (en) Analyte Enrichment Methods and Compositions
US20020037506A1 (en) Aptamer based two-site binding assay
EP2238459A2 (fr) Instrument de synthèse et d'amplification intégré
CN112195219B (zh) 小panel探针靶向捕获富集测序文库分子的降噪方法
EP2217729A1 (fr) Procédé de concentration de molécules d'acide nucléique
WO2022008641A1 (fr) Appareil de synthèse split-pool et procédés de réalisation d'une synthèse split-pool
WO2001036585A1 (fr) Micro-reseau et procede d'etiquetage sequentiel destines a la detection de plusieurs proteines au moyen de procedes adn
SI24499A (sl) Kontrolirana selektivna elucija v afinitetni kromatografiji

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 32092/00

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2360748

Country of ref document: CA

Ref country code: CA

Ref document number: 2360748

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2000 594942

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1020017009027

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: PA/a/2001/007352

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 2000909909

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000909909

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1020017009027

Country of ref document: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWR Wipo information: refused in national office

Ref document number: 32092/00

Country of ref document: AU

WWR Wipo information: refused in national office

Ref document number: 1020017009027

Country of ref document: KR

WWW Wipo information: withdrawn in national office

Ref document number: 2000909909

Country of ref document: EP