EP1040352A1 - Embouts en phase solide et utilisations correspondantes - Google Patents

Embouts en phase solide et utilisations correspondantes

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Publication number
EP1040352A1
EP1040352A1 EP98965546A EP98965546A EP1040352A1 EP 1040352 A1 EP1040352 A1 EP 1040352A1 EP 98965546 A EP98965546 A EP 98965546A EP 98965546 A EP98965546 A EP 98965546A EP 1040352 A1 EP1040352 A1 EP 1040352A1
Authority
EP
European Patent Office
Prior art keywords
tip
solid
tip structure
biomolecule
chemical layer
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Withdrawn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
EP98965546A
Other languages
German (de)
English (en)
Inventor
Lori K. Garrison
John C. Tabone
Jeffrey Van Ness
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.)
Qiagen Genomics Inc
Original Assignee
Qiagen Genomics 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
Application filed by Qiagen Genomics Inc filed Critical Qiagen Genomics Inc
Publication of EP1040352A1 publication Critical patent/EP1040352A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid 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
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • 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
    • B01J2219/00387—Applications using probes
    • 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
    • B01J2219/00457—Dispensing or evacuation of the solid phase support
    • B01J2219/0047—Pins
    • 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
    • B01J2219/00457—Dispensing or evacuation of the solid phase support
    • B01J2219/0047—Pins
    • B01J2219/00472—Replaceable crowns
    • 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
    • B01J2219/00504—Pins
    • 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
    • B01J2219/00504—Pins
    • B01J2219/00506—Pins with removable crowns
    • 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
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • 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
    • B01J2219/00725—Peptides
    • 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
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, 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

  • the present invention relates generally to the application of solid-phase techniques to synthesize and to analyze nucleic acid molecules.
  • the present invention relates to improved solid-phase supports that can be used to perform a variety of bimolecular procedures.
  • Nucleic acid hybridization provides specificity for the recognition of biomolecules, such as DNA and RNA sequences, and has become a powerful technique in medical diagnosis.
  • nucleic acid hybridization methods have been applied to analysis of genetic polymorphisms, diagnosis of genetic disease, cancer diagnosis, detection of viral and microbial pathogens, screening of clones, and ordering of genomic fragments (for a review, see Chetverin and Kramer, Bio/Technology 72: 1093, 1994).
  • the development of automated synthesis of oligonucleotide probes has also promoted the development of rapid, simple and inexpensive diagnostic assays based on nucleic acid hybridization.
  • the use of DNA probes in analytical techniques has been reviewed by Matthews and Kricka, Anal. Biochem. 169 ⁇ .
  • nucleic acid hybridization requires immobilization of target nucleic acid on solid supports, such as nitrocellulose filters and nylon membranes, followed by hybridization with a detectable nucleic acid probe.
  • solid supports such as nitrocellulose filters and nylon membranes
  • the disadvantage of such methods is that the immobilized nucleic acid typically is not tightly bound, resulting in loss of target material from the support. Moreover, only a small amount of nucleic acid molecules are available for hybridization.
  • PEI Poly(ethyleneimine)
  • Spring probes have become generally well known since they were introduced early in the development of the printed circuit board industry. They are mechanical devices designed to meet the need for precision and reliability in the construction and testing of a variety of electronic components and their connections when being assembled into functioning circuit boards. Spring probes are essentially electro-mechanical devices, typically consisting of a tubular housing encasing a compression spring, ball and plunger. Some probes are specifically designed to carry electrical current flow, while others are used to drill, crimp, and secure components to a circuit board, and yet others are designed to perform soldering. There is nothing in the design or marketing of spring probes that suggests their potential utility as a mechanical devise for the transferring and arraying of solutions onto solid support for use in the fields of microbiology, biochemistry, or molecular biology.
  • the present invention provides a solid-phase sample-retaining assembly that overcomes the drawbacks experienced by the prior art and provides further related advantages.
  • a solid-phase sample-retaining tip is provided that is usable in a procedure in synthesizing or detecting a biomolecule.
  • the sample-retaining tip includes a solid support tip structure that is connectable to a support pin, and a chemical layer coats at least a portion of the tip structure.
  • the chemical layer is bindable to the biomolecule to form a solid-phase sample of the biomolecule on the tip structure.
  • the tip structure is removably connected to the support pin.
  • the tip structure has a partially conical shape with a plurality of flutes formed therein. These flutes define a plurality of heat exchange fins that enable the tip structure to quickly heat up and cool down during selected thermocycling procedures in the synthesizing or detecting of biomolecules.
  • a sample-retaining assembly for use in a solid-phase procedure for synthesizing or detecting a biomolecule.
  • the sample-retaining assembly includes a support pin, a tip structure connected to the support pin, and a chemical layer coating at least a portion of the tip structure.
  • the chemical layer is bindable to the biomolecule so as to form a solid-phase sample of the biomolecule on the tip structure.
  • the support pin is a spring probe or other spring pin
  • the tip structure is a nylon 6/6 member that is removably connected to the spring probe.
  • an array of solid-phase sample- retaining assemblies wherein a plurality of support pins are connected to a base in a selected array.
  • Each support pin has an end portion that is spaced apart from the base and a plurality of tip structures are connected to the end portions.
  • the chemical layer coats at least a portion of each tip structure. The chemical layer is bindable to a biomolecule to form a solid-phase sample of the biomolecule.
  • a solid-phase sample-retaining assembly is combined with a microtiter plate.
  • the micotiter plate has a well that is shaped to contain a volume of a sample having a biomolecule therein.
  • the solid-phase sample-retaining assembly is sized to extend at least partially into the well.
  • the solid- phase retaining assembly includes a support pin, a tip structure connected to the support pin with the tip structure being removably positionable in the well, and a chemical layer coating at least a portion of the tip structure. The chemical layer is bindable to the biomolecule and the solution to form a solid-phase sample of the biomolecule.
  • the microtiter plate has a plurality of wells therein and the solid-phase sample-retaining assembly includes a plurality of support pins arranged in a selected array, and a plurality of the tip structures are connected to the support pins to form an array of solid-phase sample-retaining tips that are positionable into the plurality of wells.
  • solid-phase sample-retaining tips are combined with a microtiter plate having a plurality of wells therein.
  • the solid-phase sample-retaining tips are removably positioned within the microtiter plate's wells.
  • the microtiter plate and the sample-retaining tips are storable as a unit such that a solid- phase sample on the sample-retaining tip can be easily stored until needed for a synthesizing or analyzing procedure.
  • Another aspect of the invention provides a method of manufacturing the solid-phase sample-retaining tip for use in a solid-phase molecular biology process.
  • the method includes the steps of forming a substrate material as a tip structure that is attachable to a support pin, coating at least a portion of the substrate material with a chemical layer that is bondable to selected biomolecules to form a solid-phase sample of the biomolecule, and attaching the chemical layer to the substrate
  • the chemical layer is a poly(ethyleneimine) and the substrate material is a nylon 6/6 material
  • the step of attaching the chemical layer to the substrate material includes covalently attaching the poly(ethyleneim ⁇ ne) to the nylon 6/6 material
  • Another embodiment of the invention is directed toward a method of forming a solid-phase sample of a biomolecule
  • the method includes the steps of immersing a portion of a tip assembly into a solution having a biomolecule therein
  • the tip assembly has a substrate portion and a chemical layer on the substrate portion, with the chemical layer being bindable to the biomolecule
  • the biomolecule is allowed to bind to the chemical layer to form a solid-phase sample of the biomolecule on the tip assembly, and the tip assembly is removed from the solution after the biomolecule has bonded to the chemical layer
  • the method includes the step of storing the solid-phase tip assembly in a retaining member after a biomolecule has bonded to the chemical layer
  • the retaining member in one embodiment of the invention is a microtiter plate with a well therein
  • the step of storing includes placing the tip assembly in the well after the biomolecule has bonded to the chemical layer, and placing the microtiter plate and tip assembly as a unit in a storage location
  • Figure 1 is an isometric view of an array of solid-phase sample-retaining assemblies in accordance with the exemplary embodiment of the present invention
  • Figure 2A is an enlarged cross-sectional view of a solid-phase sample- retaining assembly taken substantially along line 2-2 of Figure 1
  • Figure 2B is a cross-sectional view of a solid-phase sample-retaining assembly of an alternate embodiment
  • Figure 3 is an enlarged partially cut away view of a tip structure of a sample-retaining assembly of Figure 1
  • Figure 4 is an enlarged cross-sectional view of the tip structure taken substantially along line 4-4 of Figure 3
  • Figure 5 is a side elevational view of the array of Figure 1 shown in solid lines positioned above a microtiter plate with a plurality of wells with liquid biomolecule samples therein, and shown in phantom lines in lowered position with the tip structures positioned within the wells
  • Figure 6 is an enlarged side elevation view of the array of Figure 1 shown with a plurality of the tip structure positioned in the wells of a microtiter plate
  • nucleic acid or “nucleic acid molecule” refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action
  • Nucleic acids can be composed of monomers that are naturally-occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), or analogs of naturally-occurring nucleotides (e.g., ⁇ -enantiomeric forms of naturally-occurring nucleotides), or a combination of both Modified nucleotides can have modifications in sugar moieties
  • the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.
  • nucleic acid also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
  • isolated nucleic acid molecule is a nucleic acid molecule that is not integrated in the genomic DNA of an organism.
  • a DNA molecule that encodes interleukin-2 that has been separated from the genomic DNA of a mammalian cell is an isolated DNA molecule.
  • Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism.
  • biomolecule refers either to a nucleic acid molecule, or to a polymer of amino acids or amino acid analogs.
  • a "detectable tag” or “detectable label” is a molecule or atom which is conjugated to a nucleic acid molecule to produce a probe that is useful for detection methods.
  • tags or labels include photoactive agents or dyes, radioisotopes, fluorescent agents, mass spectrometer tags, or other molecules and marker moieties.
  • Suitable fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthal- dehyde and fluorescamine.
  • chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
  • Bioluminescent compounds that are useful for such tags include luciferin, luciferase and aequorin.
  • "Complementary DNA (cDNA)” is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription.
  • cDNA to refer to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.
  • expression refers to the biosynthesis of a gene product.
  • expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
  • a "cloning vector” is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, that has the capability of replicating autonomously in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign nucleotide sequences can be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
  • Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
  • An “expression vector” is a nucleic acid molecule encoding a gene that is expressed in a host cell. Typically, gene expression is placed under the control of a promoter, and optionally, under the control of at least one regulatory element. Such a gene is said to be “operably linked to” the promoter. Similarly, a regulatory element and a promoter are operably linked if the regulatory element modulates the activity of the promoter.
  • a "recombinant host” may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
  • hybotrope refers to any chemical or any mixture of a chemical in an aqueous or organic environment with buffers, chelators, salts and/or detergents that changes the enthalpy of a nucleic acid duplex by at least 20% when referenced to a standard salt solution (0 165 M NaCl, 0 01 M Tris pH 7 2, 5 mM EDTA and 0 1% SDS) That is, the energy content of the nucleic acid duplex is decreased
  • the reference oligonucleotide is 5'-GTCATACTCCTGCTTGCTGATCCACATCTG- 3' [SEQ ID NO 9] as the immobilized oligonucleotide and 5'- TGTGGATCAGCAAGCAGGAGTATG-3' [SEQ ID NO.10] as the solution nucleotide which is typically labeled at the 5 '-end with a fluorochrome such as Texas Red
  • the oligonucleotide duplex (24 nucleotides in
  • T m is the temperature at which half the molecules of a nucleic acid duplex are single stranded T m is measured in solution, while T d is measured for the duplex affixed to a solid support, both terms indicate the temperature at which half of a duplex are single stranded
  • stringency is the percentage of mismatched base pairs that are tolerated for hybridization under a given condition
  • discrimination is the difference in T d between a perfectly base-paired duplex and a duplex containing a mismatch
  • a discrimination temperature is a temperature at which a hybridization reaction is performed that allows detectable discrimination between a mismatched duplex and a perfectly matched duplex
  • the array 10 includes a plurality of sample-retaining assemblies 12 attached to a base structure 14
  • Each sample-retaining assembly 12 includes a support pin 16 securely fixed at one end 18 to the base 14, and a sample-retaining tip structure 20 is attached to the other end 22 of the support pin 16
  • Each tip structure 20 in the exemplary embodiment is a Nylon 6/6 solid support structure, and the Nylon 6/6 is coated with a poly(ethylene ⁇ n ⁇ ne) (PEI) layer 24 or other selected chemical layer
  • PEI layer 24 or other selected chemical layer is adapted to bind to a selected biomolecule to form a solid phase sample that is used in a procedure for synthesizing or detecting one or more nucleic acids
  • the array 10 of the illustrated embodiment includes eight substantially parallel rows of twelve sample-retaining assemblies 12 to define an array with ninety- six sample-retaining assemblies equally spaced along the base structure 14
  • Each sample-retaining assembly 12 has approximately the same length so the tip structures 20 are equally spaced from the base, thereby defining a substantially coplanar array of solid-phase sample-retaining tip structures
  • the tip structures 20 are spaced apart to mate with a conventional 96-well Cetus plate or microtiter plate that is adapted to receive and retain selected liquid samples of biomolecules or nucleic acids
  • the exemplary embodiment has an 8 x 12 array of sample-retaining assemblies 12, alternate embodiments have other configurations, including a 1 x 8 array, a 1 x 12 array, and a 4 x 12 array, as well as larger arrays such as a 16 x 24 array
  • the ninety-six tip structures 20 are adapted to be dipped into the wells of the Cetus plate with the biomolecules therein such that the biomolecules chemically bind to the PEI layer 24
  • the tip structures 20 are removed from the sample, the biomolecules are adhered to the PEI layer, thereby forming the solid-phase sample of the biomolecule
  • the tip structures 20 with the solid phase sample thereon can then be used in synthesizing or analyzing procedures, such as a solid-phase nucleic acid assay and detection process as described in greater detail below
  • the array 10 is installed in a robotic or automatic actuator so the base 14 is clamped into the actuator and the sample-retaining assemblies 12 project away from the base
  • the actuator quickly and accurately moves the array 10 during automated testing to selected controlled positions or stations in accordance with a predetermined testing, synthesizing, or analyzing process
  • Such automated testing with the array 10 and the ninety-six solid phase samples allows for substantially faster testing, synthesizing, or analyzing procedures.
  • each support pin 16 is a spring probe that is typically used for construction and testing of electronic components, but has been adapted for use in the present invention.
  • the spring probe generally includes a housing 28 encasing a biasing member 30.
  • a plunger 32 extends into the housing 28 so a first end 34 of the plunger is within the housing 28 adjacent to the biasing member and a second end 36 is exterior of the housing.
  • the biasing member 30 in the exemplary embodiment is a compression spring that pushes axially against the plunger 32 toward the base 14.
  • the plunger's first end 34 has a shoulder 38 that engages a stop 40 projecting radially inwardly from the housing 28 to limit the maximum extension of the plunger 32 with respect to the housing.
  • the plunger's second end 36 is fixedly attached to the base 14, and the plunger 32 projects substantially perpendicularly away from the base.
  • the housing 28 includes concentric inner and outer tubular barrels 42 and 44, wherein the biasing member 30 and the plunger's first end 34 are contained within the inner barrel.
  • the outer barrel 44 removably receives the inner barrel 42 therein and frictionally engages the inner barrel such that the inner and outer barrels are removably attached to each other.
  • the outer barrel 44 terminates at a distal end portion 46 that is spaced away from the base 14 and that connects to the tip structure 20. Accordingly, the housing's outer barrel 44 and the tip structure 20 are removable as a unit from the inner barrel 42 and plunger 32, which remain fixed to the base 14.
  • an outer barrel 44 and tip structure 20 can be easily and quickly replaced as a unit without having to replace the entire spring probe.
  • Suitable spring probes are available from Everett Charles (Pomona, California), Interconnect Devices, Inc. (Kansas City, Kansas), Test Connections, Inc. (Upland, California), and other manufacturers. While the exemplary embodiment utilizes spring probes as the support pins 16, other support pins, including biased or unbiased support pins, are used in alternate embodiments of the invention. As best seen in Figure 2B, an alternate embodiment of the invention includes the spring probe as the support pin 16, but the spring probe is oriented 180° from the embodiment described above and illustrated in Figure 2A. For example, the distal end portion 46 of the outer barrel 44 is fixedly attached to the base 14 and the second end 36 of the plunger 32 is spaced away from the base and connected to the tip structure 20.
  • the spring probes provide a safety feature that protects the array 10 from being damaged during operation. During a sampling or analyzing process, for example, wherein the array 10 is moved to selected positions and the tip structures 20 are dipped into Cetus plate wells or the like, and if the support pins 16 or tip structures inadvertently impacts a surface or other object, the spring probe will compress axially to absorb the impact and then return to the uncompressed position.
  • the tip structure 20 of the exemplary embodiment has a truncated-conical shape with a plurality of channels or flutes 50 formed therein.
  • the tip structure is connectable to a support pin, or may be unitary with the support pin.
  • the flutes 50 are V-shaped flutes that extend axially between a flat distal face 48 and a flat proximal face 54.
  • the flutes 50 have veins or ridges 52 that converge from the proximal face 54 toward the distal face 48 at a selected angle.
  • the truncated conical shape of the tip structure 20 is selected so it virtually identically matches the lower cross-sectional shape of a Cetus plate well. Accordingly, the tip structure 20 is shaped and sized to fit in a very precise position within the Cetus plate well.
  • the tip structure 20 includes a pin-receiving aperture 56 with an open proximal end 58 in the proximal face 54 and a closed distal end 60 at a mid-portion between the tip structure's proximal and distal faces 54 and 48, respectively.
  • the pin- receiving aperture 56 is shaped and sized to removably receive the support pin's distal end portion 46. Accordingly, the tip structure 20 is removably connected to the support pin 16.
  • the tip structure could alternatively be permanently connected to the pin, and in fact the pin and tip structure could be a single unitary structure.
  • the pin-receiving aperture 56 is coaxially aligned with the tip structure's longitudinal axis.
  • the aperture's proximal portion 59 is generally funnel-shaped such that the aperture's open proximal end 58 has a larger diameter than the closed distal end 60.
  • the funnel-shaped proximal portion 59 is adapted to receive the support pin's distal end portion 46 therein. In the event the support pin is slightly misaligned relative to the aperture 56 during an installation procedure, the funnel-shaped proximal portion 59 will receive and direct the support pin 16 into a position such that the spring probe is coaxially aligned with the tip structure 20.
  • the aperture 56 in the exemplary embodiment is defined by an axial interior wall 61 of the tip structure 20 and has a substantially circular cross-sectional shape.
  • the spring probe's distal end portion 46 has a substantially square cross-sectional shape with four corners 63.
  • the spring probe's distal end portion 46 is sized such that the corners 63 frictionally engage the tip structure's interior wall 61 so as to frictionally retain the tip structure 20 on the support pin 16.
  • the end portion has a polygonal-shaped cross-sectional area with a plurality of corners that engage the tip structure's interior wall 61.
  • an octagonal-shaped cross-sectional area having the eight corners that frictionally engage the interior wall 61.
  • the support pin's distal end portion has a circular cross-sectional shape that substantially corresponds to the circular cross-sectional area of the pin- receiving aperture 56 such that the tip structure 20 is press-fit onto the spring probe's distal end portion 46 and is frictionally retained thereon.
  • the tip structure 20 is adhered to the distal end portion 46 with a conventional adhesive such that the tip structure is permanently affixed to the support pin 16.
  • the flutes 50 and ridges 52 define the truncated conical-shaped tip structure 20 with a generally star-shaped cross-sectional area.
  • the tip structure 20 has an enlarged exterior surface 62 so a greater amount of biomolecules can attach to the PEI layer 24 during formation of the solid-phase sample.
  • the flutes 50, ridges, distal face 48 and proximal face 54 of the tip structure 20 define a high-surface area, Nylon 6/6 solid support that is covalently bonded to the PEI layer 24.
  • the tip structure 20 is made of a solid substrate, such as glass or silicon and the PEI layer 24 is covalently bound to the solid substrate using sililating chemistry, as discussed in greater detail below.
  • the exterior surface 62 of the tip structure 20 along the flutes 50 and ridges 52 is dimpled so as to provide a further increased surface area along which the PEI layer 24 will bind.
  • the dimples are generally microscopic, and in an alternate embodiment, the dimples are macroscopic. Accordingly, the dimpled tip structure 20 provides a larger reaction surface for greater efficiency in the synthesizing or analyzing procedures.
  • the tip structure 20 is thermocycled, wherein the tip structure 20 is cycled between high and low temperatures.
  • the ridges 52 of the tip structure 20 form a plurality of heat exchange fins 64 that allow for faster temperature change of the tip structure during the thermocycling. As a result, the thermocycling can be done faster and more efficiently.
  • the array 10 is adapted to be combined and used with a Cetus or microtiter plate 70 having a plurality of wells 72 therein.
  • the shape of a portion of the well 72 substantially matches the truncated conical shape of the tip structure 20. Accordingly, the ridges 52 substantially engage sidewalls 74 of the well 72 and the tip structure's flat distal face 48 is positioned against the bottom 76 of the well.
  • the microtiter plate 70 has an array of wells formed by eight substantially parallel rows of twelve wells 72 to form the ninety-six well configuration that mates with the tip structures of the array 10.
  • the microtiter plates 70 have arrays of 1 x 8 wells, 1 x 12 wells, and 4 x 12 wells, as well as larger arrays such as a 16 x 24 well format.
  • the array can be automatically or manually moved from a raised position, shown in solid lines in Figure 5 with the tip structures 20 being out of the wells 72, to a lowered position, shown in phantom lines with the tip structures being positioned within the wells 72.
  • the wells 72 contain a liquid sample with the selected biomolecules therein.
  • the chemical reaction occurs between the PEI layer 24 and the biomolecule, so as to form the selected solid-phase sample of the biomolecule.
  • the well 72 has a depth that is approximately 33% larger than that of the tip structure 20, so when the tip structure is dunked into the well, the liquid sample flows over the entire tip structure to bind as much of the biomolecule as possible.
  • the array 10 of sample-retaining assemblies 12 is also usable by positioning the tip structures 20 within the wells 72 and separating the tip structures from the support pins 16, as shown in solid lines, so the tip structures remain in the wells.
  • the base 14 and support pins 16 are then moved as a unit away from the microtiter plate 70.
  • the microtiter plate 70 with the ninety-six tip structures 20 retained or stored within the wells 72 can be moved as a unit and, as an example, placed in cold storage or other suitable storage locations until the solid-phase samples are needed for a selected synthesizing or analyzing procedure.
  • the wells 72 retain the tip structures 20 in a very precise location relative to the microtiter plate 70 so the tip structures can be easily and substantially simultaneously installed onto the support pins 16.
  • the microtiter plate 70 is held in a known and fixed location, and the base 14 and support pins 16 are moved as a unit, either automatically or manually to a selected position above the wells 72 such that the support pins substantially coaxially align with the pin-receiving aperture 56 in the tip structures.
  • the base 14 and support pins 16 are then moved toward the microtiter plate 70 such that the support pins 16 are pressed into the apertures in the tip structures, thereby releasably connecting the tip structures to the support pins.
  • the base 14, support pins 16, and tip structures 20 are then moved as a unit away from the microtiter plate 70, thereby removing the tip structures 20 from the wells 72.
  • the sample-retaining tip assemblies 12 with solid phase samples thereon can be moved to a predetermined location and subjected to selected solid-phase procedures for analyzing or synthesizing a nucleic acid.
  • the solid supports of the present invention can be used in parallel and are preferentially configured in a 96-well or 384-well format.
  • the solid supports can be attached to pegs, stems, or rods in a 96-well or 384-well configuration, the solid supports either being detachable or alternatively integral to the particular configuration.
  • the particular configuration of the sold supports is not of critical importance to the functioning of the assay, but rather, affects the ease of adapting the assays to automated systems.
  • the tips described herein are useful in a variety of methods requiring attachment of a nucleic acid molecule, peptide, polypeptide, or protein to a solid support. Examples of solid-phase assays and detection methods that can be performed with such tips are described below.
  • nucleic acid molecules modified at their 5 '-ends with an aldehyde or carboxylic acid, can be attached to a solid support having hydrazide residues (see, for example, Kremsky et al., Nucleic Acids Res. 75:2891, 1987).
  • 5'- aminohexyl phosphoramidate derivatives of oligo nucleotides can be coupled with a solid support bearing carboxyl groups in a carbodiimide-mediated coupling reaction (see, for example, Ghosh et al, Nucleic Acids Res. 15:5353, 1987).
  • the solid supports are preferentially coated with an amine-polymer such as polyethylene(imine), acrylamide, amine-dendrimers, etc.
  • the amines on the polymers are used to covalently immobilize nucleic acids.
  • nucleic acids are bound to the solid supports described herein using poly(ethyleneimine) (PEI) coatings.
  • PEI poly(ethyleneimine)
  • the chemistry used to adhere a layer of PEI to the substrate depends, in substantial part, upon the chemical identity of the substrate.
  • suitable chemistries may adhere PEI to a solid support.
  • the PEI coating may be applied by the methods disclosed in Van Ness, et al. Nucleic Acids Res.
  • the PEI coating is covalently attached to the solid substrate
  • the PEI coating may be covalently bound to the substrate using silylating chemistry
  • silylating chemistry For example, PEI having reactive siloxy endgroups is commercially available from Gelest, Inc (Tullytown, PA) Such reactive PEI may be contacted with a glass or silicon tip, and after gentle agitation, the PEI will adhere to the substrate
  • a bi-functional silylating reagent may be employed According to this process, the glass or silicon substrate is treated with the bi-functional silylating reagent to provide the substrate with a reactive surface PEI is then contacted with the reactive surface, and covalently binds to the surface through the bi-functional reagent
  • PEI coatings are preferably used to immobilize nucleic acids to the nylon tips, described herein
  • One suitable method of coating nylon-6/6 with PEI has been described by Van Ness et al , Nucleic Acids Res. 19 3345, 1991 Briefly, the nylon substrate is ethylated using triethyloxonium tetrafluorob orate to form amine- reactive imidate esters on the nylon surface
  • the activated nylon is then reacted with PEI to form a polymer coating that provides an extended amine surface
  • the modified oligonucleotides are covalently attached to the nylon surface via the triazine moiety
  • nucleic acid polymers are "amine-modified" in that they have been modified to contain a primary amine at the 5 '-end of the nucleic acid polymer, preferably with one or more methylene groups disposed between the primary amine and the nucleic acid portion of the nucleic acid polymer Six is a preferred number of methylene groups
  • Nucleic acid molecules can be modified by addition of amine moieties using standard techniques Products of a polymerase chain reaction, for example, can be arrayed using 5'-hexylamine-modified primers Nucleic acid duplexes can be arrayed after the introduction of amines by nick translation using amine allyl-dUTP (Sigma, St Louis, MO) Amines can also be introduced into nucleic acids by polymerases such as terminal transferase with amino allyl-dUTP or by ligation of short amine-containing nucleic acid polymers onto nucleic acids by ligases
  • the nucleic acid polymer is activated prior to be contacted with the PEI coating This can be conveniently accomplished by combining amine- functionahzed nucleic acid polymer with a multi-functional amine-reactive chemical such as cyanuric chloride
  • cyanuric chloride can be added to a solution containing the nucleic acid polymer solution
  • the solution would contain
  • biomolecule-containing arraying solutions may be deposited onto a PEI coating even though that arraying solution contains a significant amount of the multi-functional amine-reactive chemical This provides a significant advantage over methods wherein coupling agent needs to be removed from an arraying solution prior to an arraying process When the nucleic acid polymer is double-stranded, both strands or one of the strands contains a terminal amino group.
  • the double-stranded nucleic acid polymer may be bonded through one terminal amino group to the PEI coating to immobilize the double-stranded polymer Since only one of the two strands is covalently bonded to the PEI coating, the other strand may be removed under denaturing and washing conditions
  • This approach provides one convenient method according to the present invention of achieving an array of single-stranded nucleic acid polymers
  • the double-stranded nucleic acid polymer may be obtained, for example, as a reaction product from PCR
  • the arraying solution is buffered using a common buffer such as sodium phosphate, sodium borate, sodium carbonate, or Tris-HCl
  • a common buffer such as sodium phosphate, sodium borate, sodium carbonate, or Tris-HCl
  • a preferred pH range for the arraying solution is 7 to 9, with a preferred buffer being freshly prepared sodium borate at pH 8.3 to pH 8.5.
  • oligonucleotides are synthesized with a 5'-amine (generally a hexylamine which is a six carbon spacer-arm and a distal amine).
  • oligonucleotides are 15 to 50 nucleotides in length, and are activated with homo-bifunctional or hetero-bifunctional cross-linking reagents such as cyanuric chloride.
  • the activated oligonucleotides can be optionally purified from excess cross-linking reagent (e.g., cyanuric chloride) by exclusion chromatography.
  • the activated oligonucleotides are then mixed with the solid supports to effect covalent attachment.
  • the unreacted amines of the solid support are capped (e.g., with succinic anhydride) to eliminate the positive charge of the solid support.
  • biotinylated oligonucleotides that bind to streptavidin, which in turn, is bound to a solid support.
  • Methods for producing biotinylated nucleic acid molecules and support-bound streptavidin are well known to those of skill in the art. For example, Van Ness et al., Nucleic Acids Res. 79:3345 (1991), describe a method for biotinylation of oligonucleotides, in which oligonucleotides are treated with activated biotin.
  • biotinylated oligonucleotides can be prepared by synthesizing oligonucleotides with biotin-labeled dNTPs (see, for example, Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3rd Edition, pages 12-23 to 12-25 (John Wiley & Sons, Inc. 1995)). Methods for biotinylating nucleic acids are well known in the art and are described, for example, in Avidin-Biotin Chemistry: A Handbook (Pierce Chemical Company 1992). Standard methods that can be used to bind streptavidin to the tips of the present invention are provided, for example, by Das and Fox, Ann. Rev. Biophys. Bioeng. 5: 165, 1979, and by Wilchek and Bayer, Anal. Biochem. 171 A. 1983.
  • the tips described herein can also be used to assay peptides.
  • General guidelines for the conjugation of peptides, polypeptides, and proteins to solid supports are proved, for example, by Wong, Chemistry of Protein Conjugation and Cross- linking (CRC Press, Inc 1991), and by Partis et al , J. Protein Chemistry 2 263 (1983)
  • the use of peptides and antibodies in solid phase procedures is known from, for example, Vaughn et al Nature Biotechnology 14 309, 1996 and Huse et al Science 246 1275, 1989
  • RNA is prepared using standard techniques In the studies described in Example 1, total RNA was prepared by acid-guanidinium-phenol extraction, using well known methods (see, for example, Ausubel et al (eds ), Short Protocols in Molecular Biology, 3 rd Edition, pages 4-4 to 4-6 (John Wiley & Sons, Inc 1995), Wu et al , Methods in Gene Biotechnology, pages 33-34, (CRC Press 1997))
  • Messenger RNA is then captured on a solid support, for example, that contains oligonucleotides having oligo(dT) tails
  • a simultaneous lysis-mRNA capture protocol using a chaotrope, such as guanidinium thiocyanate or guanidine hydrochloride, for both cell lysis and hybridization
  • a chaotrope such as guanidinium thiocyanate or guanidine hydrochloride
  • Support-bound RNA is used as a template to produce the first strand of cDNA, using standard methods. Then, the second strand of cDNA is synthesized or an adapter is placed at the distal end of the first strand cDNA. As an illustration, it is possible to place three dNGs at the 3' end of the first strand using, for example, terminal transferase. Alternatively, an adaptor can be ligated onto the 3' end of the cDNA molecule. A complementary primer is then hybridized to the adaptor. The second strand cDNA strand is then synthesized using the first strand of cDNA as a template.
  • RNA can be amplified using a polymerase chain reaction (PCR).
  • PCR is a process based on a specialized polymerase, which can synthesize a complementary strand to a given DNA strand in a mixture containing deoxyribonucleotides and two DNA primers, each about 20 bases long, which flank the target sequence.
  • the mixture is heated to separate the strands of double- stranded DNA containing the target sequence and then cooled to allow the primers to bind to their complementary sequences on the separated strands.
  • the polymerase then extends the primers into new complementary strands.
  • anchor PCR A modification of PCR, called “anchor PCR,” allows amplification of full-length mRNA even though only a small amount of sequence information is available (Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3 rd Edition, pages 15-27 to 15-32 (John Wiley & Sons, Inc. 1995)).
  • This procedure requires an oligo(dT) primer that is either complementary to the poly(A) tail of mature mRNA, when amplifying downstream to the known sequence, or complementary to a synthesized homopolymer tail added to the cDNA following first-strand synthesis, when amplifying upstream to the known sequence.
  • RNA template can be digested with RNaseH, hydrolyzed in sodium hydroxide or removed by heat denaturation, leaving a single-stranded DNA template which can be used for oligonucleotide-directed second strand synthesis, PCR, random-primed probe production, or gene-expression studies using labeled oligonucleotides.
  • the bound cDNA can also be used for preparing a subtracted library or differential probes for various applications.
  • Second strand cDNA synthesis represents a second branch-point in the solid support cDNA technology.
  • the choice can be made to ligate adapters to the cDNA that can support processes such as full-length single-stranded cDNA probes, library production, in vitro transcription, and 5' RACE.
  • An important advantage to solid support cDNA synthesis is the ability to automate the process.
  • it is useful to adapt the solid support described herein to a 96-well format.
  • a robotic arm can be used to deliver 96 supports onto 96 gold-plated pins and direct cDNA synthesis in a standard 96-well Cetus plate. 5. Analysis of Gene Expression
  • the solid supports described herein can be used in high through-put methods for examining the expression of numerous genes (1-2000) in a single measurement Such methods can be performed in parallel with greater than one hundred samples per process
  • the method is applicable to drug screening, developmental biology, molecular medicine studies and the like
  • methods for analyzing the pattern of gene expression from a selected biological sample comprising the steps of (a) exposing nucleic acids from a biological sample, (b) combining the exposed nucleic acids with one or more selected detectably-labeled nucleic acid probes, under conditions and for a time sufficient for the probes to hybridize to the nucleic acids, wherein the detectable label is correlative with a particular nucleic acid probe and detectable by spectrometry, or potentiometry, (c) separating hybridized probes from unhyb ⁇ dized probes, (d) detecting the label by spectrometry or potentiometry, and (e) determining therefrom the pattern of gene expression of the biological sample
  • RNA from a target source is bound to a solid support through a specific hybridization step (e g , capture of poly(A) mRNA by a tethered ohgo(dT) capture probe)
  • the solid support is then washed and cDNA is synthesized on the solid support using standard methods (/ e , reverse transc ⁇ ptase)
  • the RNA strand is then removed via hydrolysis
  • the result is the generation of a DNA population, covalently immobilized to the solid support, which reflects the diversity, abundance, and complexity of the RNA from which the cDNA was synthesized
  • the solid support is then hybridized with one to several thousand probes which are complementary to a gene sequence of interest Each probe type is labeled with a tag detectable by spectromet ⁇ c method, such as mass spectrometry After the interrogation step, excess or unhyb ⁇ dized probe is washed away, the solid support is placed,
  • tissue, primary or transformed cell lines, isolated or purified cell types or any other source of biological material in which determining genetic expression is useful can be used as a source of RNA.
  • the biological source material is lysed in the presence of a chaotrope to suppress nucleases and proteases, and to support stringent hybridization of target nucleic acid to the solid support.
  • Tissues, cells and biological sources can be effectively lysed in one to six molar chaotropic salts (guanidine hydrochloride, guanidine thiocyanate, sodium perchlorate, etc.).
  • the solution is mixed for 15 minutes to several hours with the solid support to effect immobilization of the target nucleic acid.
  • the capture of the target nucleic acid is achieved through complementary base pairing of target RNA and the capture probe immobilized on the solid support.
  • One permutation utilizes the 3' poly(A) stretch found on most eukaryotic messenger RNAs to hybridize to a tethered oligo(dT) on the solid support.
  • Another permutation is to utilize a specific oligonucleotide or long probes (greater than 50 bases) to capture an RNA containing a defined sequence.
  • RNA samples can be reversed-transcribed with each of four sets of degenerate anchored oligo(dT) primers, having the formula 5'-T 12 MN-3', where M can be G, A or C, and N is G, A, T, and C (Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3 rd Edition, pages 15-35 to 15-40 (John Wiley & Sons, Inc. 1995)). Each primer set is dictated by the 3 '-base, with degeneracy in the M position.
  • Hybridization times are guided by the sequence complexity of the RNA population and the type of capture probe employed. Hybridization temperatures are dictated by the type of chaotrope employed and the final concentration of chaotrope. General guidelines are provided, for example, by Van Ness and Chen, Nucleic Acids Res. 79:5143, 1991.
  • the lysate is preferentially agitated with the solid support continually to effect diffusion of the target RNA. After capturing the target nucleic acid, the lysate is washed from the solid support and all chaotrope or hybridization solution is removed. The solid support is preferentially washed with solutions containing ionic or non-ionic detergents, buffers and salts.
  • the next step is the synthesis of DNA complementary to the captured RNA, in which the tethered capture oligonucleotide serves as the extension primer for reverse transcriptase.
  • the reaction is generally performed at 25 to 37°C, and preferably agitated during the polymerization reaction.
  • the cDNA is synthesized, it becomes covalently attached to the solid support since the capture oligonucleotide serves as the extension primer.
  • the RNA is then hydrolyzed from the cDNA/RNA duplex.
  • the step can be effected by the use of heat which denatures the duplex or the use of base (i.e., 0.1 N NaOH) to chemically hydrolyze the RNA.
  • the objective of this step is to make the cDNA available for subsequent hybridization with defined probes.
  • the solid support or set of solid supports are then further washed to remove RNA or RNA fragments.
  • the solid support contains an approximate representative population of cDNA molecules that represents the RNA population in terms of sequence abundance, complexity, and diversity.
  • the next step is to hybridize selected probes to the solid support to identify the presence or absence and the relative abundance specific cDNA sequences. Probes are preferentially oligonucleotides in length of 15 to 50 nucleotides. The sequence of the probes is dictated by the end-user of the assay.
  • probes would be selected to be complementary to numerous cytokine mRNAs, RNAs that encode enzymes that modulate lipids, RNAs that encode factors that regulate cells involved in an inflammatory response, etc.
  • cytokine mRNAs RNAs that encode enzymes that modulate lipids
  • RNAs that encode factors that regulate cells involved in an inflammatory response etc.
  • the oligonucleotides are then hybridized to the cDNA on the solid support under appropriate hybridization conditions After completion of the hybridization step, the solid support is washed to remove any unhyb ⁇ dized probe The solid support or array of supports are then placed in solutions which effect the cleavage of the detectable tags The presence (and abundance) of an expressed mRNA is determined by measuring the amount of detectable tags For example, mass spectrometer tags are examined using a mass spectrometer
  • differential expression can be examined using a subtracted library
  • Subtraction of redundant messages to reveal patterns of gene expression representative of particular states of activation or development is a desirable capability of any gene discovery program
  • the use of a solid support to capture mRNA allows the message source representing the background to be subtracted Desired mRNA species are reverse transcribed and RNA templates are destroyed with alkali
  • the resulting "subtraction template" can be re-used indefinitely
  • RNA from the source under investigation is heat-denatured then hybridized to the first strand of cDNA on the subtraction template Unbound RNA is then washed away and either captured directly or hybridized a second time after all the bound, subtracted RNA has been eluted from the subtraction template After capture, cDNA synthesis continues as described previously
  • subtractive cDNA probes are prepared by hybridizing single-stranded cDNA from one cell type with immobilized mRNA from a closely-related cell type, and isolating the small fraction of unhyb ⁇ dized cDNA
  • DNA fragments of subtractive cDNA can be used to identify cDNA clones containing differentially expressed sequences
  • Subtractive cDNA can also be used to prepare subtractive cDNA libraries PCR can be used to amplify subtractive cDNA for use as probes or for cloning (see, for example, Kuel and Battey, "Generation of a Polymerase Chain Reaction Renewable Source of Subtractive cDNA," in PCR Protocols Current Methods and Applications, White (ed ), pages 287-304 (Humana Press, Inc. 1993); Wu et al., Methods in Gene Biotechnology, pages 29-65, (CRC Press 1997)).
  • a biotinylated oligo(dT)MN molecule is used to bind mRNA to a solid support, and to prime first strand synthesis (see, for example, R ⁇ sok et al., BioTechniques 27: 114, 1996).
  • the solid-phase cDNA is used as a template in a polymerase chain reaction, with the biotinylated oligo(dT)MN and an arbitrary decamer as primers.
  • PCR products obtained from two cell populations are then compared following fractionation by polyacrylamide gel electrophoresis, PCR bands of interest are eluted from the gel, and purified PCR products are used as templates for an additional polymerase chain reaction, which amplifies the selected bands.
  • the products of the second polymerase chain reaction can be used as probes, or can be further examined by sequence analysis.
  • Restriction endonucleases recognize short DNA sequences and cleave DNA molecules at those specific sites. Certain restriction enzymes cleave DNA very infrequently, generating a small number of very large fragments (several thousand to a million base pairs). Most restriction enzymes cleave DNA more frequently, thus generating a large number of small fragments (less than a hundred to more than a thousand base pairs). On average, restriction enzymes with four-base recognition sites will yield pieces 256 bases long, six-base recognition sites will yield pieces 4000 bases long, and eight-base recognition sites will yield pieces 64,000 bases long. Since hundreds of different restriction enzymes have been characterized, DNA can be cleaved into many different small fragments.
  • tandem repeat polymorphisms Although a few known human DNA polymorphisms are based upon insertions, deletions or other rearrangements of non-repeated sequences, the vast majority are based either upon single base substitutions or upon variations in the number of tandem repeats. Base substitutions are very abundant in the human genome, occurring on average once every 200-500 base pairs. Length variations in blocks of tandem repeats are also common in the genome, with at least tens of thousands of interspersed polymorphic sites, or "loci.” Repeat lengths for tandem repeat polymorphisms range from one base pair in (dA) n (dT) n sequences to at least 170 base pairs in -satellite DNA.
  • Tandem repeat polymorphisms can be divided into two major groups which consist of minisatellites/variable number of tandem repeats (VNTRs), with typical repeat lengths of tens of base pairs and with tens to thousands of total repeat units, and microsatellites, with repeat lengths of up to six base pairs and with maximum total lengths of about 70 base pairs. Most of the microsatellite polymorphisms identified to date have been based on (dC-dA) n or (dG-dT) n dinucleotide repeat sequences.
  • microsatellite polymorphisms involves amplification by the polymerase chain reaction of a small fragment of DNA containing a block of repeats followed by electrophoresis of the amplified DNA on denaturing polyacrylamide gel.
  • the PCR primers are complementary to unique sequences that flank the blocks of repeats.
  • Polyacrylamide gels, rather than agarose gels, are traditionally used for microsatellites because the alleles often only differ in size by a single repeat.
  • RFLP restriction fragment length polymorphism
  • the agarose gels do not usually afford the resolution necessary to distinguish minisatellite/VNTR alleles differing by a single repeat unit, but many of the minisatellites/VNTRs are so variable that highly informative markers can still be obtained. (Vos et al., Nuc. Acids Res. 23:4401, 1995).
  • Solid-phase techniques have enhanced the ability to detect polymorphisms
  • a biotinylated primer is used with allele-specific PCR primers to amplify one form of an allele
  • the PCR products can be detected by solution hybridization to a fluorophore-labeled probe, and hybrids are captured on a solid-phase support bearing streptavidin (see, for example, Syvanen and Landegren, Human Mutation 3712, 1994)
  • solid-phase minisequencing a biotinylated amplification product is immobilized by a streptavidin-coated support
  • the amplification product is then used as a template in a sequence-specific extension reaction in the presence of a single nucleoside triphosphate complementary to one of the sequence variants to be distinguished (see, for example, Syvanen and Landegren, Human Mutation 3712, 1994, Syvanen, Clin. Chem. Acta 226 225, 1994, Jarvela et al , J. Med. Genet. 33 1041, 1996)
  • oligonucleotide-ligation assay two differentially-labeled allele- specific oligonucleotides are compared for their ability to ligate to a biotinylated downstream oligonucleotide (see, for example, Nickerson et al , Proc. Natl. Acad. Sci.
  • the unlabeled oligonucleotide is immobilized on an avidin-coated solid support that projects into a test well
  • the presence of the target sequence is determined by measuring the particular signal generated by the bound labeled oligonucleotide
  • the allele-specific oligonucleotides can be labeled with different fluorophores, and the presence of the target is determined by measuring fluorescence
  • Nepom et al J. Rhematol.23 5 (1996), describe a method for genotyping analysis in which a selected sequence is amplified using PCR with a biological sample of nucleic acid and a biotinylated primer A small amount of amplified product is transferred to an automated processor instrument that performs allele-specific hybridization and detection
  • DNA fingerprinting represents another aspect of polymorphism detection
  • a variety of DNA fingerprinting techniques are presently available, most of which use PCR to generate fragments (see, for example, Jeffries et al , Nature 314 67, 1985, Welsh and McClelland, Nucleic Acids Res.
  • DNA fingerprinting can be performed by synthesizing full- length cDNA on a tip of the present invention
  • the cDNA is then digested with restriction endonucleases, and the unbound material is rinsed from the tip Adapters are then ligated onto the bound cDNA, and the product can be amplified and analyzed
  • RAPD random amplified polymorphic DNA
  • DAF DNA amplification fingerprinting
  • AP-PCR arbitrarily primed PCR
  • genomic DNA is digested with restriction endonuclease and ligated with oligonucleotide adapters, PCR provides selected amplification of sets of restriction fragments, and the amplified fragments are analyzed following fractionation by polyacrylamide gel electrophoresis
  • genomic DNA is covalently immobilized on a tip
  • Bound genomic DNA is then digested with restriction endonucleases, and the unbound material is washed from the tip.
  • Adapters are then ligated to bound genomic DNA fragments, and the bound DNA molecules are amplified and analyzed.
  • RNA fingerprinting has also been applied to mRNA fingerprinting (Habu et al., Biochem. Biophys. Res. Commun. 234:5X6, 1997).
  • double-stranded cDNA is synthesized with anchored oligo(dT) primers, and digested with Tag], which recognizes a four-base sequence.
  • a Taql adapter is then ligated to the ends of the cDNA fragments, and PCR amplification is performed with selected primers, following the general methods of AFLP-based genomic fingerprinting.
  • mRNA fingerprinting by the AFLP technique is performed with the solid supports described herein to anchor the oligo(dT) primers.
  • Mutations can also be identified via their destabilizing effects on the hybridization of short oligonucleotide probes to a target sequence (see, for example, Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:221, 1991).
  • this technique of allele-specific oligonucleotide hybridization involves amplification of target sequences and subsequent hybridization with short oligonucleotide probes.
  • An amplified product can be scanned for many possible sequence variants by determining its hybridization pattern to an array of immobilized oligonucleotide probes. Illustrations of this approach are provided by Examples 6 and 7.
  • DNA probes can be used to detect the presence of infectious agents or diseased cells, such as tumor cells expressing tumor-associated antigens.
  • a test biological sample is subjected to a lysis step using ionic detergents or choatropes to release nucleic acid targets.
  • Typical nucleic acid targets include mRNA, genomic DNA, plasmid DNA or RNA, and rRNA viral DNA or RNA.
  • the target requires some type of immobilization.
  • nucleic acids are immobilized on a solid support or substrate which possesses some affinity for nucleic acid.
  • the solid supports are then probed with tagged oligonucleotides of pre-determined sequence to identify the target nucleic acid of interest. Unhybridized probe is removed is a washing step, the tags are cleaved form their respective probes, and then measured (for a review, see Reischl and Kochanowski, Molec. Biotech. 3:55, 1995).
  • oligonucleotides representing a characteristic part of an amplified sequence are attached to a solid support.
  • the attachment may be covalent or via a biotin:streptavidin, or similar, type of linkage.
  • Target nucleic acid is used as a template to produce detectably labeled PCR products.
  • PCR products are hybridized with the capture oligonucleotides, and the presence of the PCR products is determined by a label-mediated detection reaction.
  • biotin-labeled PCR products are attached to a streptavidin-coated solid support. Immobilized PCR products are hybridized with a labeled probe complementary to internal sequences of the amplification product.
  • radiometric detection is another alternative. Suitable radiolabels for radiometric detection include 3 H, l2 % 131 1, 35 S, 14 C, 32 P, and the like. Fluorescently- labeled molecules provide yet another means of detection.
  • RNA was prepared by acid-guanidinium-phenol extraction, using standard techniques (see, for example, Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3 rd Edition, pages 4-4 to 4-6 (John Wiley & Sons, Inc. 1995)). Polyadenylated mRNA was captured on tips by first heating the mixture of isolated total RNA to 70°C, adding a high salt hybridization solution to the RNA, adding the RNA to the tip solid support having oligo(dT), and then placing the support on a moving platform, such as a rotary mixer.
  • a moving platform such as a rotary mixer.
  • RNA capture was accomplished by a variety of instruments including continual vortexer, orbital shaker, rotary rocker, and a hybridization oven equipped with a rotator.
  • the time required for RNA capture was found to be dependent on the quantity of input RNA.
  • 10 ⁇ g of total RNA were sufficient to achieve 90% saturation of a tip in approximately two hours. In a typical resting cell, this amount of total RNA corresponds to approximately 100 ng poly(A)+ mRNA bound per tip. Forty micrograms of total RNA from the same source gave the same level of saturation in 30 minutes.
  • RNA source In another series of experiments, a stimulated human T-cell line was used as the RNA source and saturation was reached in one hour under the same capture conditions using a 10 ⁇ g RNA input.
  • additional RNA sources have been captured including mouse, hamster and human cell lines, and all tend to fall within this range of 90% capture in 1-2 hours per 10 ⁇ g total RNA, defining a maximum and minimum incubation time.
  • MMLV-RT MMLV-reverse transcriptase
  • Second Strand cDNA Synthesis After reverse transcription, the tip was washed three times to remove reactants and enzyme. Second strand synthesis was performed in a 40 ⁇ l volume using one unit of RNaseH per 25 units E. coli DNA polymerase I. The reaction was incubated at room temperature on a rotary rocker for six hours or overnight.
  • Unevenly extended ends were "polished" for the subsequent ligation step by removing the second strand reaction and adding T4 DNA polymerase and dNTP's . This incubation proceeded for 30 minutes at 37°C on the rotator in a hybridization oven. Products are visualized directly by running the reaction in the presence of a 32 P- labelled dNTP, and either boiling the second strand products, or by cleavage from the support with the restriction enzyme Ascl. The radiolabelled products are run on a gel and placed on film for visualization. From a typical input of 10 ⁇ g total RNA, it is possible to recover 50-120 ng double-stranded cDNA, depending on the particular RNA.
  • thermostable DNA polymerase after reverse transcription with an enzyme possessing RNaseH activity, such as MMLV-RT is also a possibility .
  • the thermostable polymerase digests the RNA template, eliminating the need to remove RNA via a thermal denaturation step.
  • Hemi-phosphorylated adapters were ligated to the double-stranded cDNA in a 30 ⁇ l volume with a 5-10: 1 molar ratio of adapter:cDNA ends.
  • a preferred ligation buffer contained 10% PEG.
  • the adapter-cDNA mixture was incubated with T4 DNA ligase overnight at room temperature on a rotary rocker. The solid support was then washed three times to remove excess linkers. After ligation, the 5'-hydroxyl group on the adapter was phosphorylated with T4 polynucleotide kinase and ATP for one hour at 37°C on a hybridization oven rotating rack. This reaction was stopped by washing the tip three times in TE buffer. If the cDNA will be used for another application, the phosphorylation step may not be necessary.
  • second strand synthesis was specifically primed from an adapter added immediately after reverse transcription.
  • a partially single-stranded, heteroduplex adapter would be ligated using T4 RNA ligase.
  • the partial double-stranded nature of this adapter would provide a 3'-hydroxyl group from which second strand synthesis could begin, and would prevent concatemerization of the adapter during ligation since T4 RNA ligase cannot ligate double-stranded nucleic acids.
  • a vector was ligated to cDNA that had been synthesized on a tip.
  • cDNA or vectorxDNA was cleaved from the solid support in a 40 ⁇ l volume with Asc ⁇ at 37°C in a hybridization oven rotator rack for four hours. Cleaved DNA was then heated to 70°C for 20 minutes with or without mixing.
  • VectorxDNA was recircularized directly by bringing the volume to 50 ⁇ l with the addition of ligation buffer and T4 DNA ligase.
  • cDNA not previously ligated to vector was split into several aliquots for test ligations to determine which vector: insert ratio gives the best result in subsequent transformations. In this case, since the total amount of cDNA is small (50-120 ng total in 40 ⁇ l volume), the entire ligation must be transformed, making some type of desalting step necessary.
  • Transformation cDNA clones were propagated by electroporation transformation of electrocompetent E. coli with DNA aliquots from a ligation. Transformation frequencies can vary between 10 9 - 10 10 cfu per ⁇ g DNA. As those of skill in the art know, the major limitation in this procedure is the salt sensitivity of the electroporation. Only 1-2 ⁇ l of a standard ligation (5-10% of the total volume) can be electroporated per aliquot of electrocompetent cells before the threshold salt tolerance is exceeded and the applied current arcs between the electrodes, vaporizing the cells and wasting the portion of ligation used. Many separate aliquots of cells can be electroporated to keep this from happening, but this is both laborious and wasteful.
  • a better scheme is to use a desalting step that can be added to the current cDNA methodology which is flexible enough to be adapted to an in-line or scaled-down version of the technology without sacrificing yield.
  • Transformants were plated on standard growth medium, such as LB agar containing antibiotics. Only bacteria harboring the gene for antibiotic resistance form colonies on media containing that antibiotic. Differentiating between true recombinants and colonies containing only vector sequences is often accomplished by blue-white color selection which is a result of the expression of the lacZ gene which runs through the multiple cloning sites of many common plasmid vectors. Expression of the lacZ gene product is interrupted by an insert ligated into the multiple cloning site resulting in a white colony phenotype. In this scenario, recombinants can be visually identified from non-recombinants, but must either be picked from the non-recombinants or a certain level of background non-recombinants must be tolerated in the final product.
  • EXAMPLE 2 SCALING DOWN INPUT RNA FOR CDNA SYNTHESIS ON THE SOLID SUPPORT
  • RNA levels By minimizing loss of material during the many manipulations involved in standard cDNA library production, it is possible to scale down the input RNA levels by 10-100 fold. For example, roughly 100 ng of double-stranded cDNA can be synthesized on a solid support from 10 ⁇ g total RNA, which is 50-fold less starting material than that called for in commercially available kits. It may be possible to scale this down as much as 10-fold without changing the protocol described above significantly, although this will probably require the addition of an amplification step.
  • RNA pool An alternative would be to follow cDNA synthesis through adapter ligation using a T7 promoter-adapter. This would allow the amplification by in vitro transcription rather than by PCR, which may produce a more representative library by eliminating length bias inherent in PCR amplifications. In vitro synthesized transcripts could then be captured and processed just as any other RNA source. The success of this amplification would rely on careful optimization of the in vitro transcription conditions to insure full-length transcripts. Uncaptured transcripts could be polyadenylated using yeast poly(A) polymerase, then captured by adding them back to the same support. Although the re-adenylated transcripts would not be full-length, not all their information would be lost from the RNA pool.
  • a T7 promoter-adapter was synthesized and ligated to double-stranded cDNA from 10 ⁇ g total RNA on a tip.
  • a significant amount of transcripts were produced that ranged in size from 300 base pairs to 2,000 base pairs. Although these sizes are not optimal for an effective amplification step, the study shows that producing in vitro transcripts is possible on a solid support.
  • Another strategy for scale-down is to take advantage of asymmetric (linear) PCR in the same manner as in vitro transcripts to amplify and recapture the product. Since the amplified product is DNA rather than RNA, a DNA polymerase can be used to generate double-stranded cDNA.
  • Poly(A)+ mRNA from 10 ⁇ g total RNA was captured, reverse transcribed, second strand cDNA was synthesized, and a T7 promoter-adapter was ligated to the cDNA.
  • the solid support was placed in a standard Cetus PCR tube (ABI, Foster City, CA) and cycled for 35 rounds using a long PCR polymerase (Ex-Taq,TAKARA) in a format where the 70°C extension steps were increased in length one minute for every five cycles. Only one primer used in the PCR was complementary to the adapter so that amplification would be primed from the 5'- most end of the bound cDNA's, producing many copies of the (+) strand.
  • PCR products were heat denatured and run on an agarose gel to visualize the range of lengths of the single-stranded cDNA. Sizes ranged from roughly 500 base pairs to over 20 kilobase pairs, which was in good agreement with the sizes of double- stranded cDNAs labelled, cleaved, electrophoresed and visualized by autoradiography in parallel control experiments
  • PCR primers were designed for several genes known to be present at a high level, low level, or not at all The design of the primers was such that all products would be approximately the same length (400- 600 base pairs) and would be situated at or near the 5'-end of the cDNA This primer design provided a good approximation of the quality of the first strand synthesis Since the RNA source was the human Jurkat T-cell line, IL-2, IL-4, GM-CSF, GAPDH, CTLA4, c-fos, and Werner's helicase sequences were used for the primers Mouse guanylate kinase was used as a negative control
  • Rapid amplification of cDNA ends is also a technique that is adaptable to the solid support cDNA technology, described herein Either 5'- or 3'- RACE can be performed after adapter ligation of double-stranded cDNA using the back-end of the capture oligonucleotide (3'-RACE) or the proper 5' adapter oligonucleotide (5'-RACE) as anchors
  • Solid support RACE offers advantages over currently available techniques since little material is used in generating the cDNA and the product can be re-used This application would rely on the ability to run a PCR directly on the solid support described above Initial experiments have shown that this can be done for a high copy number housekeeping gene, GAPDH, and that the end product is directly proportional to the quality of the captured RNA and subsequently synthesized cDNA. Standard methods for performing 3'-RACE and 5'-RACE are well- known to those of skill in the art (see, for example, Wu et al., Methods in Gene
  • oligonucleotide 5'- ACTACTGATCAGGCGCGCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • a capture oligonucleotide (36-mer) was covalently linked to a polyethyleneimine-coated nylon pin assembly via a C6-amine tail
  • Shorter oligonucleotides (18-mer) labeled via a C6 amine tail with Texas Red were hybridized to the capture oligonucleotide in a 1 5 M guanidinium thiocyanate solution for 15 minutes at ambient temperature
  • the pin assemblies were then washed to remove unhybridized signal oligonucleotide twice with TEN buffer (0 01 M Tris (pH 7 5), 1 mM EDTA, 100 mM NaCl) and then once with TENS buffer (0 01 M Tris (pH 7 5), 1 mM EDTA, 100 mM NaCl, 0 1% SDS) followed by two washes with TEN buffer Test solutions were aliquoted into wells of a polycarbonate thermowell plate (Corning Costar Corp Cambridge, MA), and the plate was placed in
  • the level of fluorescence in each well correlates with the amount of signal oligonucleotide that has melted from the capture oligonucleotide
  • the "melting" or duplex dissociation was conducted over a temperature range of 10°C to 95°C Fluorescence was measured with a commercial fluorescence plate reader To calculate the T d , cumulative counts eluted at each temperature were plotted against temperature The temperature at which 50% of the material had been dissociated from the tip was taken as the T d
  • the helical coil transition is defined as the temperature at which a value of alpha equals 0 2 for a given oligonucleotide duplex (or nucleic acid duplex, containing or not containing a mismatch at any place in the duplex) to the temperature at which a value for alpha equals 0 8 for the same given oligonucleotide duplex (or nucleic acid duplex)
  • the data are exported into a spreadsheet and melt curves are generated for each solution From these melt
  • each test solution was placed into 16 separate wells of two thermowell plates (Corning Costar Cambridge, MA) containing 100 ul aliquots, one tube for each temperature point
  • the pin assembly was transferred to a new row of the plate before each temperature jump Just before reaching the 50°C temperature point, the first plate was removed from the thermal cycler and replaced with the second thermowell plate
  • the liquid was transferred to wells of two black microfluor plates (Dynatech) Fluorescence was measured using excitation wavelength 584 nm and emission wavelength 612 nm
  • the data were exported to a spreadsheet program for analysis
  • eight thermowell plates were cut in half to make sixteen 4 x 12 well plates A 100 ⁇ l aliquot of each test solution was placed into one well of each half-plate until all wells contained test solution All sixteen half-plates were identical in solution configuration
  • the pin assembly was transferred to a new half-plate before each temperature jump When the thermal cycling program was complete
  • This example describes the determination of the T d of wild type and mutant oligonucleotides when hybridized to a target nucleic acid It is shown that hybotrope based hybridization solutions allow the detection of single base pair mutations in a nucleic acid target with a probe up to a 30 nucleotides in length
  • EDTA "SDS/FW” is FW with 0 1% sodium dodecyl sulfate (SDS)
  • Hybridization solutions contained the text specified concentration of hybotrope, 2% N- lauroylsarcosine (sarcosyl), 50 mM Tris (pH 7 6) and 25 mM EDTA
  • Formamide hybridization solution contained 30% formamide, 0 09 M NaCl, 40 mM Tris-HCl (pH 7 6), 5 mM EDTA, and 0 1% SDS Guanidinium thiocyanate was purchased from Kodak (Rochester, NY) GuCl, lithium hydroxide, trichloroacetic acid, NaSCN, NaClO 4 and KI, were purchased from Sigma (St Louis, MO) Rubidium hydroxide was purchased from CFS Chemicals (Columbus, OH) CsTFA was purchased from Pharmacia (Piscataway, NJ) LiTCA and TMATCA, and TEAT
  • Oligonucleotides were synthesized on a commercial synthesizer using standard cyanoethyl-N,N-diisorpropylamino-phosphoramidite (CED-phosphoramidite) chemistry. Amine tails were incorporated onto the 5'-end using the commercially available N-monomethoxytritylaminohex-6-yloxy-CED-phosphoramidite.
  • oligonucleotides were commercially purchased (Midland Certified Reagents, Midland, Tx.).
  • Table I shows the oligonucleotides that were used to measure the difference in T d between a wild type oligonucleotide and a mutant oligonucleotide.
  • the wild type oligonucleotide represents fully and perfectly base-paired duplex and a mutant oligonucleotide represents a single base pair mismatch (generally in the middle of the oligonucleotide).
  • Oligonucleotides were bound to the tips described herein. In these studies, the oligonucleotides were attached to the tips using the approach described by Van Ness et al , Nucl. Acids Res. 19 3345, 1991 The oligonucleotide-tips contained 0 1 to 1 2 ⁇ g/tip of covalently immobilized oligonucleotide
  • thermodynamic properties of oligonucleotide duplexes A high throughput method for the measurement of the thermodynamic properties of oligonucleotide duplexes has been developed
  • the method allows thousands of solution samples to be scanned for their ability to modulate the thermodynamic parameters of the helical to coil transition of oligonucleotide duplexes
  • This method employs a solid support which has been designed to fit in a Cetus plate (or the well of a plate designed for 96 well PCR format) and requires about 40 ⁇ l of volume to be completely covered by liquid
  • the design of the tip is shown in Figure 1
  • This tip is also designed to be compatible with the square end of a spring probe that can be used as an attachment site in order to array the nylon tips in a 1x8, 1x12, 4x8, 4x12, or 8x12 format
  • a depiction of such a device is shown in Figure 2
  • oligonucleotide duplex is immobilized on the nylon tip as described by Van Ness and Chen, Nucleic Acids Res. 79 5143, 1991
  • a hybridization step is then used to form the oligonucleotide duplexes on a tip
  • the hybridization step can be performed en masse in a single container or individually in the wells of a plate used for the PCR It is therefore possible for every tip of a 96 member array of tips to possess a different oligonucleotide duplex
  • the tips are washed and then placed in a PCR plate mounted on a thermocycler
  • the tips are then moved through a series of wells each time the temperature is increased by 5°C Typically, the temperature increments are in 5°C steps and the period of the melting at each temperature is 1 to 5 minutes
  • tips in a 1x12 format are placed in row H at 10°C
  • the thermocycler is then programmed to ramp through
  • Fluorescent probes are commonly used in this format and have little effect on the measured T d values described herein
  • the use of radiolabeled or fluorescent probes permit a wide variety of solutions to be measured since there is no requirement of optical clarity, in contrast to the case for melt curves derived by UV spectrometry (hyperchromicity shifts) Fluorescence is measured with a microtiter plate fluorescence reader, the data are directly imported into a spreadsheet program, such as Excel, which then calculates the stability, enthalpy, helical coil transition, and temperature range, and then graphs the results Typically, a 1x12 format that measures 12 solutions at once can be completed within one hour, including set up and data reduction
  • oligonucleotide/oligonucleotide T d is incubated in various hybridization solutions with a complementary oligonucleotide immobilized on oligonucleotide-tips From 5 to 5000 ng of oligonucleotide are hybridized in 300-400 ⁇ l volumes at various temperatures (19-65°C) for 5 to 30 minutes
  • the tips are washed three times with one millihter of the respective hybridization solution, and then once with the respective melting solution at the starting temperature of the melting process
  • the tips in 100 ⁇ l of the respective melting solution are then placed on top of a thermocycler At one to five minute intervals, the temperature is raised 5°C, and the tip is moved into a new well of the microtiter plate
  • the melting, or duplex dissociation is conducted over a temperature range of 10°C to 95°C Fluorescence is measured with a
  • T d cumulative relative fluorescent units (RFUs) eluted at each temperature were platted against temperature
  • the temperature at which 50% of the material had been dissociated from the tip is the T d or T m
  • the helical coil transition is defined as the temperature at which a value of a equals 0 2 for a given oligonucleotide duplex (or nucleic acid duplex, containing or not containing a mismatch at any place in the duplex) to the temperature at which a value for a equals 0 8 for the same given oligonucleotide duplex (or nucleic acid duplex)
  • T d s were obtained in the hybridizations described below
  • na indicates not applicable or too large to accurately determine
  • T d (wt) is the T d of a perfectly base-paired oligonucleotide duplex
  • T m (mt) is the T d of a oligonucleotide duplex containing a single mismatch
  • the values shown are for a 24-mer duplex of the sequence described above From the data presented in Table III, the stringency factor is directly proportional to the difference between a perfectly base paired duplex and a duplex containing a mismatch That is, the stringency factor predicts the ability of given hybridization solution to discriminate mismatched duplexes

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Abstract

Les techniques de dosage en phase solide ont apporté une approche efficace à l'analyse des biomolécules dans le diagnostic médical et la recherche de base. Des procédés d'hybridation d'acides nucléiques en phase solide ont par exemple été appliqués à l'analyse des polymorphismes génétiques, au diagnostic des maladies génétiques, au diagnostic du cancer, à la détection des pathogènes viraux et microbiens, au criblage des clones et au classement des fragments génomiques. Cette invention présente un nouvel embout de collecte d'échantillons en phase solide qui permet d'améliorer les techniques de synthèse ou de détection des biomolécules. Ces embouts peuvent être utilisés pour mettre au point des systèmes de collecte d'échantillons, qui, à leur tour, peuvent être combinés pour former des réseaux matriciels de systèmes de collecte d'échantillons en phase solide, utiles dans des processus automatisés. Ces embouts peuvent être reliés à une tige de support sollicitée par ressort et ils sont également recouverts d'une couche chimique à laquelle une biomolécule peut se fixer.
EP98965546A 1997-12-31 1998-12-30 Embouts en phase solide et utilisations correspondantes Withdrawn EP1040352A1 (fr)

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CA2315296A1 (fr) 1999-07-08
BR9814604A (pt) 2000-10-17
AU2098999A (en) 1999-07-19
WO1999034214A1 (fr) 1999-07-08
HUP0100697A2 (hu) 2001-06-28

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