WO2002092615A2 - Compositions and methods for array-based genomic nucleic acid analysis of biological molecules - Google Patents
Compositions and methods for array-based genomic nucleic acid analysis of biological molecules Download PDFInfo
- Publication number
- WO2002092615A2 WO2002092615A2 PCT/US2001/015446 US0115446W WO02092615A2 WO 2002092615 A2 WO2002092615 A2 WO 2002092615A2 US 0115446 W US0115446 W US 0115446W WO 02092615 A2 WO02092615 A2 WO 02092615A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- group
- biological molecule
- article
- manufacture
- Prior art date
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
-
- 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
- B01J2219/00497—Features relating to the solid phase supports
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00585—Parallel processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00596—Solid-phase processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00614—Delimitation of the attachment areas
- B01J2219/00617—Delimitation of the attachment areas by chemical means
- B01J2219/00619—Delimitation of the attachment areas by chemical means using hydrophilic or hydrophobic regions
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00623—Immobilisation or binding
- B01J2219/00626—Covalent
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00623—Immobilisation or binding
- B01J2219/0063—Other, e.g. van der Waals forces, hydrogen bonding
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00659—Two-dimensional arrays
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00677—Ex-situ synthesis followed by deposition on the substrate
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/0068—Means for controlling the apparatus of the process
- B01J2219/00702—Processes involving means for analysing and characterising the products
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00718—Type of compounds synthesised
- B01J2219/0072—Organic compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/11—Compounds covalently bound to a solid support
-
- 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
- TECHNICAL FIELD The present invention claims a closely related family of compounds, devices, and methods relating to techniques for immobilizing biological molecules, e.g., nucleic acids, to a solid support for the purpose of conducting scientific investigation or routine testing upon the bound molecule (e.g., nucleic acid) samples in areas such as genome-wide genetic mapping and gene expression studies, protein interaction studies, peptide interaction studies and small molecule interactions with larger macromolecules.
- biological molecules e.g., nucleic acids
- DNA is a water-soluble compound, that if left in solution (i.e., a water-based solution), is likely to degrade, through hydrolysis, and so forth. Obviously this frustrates any investigation involving DNA, and so therefore, accurate and reliable study involving DNA requires a method or device to ensure the integrity of DNA.
- immobilizing DNA means fixing one end of the strand to the solid support so that the remainder of the strand is unmodified and free to undergo further reaction depending upon the particular study. Indeed, this is a widely used method to conduct laboratory studies involving DNA.
- the particular immobilized strand to which the probe reacts reveals the nucleotide sequence of the previously unknown immobilized strand.
- probe studies are severely confounded by electrostatic sticking of the probe to the derivatized (hence electrostatically charged) glass surface.
- the probe is often radiolabeled so that its presence can be detected by an ordinary radiation detector.
- the location of the probe on the glass surface as evidenced by the detector, reveals the chemical identity or sequence of the immobilized DNA strand at that particular location on the glass surface (which is known and designated in advance).
- the radiation detector is unable to distinguish between probe that is chemically bound to a complementary strand of DNA affixed to the solid support, and probe that is simply electrostatically stuck to the glass surface (but not to a DNA strand).
- poly-L-lysine One very common substance used to prepare a glass surface to receive a nucleic acid sample is poly-L-lysine. See, e.g., DeRisi (1996) 14 Nature Genetics 457; Shalon (1996) 6 Genome Res. 639; and Schena (1995) 270 Science 467.
- Other types of pre-derivatized glass supports are commercially available (e.g., sialylated microscope slides). See, e.g., Schena (1996) 93 Proc. Natl. Acad. Sci. USA 10614.
- U.S. Pat. No. 5,610,287 discloses coating a solid support with a salt or cationic detergent to non-covalently bond nucleic acids to the support.
- U.S. Pat. No. 5,024,933 discloses coating a solid support with an isolate of naturally occurring mussel adhesive protein.
- U.S. Pat. No. 4,937,188 discloses covalently bonding an enzyme to a solid support via molecular chain which acts as a substrate for the enzyme.
- 4,818,681 discloses coating a solid support with a nucleoside phosphate through the heterocyclic moiety of the nucleoside; the nucleic acid is then immobilized upon the solid support by enzymatic coupling.
- U.S. Pat. No. 4,806,631 discloses activating a nylon solid support by partially solvolyzing the amine groups (e.g., by treating with an alkylating group) on the nylon surface.
- Another approach to this problem involves derivatizing both the solid support and the nucleic acid sought to be immobilized. See, e.g., U.S. Pat. No. 5,641,630, discloses coating a solid support with a complexing agent that binds to another complexing agent to which the nucleic acid sought to be bound is likewise bound. U.S. Pat. No. 5,554,744, discloses contacting a solid support with dusopropylcarbodiimide and an acid catalyst and a succinylated nucleoside to immobilize the nucleoside. U.S. Pat. No.
- 5,514,785 discloses coating a solid support with, preferably, primary and secondary amines, followed by activation of the nucleic acid using cyanuric chloride.
- U.S. Pat. No. 5,215,882 discloses modifying the nucleic acid sought to be immobilized with a primary amine or equivalent, followed by reaction of the modified nucleic acid with the solid support (the support must have free aldehyde groups) in the presence of a reducing agent.
- the solid support in order to effectively immobilize nucleic acids onto solid surfaces, the solid support must first be derivatized, or made chemically labile, so that the nucleic acid can then be reacted with solid support.
- epoxides are known mutagens; that is, they are known to damage nucleic acids, particularly DNA.
- compositions and methods for affixing biological molecules to solid supports It demonstrates that any biological molecule can be modified and affixed to an unmodified solid support. A skilled artisan will recognize the significance of first modifying a molecule to enhance its binding affinity by appropriate modifications; thus, this modified molecule can be immobilized to an unmodified solid surface to generate a fully functional array of molecules for a spectrum of specific applications.
- the invention provides any biological molecule, e.g., DNA and nucleic acids more generally, that are modified such that they readily adhere to an unmodified or underivatized glass surface.
- epoxide-modified nucleic acids, particularly DNA are readily affixed to an unmodified solid support.
- the invention provides a modified biological molecule comprising a biological molecule modified by reaction with a compound having the formula:
- Ri — X — R 2 wherein R t is a cyclic ether group or an amino group, R is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group.
- the Ri cyclic ether is a compound comprising an epoxide group, such as an ethylene oxide, or equivalent.
- the cyclic ether is an oxirane group, or equivalent, or a compound comprising an aromatic hydrocarbon epoxide group.
- the Ri group reacts with the biological molecule such that the modified biological molecule is linked to the compound through Ri group.
- the linkage, or association, of the R group to the biological molecule can be such that the Ri group is covalently or non-covalently bound to the biological molecule.
- the biological molecule comprises a nucleic acid (e.g., a oligonucleotide), a lipid, a polysaccharide, a polypeptide (e.g., a peptide), or an analog or a mimetic thereof, or a combination thereof.
- the nucleic acid can comprise a DNA (e.g., a genomic DNA or a cDNA), an RNA (e.g., an mRNA, rRNA, and the like) or an analog or a mimetic thereof or a combination thereof.
- the nucleic acid can further comprise a telomeric structure or a chromatin structure.
- the nucleic acid is attached to the compound by the R group, i.e., the nucleic acid reacts with the Ri group at its 5' end.
- the cyclic ether is an epoxide group and the alkoxysilane is — Si(OCH 3 ) 3 , — Si(OC 2 H 5 ) 3 , — Si(OCH 3 )H 2 , — Si(OCH 3 )(CH 3 ) 2 , or — Si(OCH) 3 ) 2 CH 3 .
- the cyclic ether is an epoxide group and the compound is 3- glycidoxypropyltrimethoxysilane.
- the Ri group is a primary amino group. In one aspect, the Ri group is an amino group and the alkoxysilane is selected from the group consisting of — Si(OCH 3 ) 3 , — Si(OC 2 H 5 ) 3 and
- R l5 R 2 and R 3 are selected from the group consisting of — H, — CH 3 , — OCH , and — OC 2 H 3 , and provided that at least one of R 1; R or R 3 is either — OCH 3 or— OC 2 H 3 .
- the Ri group is an amino group and the compound is 3- aminopropyltriethoxysilane.
- the invention provides an article of manufacture comprising an arrayed plurality of biological molecules covalently bound to a surface, wherein, before attachment to the surface, the biological molecules are modified by reaction with a compound having the formula: R] — X — R 2 , wherein Ri is a cyclic ether group or an amino group, R 2 is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group, and upon attachment to the surface the modified biological molecules are covalently bound to the surface; wherein each biological molecule is attached to the surface on at least one discrete and known location to form a cluster of substantially identical biological molecules.
- the surface is a glass, a mica, a quartz, or a metal oxide surface.
- the metal oxide surface can be an alumina (Al 2 O 3 ), a titania (TiO 2 ), a SnO 2 , a RuO 2 , or a PtO 2 , or an equivalent thereof.
- the surface of the article of manufacture can comprise a polystyrene, a polyester, a polycarbonate, a polyethylene, a polypropylene or a nylon.
- the modified biological molecules are covalently bound to the surface via the R 2 group.
- the biological molecules can comprise a nucleic acid, a lipid, a polypeptide, a polysaccharide, or an analog or a mimetic thereof, or a combination thereof.
- the biological molecules are derived from a virus, a bacteria, a yeast, a plant, an insect, a mammal, such as a human or a mouse.
- the biological molecules can comprise nucleic acids or analogs or mimetics thereof.
- the nucleic acids can comprise DNA, RNA or analogs or mimetics thereof or a combination thereof.
- the nucleic acids can be oligonucleotides.
- the nucleic acids react with the Rj[ group at their 5' end.
- the nucleic acids immobilized on the article of manufacture can comprise a plurality of fragments of a genomic nucleic acid.
- the biological molecule e.g., a genomic nucleic acid or RNA
- the genomic DNA is derived from a virus, a bacteria, a yeast, a plant, an insect, a mammal, such as a human or a mouse.
- the fragments of nucleic acid e.g., genomic nucleic acid, further comprise a cloning vehicle.
- the cloning vehicle can comprise a bacterial artificial chromosome (BAC).
- the cloning vehicle comprises a plasmid, a cosmid, a bacteriophage PI -derived vector (PAC), a yeast artificial chromosome (YAC) or a mammalian artificial chromosome (MAC).
- the nucleic acid comprises a plurality of CpG island tags.
- the fragments of genomic nucleic acid comprise sequences representing at least one substantially complete chromosome or at least one defined section of a chromosome.
- each genomic nucleic acid fragment has been mapped to a known location on a chromosome.
- the nucleic acid e.g., the genomic nucleic acid fragments, have a size no more than about 1.5 megabase, no more than about 1.2 megabase, no more than about 1.0 megabase, and, no more than about 0.75 megabase in size.
- each cluster of substantially identical biological molecules consists of between about 5 and about 400, or, between about 10 and about 200, or, between about 50 and 100, substantially identical copies of a biological molecule.
- the surface can consist of less than about 800, about 600, about 500, about 400, about 300, about 200 or about 100 clusters per square centimeter.
- each cluster of substantially identical biological molecules is about 100 microns, about 50 microns, about
- the invention provides an article of manufacture (e.g., array or biochip) comprising an array of cloned genomic nucleic acid fragments representing a defined subsection of or a substantially complete chromosome, wherein, before attachment to the surface, the cloned fragments are modified by reaction with a compound having the formula:
- Ri is an epoxide group
- R is an alkoxysilane group
- X is a moiety chemically suitable for linking the epoxide group and the alkoxysilane group
- each array- bound cloned fragment has been mapped to a known location on a chromosome.
- the invention provides a kit comprising an article of manufacture of the invention, as described herein, and printed matter, wherein the printed matter comprises instructions on hybridizing a sample of nucleic acid to an array-bound nucleic acid.
- the invention provides a method for identifying a specific binding partner, comprising: (a) providing an article of manufacture comprising an arrayed plurality of biological molecules covalently bound to a surface, wherein, before attachment to the surface, the biological molecules are modified by reaction with a compound having the formula: Ri — X — R 2 , wherein Ri is an cyclic ether group or an amino group, R 2 is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group, and upon attachment the modified biological molecules are covalently bound to the surface, wherein each biological molecule is attached to the surface on at least one discrete and known location to form a cluster of identical biological molecules; (b) providing a sample of biological molecules; (c) contacting the sample of step (b) with the array-bound biological molecules as set forth in step (a) under conditions permissive for specific binding of a molecule in the sample of step (b) to an array-bound biological
- the invention provides a method for generating a molecular profile of a nucleic acid sample, comprising the following steps: (a) providing an article of manufacture comprising an array of biological molecules, wherein, before attachment to the surface, the biological molecules are modified by reaction with a compound having the formula: Ri — X — R 2 , wherein Ri is a cyclic ether group, R 2 is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group and the alkoxysilane group, and the modified biological molecules are covalently bound to the surface; (b) providing a sample comprising a nucleic acid; and (c) contacting the nucleic acid with the array-bound biological molecules as set forth in step (a) under conditions, allowing binding of the sample nucleic acid to the array-bound biological molecules, and detecting binding of the sample nucleic acid to the array-bound biological molecules, thereby generating a molecular profile of the sample nucleic acid.
- the array-bound biological molecules comprise a nucleic acid, such as a DNA corresponding to, or derived from, a genomic
- the binding can comprise hybridization of the sample nucleic acid to the array-bound nucleic acid.
- the array-bound nucleic acid can represent a section of at least one a chromosome or at least one substantially complete chromosome.
- the chromosome can be a viral, a bacterial, a yeast, a plant, an insect, or a mammalian, such as a human or a mouse, chromosome.
- the array-bound nucleic acids have been mapped to a known location on a chromosome.
- the molecular profile is a comparative genomic hybridization (CGH).
- the molecular profile can comprise detection of a genomic DNA amplification, a genomic DNA deletion, or a genomic DNA insertion.
- the molecular profile can comprise detection of a point mutation.
- the molecular profile is the identification of a single or multiple point mutations, such as a single-nucleotide polymorphism (SNP).
- the detection of a point mutation can further comprise use of a primer extension assay.
- the modified nucleic acid of the invention, or the array-bound nucleic acids comprise on or more CpG island tags.
- the molecular profile is generated by a differential methylation hybridization (DMH) reaction.
- the sample nucleic acids can comprise genomic DNA digested with at least one methylation-sensitive restriction endonuclease and the molecular profile comprises detection and mapping of hypermethylated regions of the genome.
- the methylation- sensitive restriction endonuclease can be selected from the group consisting of Notl, Smal, SacII, Eagl, Mspl, Hpall and BssHII.
- the molecular profile comprises detection of transcriptionally active regions of a genome.
- the sample of nucleic acid can be derived from a nuclear run-off assay; tins sample can be modified by the methods of the invention, and, in one aspect, these modified nucleic acids are immobilized onto an array as set forth in the invention.
- the molecular profile comprises an analysis of a chromatin structure.
- the modified nucleic acids of the invention, or the array-bound biological molecule can comprise a chromatin structure.
- the molecular profile comprises an analysis of a telomeric structure.
- the molecular profile of a telomeric structure can comprise an analysis of telomeric erosion or telomeric addition.
- the modified nucleic acids of the invention, or the array-bound biological molecule e.g., nucleic acid
- the invention provides a method for making a modified biological molecule comprising (a) providing a biological molecule; (b) providing a compound having the formula: R t — X — R 2 , wherein Ri is a cyclic ether group or an amino group, R 2 is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group; and (c) reacting the biological molecule with the compound, thereby modifying the biological molecule with the compound.
- the invention provides a method for making an article of manufacture (e.g., array or biochip) comprising an arrayed plurality of biological molecules covalently bound to a surface comprising (a) providing a biological molecule; (b) providing a compound having the formula: Ri — X — R 2 , wherein Ri is a cyclic ether group or an amino group, R 2 is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group; (c) providing a surface comprising hydroxyl groups; (d) reacting the biological molecule with the compound, thereby modifying the biological molecule with the compound; and, (e) depositing a plurality of modified biological molecules on the surface as discrete clusters, wherein a modified biological molecule is attached to the surface on at least one discrete and known location to form at least one cluster of substantially identical biological molecules; the array comprises at least two, a plurality of, clusters.
- Ri
- the compound used to modify the biological molecule is a 3-glycidoxy- propyltrimethoxysilane.
- the biological molecule is a nucleic acid and the reaction with the 3-glycidoxypropyltrimethoxysilane is at a basic pH, thereby generating a modified nucleic acid.
- the pH can be above about pH 9.5.
- a 3- glycidoxy-propyltrimethoxysilane modified nucleic acid can be deposited on an underivatized glass surface at about a neutral pH.
- the compound used to modify the biological molecule is a 3- aminopropyltriethoxy silane.
- the biological molecule is a nucleic acid and the reaction with the 3-aminopropyltriethoxysilane is at about a neutral pH. This reaction can take place in the presence of sodium bisulfate, or equivalent.
- This modified nucleic acid can be deposited on an underivatized glass surface.
- the modified biological molecules, such as a modified nucleic acid, of the invention will adhere to a solid surface to allow subsequent biochemical investigations.
- a modified biological molecule such as a modified nucleic acid
- a biological molecule e.g., a nucleic acid
- two crucial functional groups e.g., a cyclic ether group and an alkoxysilane group.
- methods for preparing the aforementioned modified biological molecules are claimed.
- the invention provides a high-density microarray comprising a glass or other inert surface.
- This array, or “biochip” can be made by printing numerous highly discrete modified biological molecule (e.g., DNA) sample spots, or “clusters,” upon the surface.
- the invention provides a modified biological molecule (e.g., a nucleic acid) prepared from a biological molecule (e.g., a nucleic acid) and a halogenated silane, or equivalent.
- a modified nucleic acid prepared by reaction of a biological molecule (e.g., a nucleic acid) with a brominated moiety, followed by reaction with an aminated silane.
- the invention provides a device that allows printing of the aforementioned high-density microarrays.
- the invention provides modified silanes that allow the skilled artisan to modulate the electrostatic properties of the solid surface to optimize sample density and detection sensitivity.
- the present invention possesses numerous advantages over the prior art. Many of the advantages derive from the fact that the solid surface, which is can be ordinary glass, remains highly chemically inert. Thus, the previously mentioned problems of probe (or other reactant) sticking to the glass as well as “spreading" are entirely eliminated. The ultimate result is, among other things, far higher detection sensitivity compared with state-of-the-art derivatized solid support.
- the biological molecule e.g., a nucleic acid
- the reaction of the epoxide derivatives of the invention is simple to execute; it occurs under mild conditions, reaction rates are quick, and equilibrium is highly favorable.
- the epoxide-modified biological molecules (e.g., nucleic acids) of the present invention are essentially permanently stable; thus they can be prepared (derivatized) and stored for later use
- FIG. 1 depicts a coupling reaction of nucleic acid (in this instance DNA) with 3-glycidoxypropyltrimethoxysilane, followed by the reaction of the newly modified DNA and the solid support (in this instance a glass surface). The final reaction product, the immobilized DNA, is shown at bottom.
- FIG. 2 depicts a coupling reaction of nucleic acid (in this instance DNA) with 3-aminoproplytriethoxysilane followed by the reaction of the newly modified DNA and the solid support (in this instance a glass surface).
- the final reaction product, the immobilized DNA is shown at bottom.
- FIG. 3 depicts a device for making a high-density microarray; both a top (FIG. 3 A) and a side view (FIG. 3B) are shown.
- FIG. 4 depicts the silanization of nucleic acid through alkylation of halogen-containing silane compounds.
- FIG. 5 a depicts the first step in the silanization of nucleic acid using amine-containing silane compounds.
- the reaction occurs preferentially at the guanine base at neutral and slightly basic pH.
- FIG. 5b depicts the first step in the silanization of nucleic acid using amine-containing silane compounds. In this case, the reaction occurs preferentially at the cytosine base at more basic pH.
- FIG. 5 c depicts the second and final step in the silanization of nucleic acid using amine-containing silane compounds.
- Figure 6 is a schematic representation of one aspect of the present invention showing silane linkers by hydrophobic linkers.
- Figure 7 is a schematic representation of an exemplary reaction wherein a biological molecule, a polypeptide, is modified, or "activated,” by a method comprising use of succinimidyl acetylthiopropionate (SATP) to introduce an active sulf ydryl functional group, as described in detail in Example 9, below.
- SATP succinimidyl acetylthiopropionate
- the invention provides modified biological molecules, such as polypeptides and nucleic acids, and articles of manufacture comprising arrays, with these modified biological molecules immobilized to the array surface.
- the invention also provides methods for making and using these compositions.
- One aspect of the invention is chemical modification of the biological molecule (e.g., nucleic acid) sought to be immobilized. This chemically modified nucleic acid is then readily reacted to a solid support such as a glass surface, rendering the biological molecule (e.g., nucleic acid) immobilized. Again, this is in direct contradiction to the prior art, which teaches modification of the solid support, rather than the nucleic acid itself.
- the modified the biological molecules (e.g., nucleic acids) of the present invention readily adhere to a variety of solid surfaces having reactive functional groups, e.g., hydroxyl groups.
- reactive functional groups e.g., hydroxyl groups.
- These include, though are not limited to: quartz glass, mica, alumina (Al O 3 ), titania (TiO ), SnO , RuO 2 , PtO , plastics such as the following polymer materials, polystyrene, polyester, polycarbonate, polyethylene, polypropylene, and nylon as well as numerous semi-conductive surfaces, such as numerous other metal oxide surfaces and equivalents.
- the chemically modified biological molecules (e.g., nucleic acids) of the present invention are so modified with compounds having two crucial functionalities: a ring ether and an alkoxysilane group.
- the biological molecule e.g., nucleic acid
- the newly modified biological molecules e.g., nucleic acids
- the otherwise inert surface e.g., glass
- the alkoxysilane group reacts with a hydroxyl-containing (e.g., hydroxyl derivatized) surface, e.g., Si ⁇ OH groups on the glass surface.
- the chemically modified biological molecules (e.g., nucleic acids) of the present invention are so modified with compounds having two crucial functionalities: an amino group and an alkoxysilane group.
- the biological molecules (e.g., nucleic acid) react with the amino group, then the newly modified biological molecules (e.g., nucleic acids) are contacted with the otherwise inert
- hydroxyl-containing e.g., hydroxyl derivatized
- the biological molecules are modified by reaction with halogenated silane compounds.
- the biological molecules e.g., nucleic acids
- the biological molecules are derivatized by a two-step process involving a final reaction with amine-containing silanes and brominated nucleic acids.
- Other aspects are directed to preparing and optimizing high-density microarrays utilizing the modified biological molecules (e.g., nucleic acids) of the other aspects of the present invention.
- compositions and methods of making and using that comprise any biological molecule or combinations of biological molecules are only one of many biological compositions, e.g., biological polymers, that can be modified by the methods of the invention and used in the methods of the invention.
- a polymer refers to a molecule that has joined prefabricated units, e.g., monomers or compositions that can be of limited diversity, linked together, usually by identical mechanisms, e.g., a cellulose is a polymer is simple sugars or polysaccharides.
- Exemplary biological molecules include but are not limited to DNA, RNA, protein, peptides, lipids, saccharides, polysaccharides and mimetics and analogs thereof.
- any biological molecule including those having a structure found in nature or a synthetic structure, including polymers, can be modified by the methods of the invention and affixed to a solid surface similar to the modified nucleic acids of the invention.
- Another aspect of the invention is the modification of biological molecules.
- One type of modification is chemical cross-linking.
- bifunctional "crosslinking" reagents contain two reactive groups, thus providing a means of covalently crosslinking two target groups.
- the reactive groups in a chemical crosslinking reagent typically belong to the classes of functional groups, e.g., succinimidyl esters, maleimides and iodoacetamides.
- Bifunctional crosslinking reagents can be divided in homobifunctional, heterobifunctional and zero-length bifunctional crosslinking reagents. In homobifunctional crosslinking reagents, the reactive groups are identical.
- reagents couple like functional groups, e.g., two thiols, two amines, two acids or two alcohols, and are predominantly used to form intramolecular crosslinks.
- the reactive groups In heterobifunctional crosslinking reagents, the reactive groups have dissimilar chemistry, allowing the formation of crosslinks between unlike functional groups.
- the "zero-length" crosslinking reagent forms a chemical bond between two groups without itself being incorporated into the product.
- water-soluble cardodiimide (ED AC) is used to couple carboxylic acids to amines.
- a noncovalent interaction between two molecules that has very slow dissociation kinetics can also function as a crosslink.
- reactive derivatives of phospholipids can be used to link the liposomes or cell membranes to antibodies or enzymes.
- Biotinylation and haptenylation reagents can also be thought of as heterobifunctional crosslinking reagents because they comprise a chemically reactive group as well as a biotin or a hapten moiety that binds with high affinity to avidin or an anti-hapten antibody, respectively.
- photoreactive crosslinking reagents are available.
- the general scheme involves photoreactive crosslinking reagents that contain a chemically reactive group as well as a photoreactive group. These crosslinkers are first chemically reacted with one molecule and then this modified molecule is coupled to a second molecule using UV illumination. Depending on the reactive properties of the chemical and photoreactive groups, these crosslinkers can be used to couple like or unlike functional groups.
- nucleic acid refers to a deoxyribonucleotide (DNA) or ribonucleotide (RNA) in either single- or double-stranded form.
- DNA deoxyribonucleotide
- RNA ribonucleotide
- the term encompasses nucleic acids containing known analogues of natural nucleotides.
- the term encompasses mixed oligonucleotides comprising an RNA portion bearing 2'-O-alkyl substituents conjugated to a DNA portion via a phosphodiester linkage, see, e.g., U.S. Patent No. 5,013,830.
- the term also encompasses nucleic-acid-like structures with synthetic backbones.
- DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of
- PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described, e.g., by U.S. Patent Nos. 6,031,092; 6,001,982; 5,684,148; see also, WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197.
- nucleic acid is used interchangeably with gene, DNA, RNA, cDNA, mRNA, oligonucleotide primer, probe and amplification product.
- polypeptide include compositions of the invention that also include “analogs,” or “conservative variants” and “mimetics” or
- peptidomimetics with structures and activity that substantially correspond to the polypeptide from which the variant was derived, as discussed in detail, below.
- small molecule means any synthetic small molecule, such as an organic molecule or a synthetic molecule, such as those generated by combinatorial chemistry methodologies. These small molecules can be synthesized using a variety of procedures and methodologies, which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY; Venuti (1989) Pharm Res. 6:867-873. Synthesis of small molecules, as with all other procedures associated with this invention, can be practiced in conjunction with any method or protocol known in the art. For example, preparation and screening of combinatorial chemical libraries are well known, see, e.g., U.S. Patent Nos. 6,096,496; 6,075,166; 6,054,047; 6,004,617; 5,985,356; 5,980,839; 5,917,185; 5,767,238.
- array or “microarray” or “biochip” or “chip” as used herein is an article of manufacture, a device, comprising a plurality of immobilized target elements, each target element comprising a “cluster” or “biosite” or defined area comprising a biological molecule (e.g., a nucleic acid molecule or polypeptide, such as an antibody) immobilized to a solid surface, as discussed in further detail, below.
- a biological molecule e.g., a nucleic acid molecule or polypeptide, such as an antibody
- sample of nucleic acid targets or “sample of nucleic acid” as used herein refers to a sample comprising DNA or RNA, or nucleic acid representative of DNA or RNA isolated from a natural source, in a form suitable for hybridization (e.g., as a soluble aqueous solution) to another nucleic acid or polypeptide or combination thereof (e.g., immobilized probes).
- the nucleic acid may be isolated, cloned or amplified; it may be, e.g., genomic DNA, mRNA, or cDNA from substantially an entire genome, substantially all or part of a particular chromosome, or selected sequences (e.g.
- the nucleic acid sample may be extracted from particular cells or tissues.
- the cell or tissue sample from which the nucleic acid sample is prepared is typically taken from a patient suspected of having a genetic defect or a genetically-linked pathology or condition, e.g., a cancer, associated with genomic nucleic acid base substitutions, amplifications, deletions and/or translocations.
- Methods of isolating cell and tissue samples are well known to those of skill in the art and include, but are not limited to, aspirations, tissue sections, needle biopsies, and the like.
- the sample will be a "clinical sample” which is a sample derived from a patient, including sections of tissues such as frozen sections or paraffin sections taken for histological purposes.
- the sample can also be derived from supematants (of cells) or the cells themselves from cell cultures, cells from tissue culture and other media in which it may be desirable to detect chromosomal abnormalities or determine amplicon copy number.
- the nucleic acids may be amplified using standard techniques such as PCR, prior to the hybridization.
- the target nucleic acid may be unlabeled, or labeled (as, e.g., described herein) so that its binding to the probe (e.g., oligonucleotide, or clone, immobilized on an array) can be detected.
- the probe e.g., oligonucleotide, or clone, immobilized on an array
- the probe an be produced from and collectively can be representative of a source of nucleic acids from one or more particular (pre-selected) portions of, e.g., a collection of polymerase chain reaction (PCR) amplification products, substantially an entire chromosome or a chromosome fragment, or substantially an entire genome, e.g., as a collection of clones, e.g., BACs, PACs, YACs, and the like (see below).
- PCR polymerase chain reaction
- the probe or genomic nucleic acid sample may be processed in some manner, e.g., by blocking or removal of repetitive nucleic acids or by enrichment with selected nucleic acids.
- the invention provides modified nucleic acids, and articles of manufacture comprising arrays that include modified nucleic acid compositions and methods for making and using these arrays.
- the nucleic acid is modified by reaction with a compound having the formula: Ri — X — 2 , where Ri is a cyclic ether group or an amino group, R 2 is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group.
- the modified nucleic acid or the immobilized nucleic acid on the array can be representative of genomic DNA, including defined parts of, or entire, chromosomes, or entire genomes.
- the arrays and methods of the invention are used in comparative genomic hybridization (CGH) reactions, including CGH reactions on arrays (see, e.g., U.S. Patent Nos. 5,830,645; 5,976,790), see discussion below.
- CGH comparative genomic hybridization
- the invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.
- RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof may be isolated from a variety of sources, genetically engineered, amplified, and or expressed/ generated recombinantly (recombinant polypeptides can be modified or immobilized to arrays in accordance with the invention).
- Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
- these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic.
- Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with a primer sequence.
- nucleic acids such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.
- genomic nucleic acid used in the methods and compositions of the invention include genomic or cDNA libraries contained in, or comprised entirely of, e.g., mammalian artificial chromosomes (see, e.g., Ascenzioni (1997) Cancer Lett. 118:135-142; U.S. Patent Nos. 5,721,118; 6,025,155) (including human artificial chromosomes, see, e.g., Warburton (1997) Nature
- yeast artificial chromosomes YAC
- bacterial artificial chromosomes BAC
- PI artificial chromosomes see, e.g., Woon (1998) Genomics 50:306-316; Boren (1996) Genome Res. 6:1123-1130); PACs (a bacteriophage PI -derived vector, see, e.g., Vietnamese (1994) Nature Genet. 6:84-89; Reid (1997) Genomics 43:366-375; Nothwang (1997) Genomics 41:370-378; Kern (1997) Biotechniques 23:120-124); cosmids, plasmids or cDNAs.
- Amplification using oligonucleotide primers can be used to generate nucleic acids used in the compositions and methods of the invention, to detect or measure levels of test or control samples hybridized to an array, and the like.
- the skilled artisan can select and design suitable oligonucleotide amplification primers.
- Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N. Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,
- LCR ligase chain reaction
- transcription amplification see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci. USA 86:1173
- self-sustained sequence replication see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874)
- Q Beta replicase amplification see, e.g., Smith (1997) J. Clin. Microbiol.
- the invention is directed to modified polypeptides and articles of manufacture comprising arrays with immobilized polypeptides, peptides and peptidomimetics.
- the polypeptide is modified by reaction with a compound having the formula: Ri — X — R 2 , where Ri is a cyclic ether group or an amino group, R 2 is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group.
- polypeptide As noted above, the terms “polypeptide,” “protein,” and “peptide,” used to practice the invention, include compositions of the invention that also include “analogs,” or “conservative variants” and “mimetics” or “peptidomimetics.”
- mimetic and “peptidomimetic” refer to a synthetic chemical compounds.
- the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
- the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetics' structure and/or activity.
- Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
- a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
- Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N- hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIG).
- glutaraldehyde N- hydroxysuccinimide esters
- bifunctional maleimides N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIG).
- a polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature.
- the skilled artisan will recognize that individual synthetic residues and polypeptides incorporating mimetics can be synthesized using a variety of procedures and methodologies, which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman, et al., supra.
- Polypeptides incorporating mimetics can also be made using solid phase synthetic procedures, as described, e.g., by U.S. Pat. No. 5,422,426.
- Peptides and peptide mimetics can also be synthesized using combinatorial methodologies.
- Various techniques for generation of peptide and peptidomimetic libraries are well known, and include, e.g., multipin, tea bag, and split-couple-mix techniques; see, e.g., al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.
- Modified polypeptide and peptides can be further produced by chemical modification methods, see, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med.
- Peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automated synthesis may be used.
- arrays or “BioChips”
- the invention provides “arrays” or “microarrays” or “biochips” or “chip” comprising the modified biological molecules of the invention, including the modified nucleic acids and polypeptides of the invention.
- Arrays are generically a plurality of target elements immobilized onto the surface of the array as defined “clusters,” or “biosites,” each target element comprising a one or more biological molecules (e.g., nucleic acids or polypeptides) immobilized a solid surface for association (e.g., specific binding or hybridization) to a sample.
- the immobilized nucleic acids can contain sequences from specific messages (e.g., as cDNA libraries) or genes (e.g., genomic libraries), including a human genome. Other target elements can contain reference sequences and the like.
- the biological molecules of the arrays may be arranged on the solid surface at different sizes and different densities. The densities of the biological molecules in a cluster and the number of clusters on the array will depend upon a number of factors, such as the nature of the label, the solid support, and the like.
- Each cluster/ biosite may comprise substantially the same biological molecule (e.g., nucleic acid or polypeptide), or, a mixture of biological molecules (e.g., nucleic acids of different lengths and/or sequences).
- a cluster/ biosite may contain more than one copy of a cloned piece of DNA, and each copy may be broken into fragments of different lengths.
- the surface onto which the modified biological molecules of the invention are immobilized can include nitrocellulose, glass, quartz, fused silica, plastics and the like, as discussed further, below.
- the compositions and methods of the invention can incorporate in whole or in part designs of arrays, and associated components and methods, as described, e.g., in U.S. Patent Nos.
- the articles of manufacture of the invention comprising arrays can have substrate surfaces of a rigid, semi-rigid or flexible material.
- the substrate surface can be flat or planar, be shaped as wells, raised regions, etched trenches, pores, beads, filaments, or the like.
- Substrates can be of any material upon which a "capture probe" can be directly or indirectly bound.
- suitable materials can include paper, glass (see, e.g., U.S. Patent No. 5,843,767), ceramics, quartz or other crystalline substrates (e.g.
- gallium arsenide metals, metalloids, polacryloylmorpholide, various plastics and plastic copolymers, NylonTM, TeflonTM, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polystyrene/ latex, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF) (see, e.g., U.S. Patent No. 6,024,872), silicones (see, e.g., U.S. Patent No. 6,096,817), polyformaldehyde (see, e.g., U.S. Patent Nos.
- PVDF polyvinylidene difluoride
- Reactive functional groups can be, e.g., hydroxyl, carboxyl, amino groups or the like.
- Silane e.g., mono- and dihydroxyalkylsilanes, aminoalkyltrialkoxysilanes, 3- aminopropyl-triethoxy silane, 3-aminopropyltrimethoxysilane
- Silane can provide a hydroxyl functional group for reaction with an amine functional group.
- the invention provides compositions and methods for generating a molecular profile of a nucleic acid sample, such as a sample of genomic DNA or a cDNA library.
- the invention provides articles of manufacture and methods for contacting array- bound nucleic acids with a sample containing nucleic acids and detecting the binding of the sample nucleic acids to the array, thereby generating a molecular profile of the sample nucleic acid.
- the molecular profile can be a comparative genomic hybridization (CGH) reaction; detection of a genomic DNA amplification, a genomic DNA deletion, or a genomic DNA insertion; detection of a point mutation, such as identification of a single-nucleotide polymorphism (SNP); differential methylation hybridization (DMH), where the array-bound nucleic acids are CpG island tags; detection of transcriptionally active regions of a genome (using, e.g., nuclear run-off assays); analysis of a chromatin structure; and analysis of a telomeric structure (such as telomeric erosion or telomeric addition). All of these procedures are well lcnown in the art, and any molecular biology procedure or analysis, can be performed using the modified biological molecules or arrays of the invention.
- CGH comparative genomic hybridization
- the arrays and methods of the invention are used in comparative genomic hybridization (CGH) reactions.
- CGH is a molecular cytogenetics approach that can be used to detect regions in a genome undergoing quantitative changes, i.e. gains or losses of copy numbers. Analysis of genomes of tumor cells can detect a region or regions of anomaly under going gains and/or losses. Differential expression of hundreds of genes can be analyzed using a cDNA array, thus facilitating characterization of gene expression in normal and diseased tissues.
- Generating a molecular profile of a nucleic acid sample by comparative genomic hybridization using methods and arrays of the invention can be practiced with methods and compositions known in the art, see, e.g., U.S. Patent Nos.
- SNPs single-nucleotide polymorphisms
- the arrays and methods of the invention are used to detect point mutations, such as single-nucleotide polymorphisms (SNPs).
- SNPs single-nucleotide polymorphisms
- Arrays can be used for high-throughput genotyping approaches for pharmacogenomics, where numerous individuals are studied with thousands of SNP markers.
- SNP mapping has accelerated complex disease gene localization; detection of multiple SNPs associated with a disease in a relatively small linkage disequilibrium region can narrow the linkage region for that disease, and, identification of susceptibility genes will enable a better understanding of the mechanisms of the disease processes and will facilitate the discovery of new and more efficacious medicines.
- Generating a molecular profile of a nucleic acid sample by the analysis and detection of SNPs using methods and arrays of the invention can be practiced with methods and compositions known in the art, see, e.g., U.S. Patent Nos. 6,221,592; 6,110,709; 6,074,831; 6,015,888; and, Kwok (2000) Pharmacogenomics 1:95-
- the arrays and methods of the invention are used in differential methylation hybridization (DMH), including, for example, CpG island analysis.
- the array-bound nucleic acids comprise CpG island tags.
- the methods and arrays of the invention are used to identify, analyze and map hypermethylated or hypomethylated regions of the genome.
- the sample nucleic acids can comprise genomic DNA digested with at least one methylation-sensitive restriction endonuclease and the molecular profile comprises detection and mapping of hypermethylated (or hypomethylated) regions of the genome.
- Any methylation-sensitive restriction endonuclease or equivalent endonuclease enzyme can be used, including, for example, Notl, Smal, SacII, Eagl, Mspl, Hpall, Sau3AI and BssHII.
- both a methylation-sensitive enzyme and its methylation insensitive isoschizomer is used; see, e.g., Robinson (2000) Chromosome Res. 8:635-643; described use of the methylation-sensitive enzyme Hpall and its methylation insensitive isoschizomer Mspl. Windhofer (2000) Curr. Genet.
- DNA methylation or the covalent addition of a methyl group to cytosine within the context of the CpG dinucleotide, has profound effects on the mammalian genome. These effects include transcriptional repression via inhibition of transcription factor binding or the recruitment of methyl-binding proteins and their associated chromatin remodeling factors, X chromosome inactivation, imprinting and the suppression of parasitic DNA sequences.
- DNA methylation is also essential for proper embryonic development, DNA repair and genome stability. For example, DNA demethylation influence on chromosome stability is modulated by a sequence-specific chromatin structure (the invention also provides modified biological molecules and arrays comprising chromatin structures) (see, e.g., Vilain (2000) Cytogenet. Cell. Genet. 90:93- 101).
- telomeres are specialized nucleoprotein structures, which are specialized nucleoprotein structures, is essential for chromosome stability. Without new synthesis of telomeres at chromosome ends the chromosomes shorten with progressive cell division. This eventually triggers either replicative senescence or apoptosis when telomere length becomes critically short.
- the regulation of telomerase activity in human cells plays a significant role in the development of cancer (telomerase is the enzyme that synthesizes the telomere ends of linear eukaryotic chromosomes).
- telomerase is tightly repressed in the vast majority of normal human somatic cells, but becomes activated during cellular immortalization and in cancers. Thus, telomerase assays are useful for cancer detection and diagnosis (see, e.g., Hahn (2001) Ann Med 33:123-129; Meyerson (2000) J. Clin. Oncol. 18:2626-2634; Meyerson (1998) Toxicol. Lett. 102-103:41-5). Using the array-based telomeric structures of the invention will accelerate understanding of telomerase biology and lead to clinically relevant telomerase-based therapies.
- Generating a molecular profile of a nucleic acid sample by the analysis of telomeric structures using methods and arrays of the invention can be practiced with methods and compositions known in the art, see, e.g., U.S. Patent Nos. 6,221,590; 6,221,584; 6,022,709; 6,007,989; 6,004,939; 5,972,605; 5,871,926; 5,834,193; 5,830,644; 5,695,932; 5,645,986.
- the arrays and methods of the invention are used in the analysis of chromatin structure, including chromatin condensation, chromatin decondensation, histone phosphorylation, histone acylation, and the like (see, e.g., Guo (2000) Cancer Res. 60:5667-5672; Mahlknecht (2000) Mol. Med. 6:623-644).
- Chromatin structure remodeling occurs in certain cancers (see, e.g., Giamarchi (2000) Adv. Exp. Med. Biol. 480:155-161).
- Chromatin structure affects nuclear processes that utilize DNA as a substrate, e.g., transcription, replication, DNA repair, and DNA organization within the nucleus.
- Chromatin structure analysis is useful in fertility assessment; for example, sperm with decondensed chromatin are infertile. DNA damage in patients with untreated cancer can be measured using a sperm chromatin structure assay (see, e.g., Kobayashi (2001) Fertil. Steril. 75:469-475). Generating a molecular profile of a nucleic acid sample by the analysis of chromatin structure using the methods and arrays of the invention can be practiced with methods and compositions known in the art, see, e.g., U.S. Patent Nos.
- the arrays and methods of the invention are used in the detection and analysis of transcriptionally active regions of nucleic acid, e.g., transcriptionally active regions of a genome.
- a sample of nucleic acid can be derived from a nuclear run-off assay and detected and analyzed by the compositions and methods of the invention.
- nuclear run-off samples are modified by the methods of the invention.
- these modified nucleic acids are immobilized onto an array as set forth in the invention.
- Generating a molecular profile of a nucleic acid sample by the detection and analysis of transcriptionally active regions of nucleic acid by, e.g., nuclear run-off assays, using the methods and arrays of the invention can be practiced with methods and compositions known in the art, see, e.g., U.S. Patent Nos. 6,200,960; 6,184,032;
- Example 1 Preparation of Modified Nucleic Acid Using 3-glycidoxypropyl- trimethoxysilane
- the following example describes making and using one aspect of modified nucleic acid of the present invention.
- the purpose of the chemical modification is to enable the nucleic acid to be readily affixed to an underivatized solid surface.
- the nucleic acid-preferably DNA ⁇ is modified by reaction with 3- glycidoxypropyl-trimethoxysilane (GPTS), according to FIG. 1.
- GPTS 3- glycidoxypropyl-trimethoxysilane
- GPTS 3- glycidoxypropyl-trimethoxysilane
- affixing the nucleic acid to the solid support consists essentially of two steps. In the first, the nucleic acid reacts with the epoxide end of the GPTS molecule; in the second step, the glass surface reacts with the other end, or the silane end of the GPTS-modified nucleic acid, thereby affixing the nucleic acid onto an underivatized glass surface.
- the entire reaction is rapid, is characterized by a favorable equilibrium, and occurs under very mild conditions using a minimum of inexpensive reagents. Though there quite obviously are numerous ways to carry out either step of the reaction, the preferred method is shown in this and the following example.
- the compound shown is 3-glycidoxypropyItrimethoxysilane or GPTS.
- DNA is reacted with GPTS at basic pH, preferably above 9.5, to form the modified DNA.
- the modified DNA is then reacted with an underivatized glass (or other silanol-containing) surface at neutral pH, thus immobilizing the DNA onto the glass surface.
- the ring ether functionality reacts with the DNA.
- the ring ether need not be ethylene oxide, as it is in GPTS, although the small ring is preferred to increase reactivity of the ether functionality, which is relatively unreactive.
- the first reaction leading to the derivatized DNA, is a ring-opening reaction likely involving carbon 5 of the ribose ring of the DNA.
- This derivatized DNA is unusually stable and can be stored for long periods of time prior to actual use.
- the second reaction immobilizing the derivatized DNA onto the glass surface, is a simple substitution reaction creating an Si ⁇ O ⁇ Si linkage in the glass surface, and removing one of the alkoxy groups from the GPTS molecule.
- Example 2 Preparation of Modified Nucleic Acid Using 3-aminopropyltriethoxysilane
- nucleic acid preferably DNA
- affixing the nucleic acid to the solid support consists essentially of two steps.
- the nucleic acid reacts with the amino end of the 3-aminopropyltrimethoxysilane molecule; in the second step, the glass surface reacts with the other end, or the silane end of the 3- aminopropyltrimethoxysilane-modified nucleic acid, thereby affixing the nucleic acid onto an underivatized glass surface.
- the entire reaction is rapid, is characterized by a favorable equilibrium, and occurs under very mild conditions using a minimum of inexpensive reagents. Though there quite obviously are numerous ways to carry out either step of the reaction, the preferred method is shown in this and the following example.
- the compound shown is 3-aminopropyltriethoxysilane.
- DNA is reacted with 3-aminopropyltriethoxysilane at neutral pH in the presence of sodium bisulfite, or equivalent.
- the first reaction leading to the derivatized DNA, is transamination reaction of the cytosine residues on nucleic acids.
- the second reaction as in Example 1, involving immobilizing the derivatized DNA onto the glass surface, is a simple substitution reaction. It creates an Si— O ⁇ Si linkage in the glass surface and removes one of the alkoxy groups from the GPTS molecule.
- Example 3 Preparation of a High Density Microarray using Modified Nucleic Acid
- modified nucleic acids of the present invention such as those described in Examples 1 and 2
- they can then be exploited in a variety of ways, including, e.g., to make a high density array. Again, these modified nucleic acids
- FIG. 3 illustrates one aspect of a device for preparing such a high-density microarray using the DNA chips of the present invention.
- the device is made from a plurality of inexpensive commercially available capillary micropipets, preferably 10 cm micropipets, although other sizes will, of course, work. As depicted in FIG.
- each 10 cm micropipet is pulled to make a taper at one end. They are arranged in a hexagonal close-packed array, bounded by a square frame. The micropipets can be glued to one another to form a stable unit within the frame. The tapered ends (FIG. 3B) are cut off and polished to optical flatness.
- the tips of the device are dipped into a multi- well container that contains the (chemically modified in accordance with the present invention) DNA samples to be tested, and whose wells are aligned with the micropipets of the device. Upon contact of the tips into the wells, a small portion of each DNA sample is deposited into the micropipet corresponding to the particular well by simple capillary action.
- the size of the spot can be carefully controlled by the size of the tapered end.
- Using this device and the DNA chips of the present invention thousands of samples can be arrayed in a narrow area, simultaneously and without the need for expensive robotics.
- the method (comprising the DNA chips and pipet device) of the present invention has been shown to be even more efficient than methods using high-speed spotting robots.
- the compounds, methods and devices of the present invention are readily incorporated into a pre-packaged kit for commercial sale.
- the high-density microarray of the present invention can also be readily incorporated into the microarray systems of the art, such as those disclosed in the art section above.
- fluorescent in situ hybridization FISH
- Shalon 1996 6 Genome Res. 639 (1996)
- Sargent, et al. U.S. Pat. No. 5,601,982 discloses a method and apparatus for determining the sequence of polynucleotides involving scanning the nucleic acids by scanning tunneling microscopy.
- this invention is not limited to using only nucleic acids.
- Other biological molecules such as biopolymers, e.g., DNA, RNA, proteins, peptides or polypeptides, and polysaccharides, can be directly activated using the methods of the invention, such as bifunctional silane compounds or other crosslinking reagents, resulting in an immobilized biologic molecule, e.g., a biopolymer, to a solid surface.
- This invention demonstrates that the target molecules to be arrayed (i.e., immobilized) are first modified so that they have gained a binding affinity for solid surfaces without losing their probing (e.g., hybridization) abilities.
- halogenated silane depicted to the left of the arrow in Fig. 4 is 8-bromocytltrichlorosilane, 8-bromo- cytltrimethoxysilane, 4-chlorobutylmethyldichlorosilane, and 3- iodopropyltrimethoxysilane.
- the conversion depicted in Fig. 4 was performed as follows.
- the halogenated silane was dissolved in dimethylformamide (DMF) at a concentration of about 30 mM.
- DMF dimethylformamide
- 3 ⁇ g to 10 ⁇ g of nucleic acid was dissolved in 100 ul of 0.01 M phosphate buffer (pH 7.0).
- 1 to 3 ⁇ g of 30 mM halogenated silane was added, the solution is then mixed well, and allowed to react at about 37°C for about 3 hours (alternatively, it can be reacted at ambient temperature overnight).
- the desired product the modified nucleic acid — is purified by ethanol precipitation; then the modified nucleic acid is dissolved in water.
- Example 5 Preparation of Arrays and Controlling Spot (Cluster) Density and Size
- the following example demonstrates an exemplary method for manufacturing the arrays, or "biochips" of the invention.
- one particular advantage of the present invention is that it allows the investigator to prepare unusually high-density microarrays to conduct nucleic acid studies.
- This example is best understood in relation to example 3, which disclosed the preparation of a high-density microarray in accordance with the present invention.
- This example discloses enhanced methods for controlling the size and density of the individual nucleic acid "spots" or "clusters" on the solid supports, in accordance with the present invention.
- Small “spot” or “cluster” size in relation to high-density microarrays, allows higher sample density (i.e., more samples per unit area) and superior detection sensitivity (because the signals are less diffuse).
- the skilled artisan faces a crucial dilemma.
- silanes of the present invention contains an Si(OH) 3 at each end, linked by a hydrophobic group. See FIG. 6. Any of a variety of hydrophobic linkers can be used.
- Particularly preferred aspects include: 1,6-Bis-trichlorosilyhexane, 1,8-Bis- trichlorosilyloctane, 1,6-Bis-trimethoxysilyhexane, and 1,4 Bis- trimethoxysilylethylbenzene.
- one end of the silane attaches to the surface, and the other end remains reactive to the modified biological molecule, e.g., nucleic acid.
- the hydrophobic linker confers hydrophobicity to the surface.
- the skilled artisan can readily see how the electrostatic properties of the surface (hydrophobic versus hydrophilic) can be readily modulated— e.g., the chain length of the linker can be adjusted to control hydrophobicity, and the surface reactivity can be controlled by adjusting the amount of silane contacted with the surface.
- the glass surface was cleaned by slowly boiling in 3 M HCl for about 2 hrs in a fume hood. Next, the surfaces were rinsed with deionized water then kept in 0.1 M HCl until ready for use. When ready for use, the surfaces were rinsed with doubly distilled deionized water to remove any extant acid, then rinsed in absolute ethanol. Next, the surfaces were immediately transferred to an ethanol solution containing 0.0005 % to 0.002 % of the bi-functional silanes of this aspect of the invention. The surfaces were then treated at room temperature for about 48 hours. The surfaces were then rinsed with ethanol and air dried. Finally, the glass surfaces were stored in a dust-free environment until ready for use.
- the modified nucleic acid is prepared by reacting pristine nucleic acids with an amine-containing silane.
- the derivatization of nucleic acid with amine-containing silanes is comprised of two steps: (1) the halogenation (or bromination, as shown) of the nucleic acid (FIG. 5a, 5b); and (2) the derivatization of the halogenated nucleic acid (FIG. 5c).
- the reaction can occur in the presence of N-bromosuccinimide under mild pH conditions; varying either of these reaction variables allows the skilled biochemist to control the reaction rate.
- the reaction normally occurs at the guanine or cytosine base depending upon the pH— i.e., neutral to slightly basic pH favors reaction at the guanine residue, more basic pH favors reaction at the cytosine residue.
- Slightly different reaction protocols are preferably used depending upon whether the nucleic acid is DNA or RNA.
- DNA 5 ⁇ g of DNA was dissolved in 100 ⁇ l of 0.1 M NaHCO 3 , to reach a pH of about 9.5. This solution is kept on ice for about 5 minutes. Contemporaneously, a fresh N-bromosuccinimide solution at concentration of about 10 mM was prepared and also chilled on ice.
- the silane-modified DNA was purified by methods well known in the art; preferably, it is purified by ethanol precipitation, or equivalent procedures.
- RNA For RNA, a similar, though slightly different protocol was used: 5 ⁇ g of RNA was dissolved in 100 ⁇ l of 0.1 M phosphate buffer, to reach a pH of about 7.5. This solution is kept on ice for about 5 minutes. Contemporaneously, a fresh N- bromosuccinimide solution at concentration of about 10 mM was prepared and also chilled on ice. Next, 1 ⁇ l of the N-bromosuccinimide solution is added to the DNA solution; the solution was then stirred vigorously (to vortex). The reaction was then allowed to proceed on ice for about 15 minutes.
- silane-modified DNA was purified by methods well known in the art; preferably, it is purified by ethanol precipitation, or equivalent procedures.
- R can be — CH 3 , or — C 2 H 5;
- Ri can be H, — CH 3 , — C 2 H 5 — OCH 3 , or — OC 2 H 5;
- R 2 can be H, — CH 3 , — C 2 H 5 — OCH 3 , or — OC 2 H 5;
- This example describes methods to modify biological molecules, e.g., nucleic acids, using bifunctional silane compounds.
- a biopolymer is modified by reaction with 3-glycidoxypropyltrimethoxysilane
- GPTS GPTS
- affixing the biopolymer to a solid surface consists essentially of two steps. In the first, the biopolymer reacts with the epoxide end of the GPTS molecule; in the second step, the glass surface reacts with the other end, or the silane end of the GPTS -modified biopolymer, thereby affixing the biopolymer onto an underivatized surface.
- the biopolymer reacts with the epoxide end of the GPTS molecule
- the glass surface reacts with the other end, or the silane end of the GPTS -modified biopolymer, thereby affixing the biopolymer onto an underivatized surface.
- bifunctional crosslinking reagents could be used in the present invention (see above).
- any biological molecule can be incorporated into the compositions and methods of the invention.
- These non- biopolymers are first crosslinked to epoxide silane-activated biopolymers, e.g., biopolymers activated according to Example 7.
- the crosslinking of these non- biopolymers which are typically small molecules, increases the size and stability of the molecule.
- an activated biopolymer e.g., polyethylene glycol (PEG) or DNA
- PEG polyethylene glycol
- Example 9 Silanization of Amine-Containing Biopolymers such as Proteins and
- Polypeptides e.g., Antibodies
- Polysaccharides e.g., Polysaccharides
- This example describes methods to modify amine-containing biological molecules by silanization.
- biological molecules include peptides and polypeptides
- Nucleic acids comprising amine groups include, e.g., nucleotide compositions containing aminooxy moieties, as described in U. S. Patent No. 6,127,533.
- Biopolymers are effectively silanized and arrayed onto glass surfaces. Biopolymers are first treated with 2-iminothiolane (commonly known as "Traut's Reagent") or N-succinimidyl S-acetylthioacetate (SATA) or succinimidyl acetylthiopropionate (SATP) to introduce an active sulfhydryl functional group.
- the activated biopolymers are silanized by reacting with the epoxide silane compound as described previously under mild conditions. A typical reaction is depicted in Figure 7; this example uses SATP and an epoxide silane comprising ⁇ Si(OCH 3 ) 3 .
- Antibodies are silanized by various methods.
- One such method is to first dissolve an antibody in 0.1 M sodium phosphate buffer (pH 7.3) with 50 mM NaCl and 10 mM EDTA at a concentration of about 1 to 3 mg/ml. Then, add 5 ⁇ l of 100 mM SATA or SATP in a DMSO solution to 1 ml antibody solution and react at room temperature (RT) overnight. Next, add 100 ⁇ M of 1 M hydroxylamine hydrochloride and react at RT for one hour. After the RT activation, add 10 ⁇ M of 0.2 M 3- glycidoxypropyltrimethoxysilane (epoxide silane) and react at RT for about 5 hours. Upon completion of all reactions, antibodies are purified by gel filtration on a Sephadex G25 column, or equivalent. The modified antibody is fixed on a glass surface by direct deposition.
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2001263094A AU2001263094B2 (en) | 1998-05-04 | 2001-05-10 | Compositions and methods for array-based genomic nucleic acid analysis of biological molecules |
EP01937348A EP1385859A2 (en) | 2001-05-10 | 2001-05-10 | Compositions and methods for array-based genomic nucleic acid analysis of biological molecules |
JP2002589498A JP2005500023A (en) | 2001-05-10 | 2001-05-10 | Compositions and methods for performing arrayed genomic nucleic acid analysis of biomolecules |
CA002446050A CA2446050A1 (en) | 2001-05-10 | 2001-05-10 | Compositions and methods for array-based genomic nucleic acid analysis of biological molecules |
PCT/US2001/015446 WO2002092615A2 (en) | 2001-05-10 | 2001-05-10 | Compositions and methods for array-based genomic nucleic acid analysis of biological molecules |
AU2008229872A AU2008229872B2 (en) | 1998-05-04 | 2008-10-10 | Compositions and methods for array-based genomic nucleic acid analysis of biological molecules |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US2001/015446 WO2002092615A2 (en) | 2001-05-10 | 2001-05-10 | Compositions and methods for array-based genomic nucleic acid analysis of biological molecules |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2002092615A2 true WO2002092615A2 (en) | 2002-11-21 |
WO2002092615A3 WO2002092615A3 (en) | 2002-12-19 |
Family
ID=21742567
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2001/015446 WO2002092615A2 (en) | 1998-05-04 | 2001-05-10 | Compositions and methods for array-based genomic nucleic acid analysis of biological molecules |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1385859A2 (en) |
JP (1) | JP2005500023A (en) |
CA (1) | CA2446050A1 (en) |
WO (1) | WO2002092615A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005064333A1 (en) * | 2003-12-25 | 2005-07-14 | National Institute Of Advanced Industrial Science And Technology | Method of analyzing interaction between protein and sugar chain |
WO2010051288A1 (en) | 2008-10-27 | 2010-05-06 | Revivicor, Inc. | Immunocompromised ungulates |
EP2527456A1 (en) | 2004-10-22 | 2012-11-28 | Revivicor Inc. | Transgenic porcines lacking endogenous immunoglobulin light chain |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5476989B2 (en) | 2007-01-31 | 2014-04-23 | 和光純薬工業株式会社 | Method for detecting amplification or deletion of genomic DNA fragment |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5851769A (en) * | 1995-09-27 | 1998-12-22 | The Regents Of The University Of California | Quantitative DNA fiber mapping |
WO1999057323A1 (en) * | 1998-05-04 | 1999-11-11 | Baylor College Of Medicine | Chemically modified nucleic acids and methods for coupling nucleic acids to solid support |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1109111C (en) * | 1998-07-17 | 2003-05-21 | 中国科学院上海原子核研究所 | Macro-fragment deoxyribonucleic acid core piece and method for mfg. same |
-
2001
- 2001-05-10 WO PCT/US2001/015446 patent/WO2002092615A2/en active Application Filing
- 2001-05-10 EP EP01937348A patent/EP1385859A2/en not_active Withdrawn
- 2001-05-10 CA CA002446050A patent/CA2446050A1/en not_active Abandoned
- 2001-05-10 JP JP2002589498A patent/JP2005500023A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5851769A (en) * | 1995-09-27 | 1998-12-22 | The Regents Of The University Of California | Quantitative DNA fiber mapping |
WO1999057323A1 (en) * | 1998-05-04 | 1999-11-11 | Baylor College Of Medicine | Chemically modified nucleic acids and methods for coupling nucleic acids to solid support |
Non-Patent Citations (2)
Title |
---|
DATABASE CAPLUS [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; LI, MINQIAN ET AL: "Method for manufacturing DNA chips with large immobilized DNA fragments" retrieved from STN Database accession no. 133:205074 XP002189763 & CN 1 242 430 A (SHANGHAI INST. OF ATOMIC NUCLEUS, CHINESE ACADEMY OF SCIENCES, PEOP. R) 26 January 2000 (2000-01-26) * |
KUMAR, ANIL ET AL: "Silanized nucleic acids: a general platform for DNA immobilization" NUCLEIC ACIDS RES. ( 2000 ), 28(14), E71, II-VI, XP002188992 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005064333A1 (en) * | 2003-12-25 | 2005-07-14 | National Institute Of Advanced Industrial Science And Technology | Method of analyzing interaction between protein and sugar chain |
US8008094B2 (en) | 2003-12-25 | 2011-08-30 | National Institute Of Advanced Industrial Science And Technology | Methods for analyzing interactions between proteins and sugar chains |
EP2527456A1 (en) | 2004-10-22 | 2012-11-28 | Revivicor Inc. | Transgenic porcines lacking endogenous immunoglobulin light chain |
WO2010051288A1 (en) | 2008-10-27 | 2010-05-06 | Revivicor, Inc. | Immunocompromised ungulates |
Also Published As
Publication number | Publication date |
---|---|
CA2446050A1 (en) | 2002-11-21 |
JP2005500023A (en) | 2005-01-06 |
EP1385859A2 (en) | 2004-02-04 |
WO2002092615A3 (en) | 2002-12-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20060275787A1 (en) | Compositions and methods for array-based genomic nucleic acid analysis of biological molecules | |
EP1075544B1 (en) | Chemically modified nucleic acids and methods for coupling nucleic acids to solid support | |
US6916621B2 (en) | Methods for array-based comparitive binding assays | |
US7615350B2 (en) | Methods for haplotyping genomic DNA | |
WO2003020898A2 (en) | Arrays comprising pre-labeled biological molecules and methods for making and using these arrays | |
US20080187912A1 (en) | Oligonucleotide arrays for high resolution HLA typing | |
JP2001502909A (en) | Method for preparing single-stranded DNA array | |
WO2001006011A2 (en) | Arrays comprising non-covalently associated nucleic acid probes and methods for making and using them | |
EP1312685A2 (en) | Oligonucleotide array for detecting the methylation state | |
JP2001108683A (en) | Dna fragment fixing solid-phase carrier, dna fragment fixing method, and nucleic-acid fragment detecting method | |
WO2002092615A2 (en) | Compositions and methods for array-based genomic nucleic acid analysis of biological molecules | |
US20050032060A1 (en) | Arrays comprising pre-labeled biological molecules and methods for making and using these arrays | |
AU2001263094B2 (en) | Compositions and methods for array-based genomic nucleic acid analysis of biological molecules | |
AU2001263094A1 (en) | Compositions and methods for array-based genomic nucleic acid analysis of biological molecules | |
US8278049B2 (en) | Selective enrichment of CpG islands | |
Wu et al. | An activated GOPS‐poly‐L‐lysine‐coated glass surface for the immobilization of 60mer oligonucleotides | |
WO2003076903A2 (en) | Articles of manufacture and methods for making hydrophobic derivatized arrays | |
JP2001178442A (en) | Method for immobilizing dna fragment on surface of solid phase carrier and dna chip | |
Kumar | Synthesis of Oligonucleotides on Nylon Supports and Hybridization with Human Genomic DNA. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
AK | Designated states |
Kind code of ref document: A3 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2001263094 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2446050 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2002589498 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2001937348 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2001937348 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |