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WO2001032927A2 - Genes specifiques de tissu a signification diagnostique - Google Patents

Genes specifiques de tissu a signification diagnostique Download PDF

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Publication number
WO2001032927A2
WO2001032927A2 PCT/US2000/030396 US0030396W WO0132927A2 WO 2001032927 A2 WO2001032927 A2 WO 2001032927A2 US 0030396 W US0030396 W US 0030396W WO 0132927 A2 WO0132927 A2 WO 0132927A2
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WIPO (PCT)
Prior art keywords
polynucleotides
protein
seq
nos
fragments
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PCT/US2000/030396
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English (en)
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WO2001032927A3 (fr
Inventor
Thierry Sornasse
Jeffrey J. Seilhamer
George A. Watson
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Incyte Genomics, Inc.
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Application filed by Incyte Genomics, Inc. filed Critical Incyte Genomics, Inc.
Priority to JP2001535607A priority Critical patent/JP2004507206A/ja
Priority to CA002388511A priority patent/CA2388511A1/fr
Priority to EP00976921A priority patent/EP1255859A2/fr
Priority to AU14628/01A priority patent/AU1462801A/en
Publication of WO2001032927A2 publication Critical patent/WO2001032927A2/fr
Publication of WO2001032927A3 publication Critical patent/WO2001032927A3/fr

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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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a composition
  • a composition comprising a plurality of polynucleotides which are cell and/or tissue specific. These polynucleotides may be used to define and direct a metabolic or developmental process, to identify or to monitor the progression of a condition, disease, or disorder, or to evaluate and monitor the efficacy of a treatment protocol.
  • array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • arrays provide a platform for examining which genes are tissue specific, direct the differentiation of a cell type or tissue, carry out housekeeping functions, function as parts of a signaling cascade, or characterize a particular genetic predisposition, condition, disease, or disorder.
  • gene expression profiling is particularly relevant to improving diagnosis and prognosis of disease.
  • tissue and cell specific genes against which genes expressed during the disease process may be compared.
  • both the levels and sequences expressed in brain tumors may be compared with the levels and sequences expressed in normal brain tissue.
  • These comparisons may be made on a single array by incorporating a particular tissue or cell specific reference set alongside novel sequences or on multiple arrays, each of which contains at least some subset of the known reference set.
  • the present invention satisfies a need in the art in that it provides such a reference set.
  • the reference set may be used in its entirety or in part to produce an expression profile that may be used to define and direct a metabolic or developmental process, to identify or to monitor the progression of a condition, disease, or disorder, or to evaluate and monitor the efficacy of a treatment protocol.
  • the present invention provides a plurality of tissue or cell specific polynucleotides which may be used on an array to produce an expression profile.
  • This profile may define expression of the polynucleotides in normal tissue, during a particular metabolic or developmental process or during the onset, progression, or treatment of a human condition, disease, or disorder.
  • these polynucleotides are selected from SEQ ID NOs:l-416.
  • the invention also provides a plurality of polynucleotides which display tissue or cell specific expression and are selected from: a) SEQ ID NOs:209-218 and 1-10, cell specific polynucleotides of heart and fragments thereof; b) SEQ ID NOs:219-249 and 11-41, cell specific polynucleotides of skeletal muscle and fragments thereof; c) SEQ ID NOs: 250-25 land 42-43, cell specific polynucleotides of uterus and fragments thereof; d) SEQ ID NOs:252-256 and 44-48, cell specific polynucleotides of 5 ovary and fragments thereof; e) SEQ ID NOs:257-263 and 49-55, cell specific polynucleotides of stomach and fragments thereof; f) SEQ ID NOs:264-283 and 56-75, cell specific polynucleotides of intestine and fragments thereof; g) SEQ ID NOs:284-293 and 76-85
  • the plurality of polynucleotides are immobilized on a substrate.
  • the expression of a plurality of polynucleotides is used to detect
  • the tissue is embryonic stem cells which are differentiating into brain, heart, kidney, liver, lung, muscle or pancreatic tissues.
  • the tissue is a biopsy from diseased brain, heart, kidney, liver, lung, muscle, ovarian, pancreatic, small intestine, stomach, or uterine tissues which is being diagnosed for a cancer or immune or inflammatory disease or subjected to forensic analysis.
  • the point of origin of a metastatic cancer is determined.
  • the polynucleotides are used in high throughput methods of screening molecules or compounds to identify a ligand, the method comprising combining a polynucleotide with molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds to the polynucleotide.
  • the molecules or compounds to be screened are selected from DNA molecules, RNA molecules, PNAs, mimetics,
  • the invention provides a substantially purified polynucleotide selected from SEQ ID NOs:212, 228, 233, 259, 271, 287, 316-319, 324, 370, 379, 380, 383, 410, and 412 or a fragment thereof, SEQ ID NO:4, 20, 25, 51, 63, 79, 108-111, 116, 162, 171, 172, 175, 202, and 204.
  • 30 316-319, 324, 370, 379, 380, 383, 410, and 412 or a fragment thereof, SEQ ID NO:4, 20, 25, 51, 63, 79, 108-111, 116, 162, 171, 172, 175, 202, and 204 is used in an expression vector transformed into a host cell to produce a protein or a portion thereof by culturing the host cell under conditions for the expression of protein and recovering the protein from the host cell culture.
  • the invention provides a protein or a portion thereof.
  • the invention provides a protein or a portion thereof.
  • 35 protein is used in a high throughput method to screen large numbers of molecules or compounds to identify at least one ligand which specifically binds the protein, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein.
  • the protein is used to purify a ligand, the method comprising combining the protein with a sample under conditions to allow specific binding, recovering the bound protein, and separating the protein from the ligand, thereby obtaining purified ligand.
  • the molecules or compounds screened or purified may be selected from DNA molecules, RNA molecules, PNAs, mimetics, peptides, proteins, agonists, antagonists, antibodies or their fragments, immunoglobulins, inhibitors, drug compounds, and pharmaceutical agents. Any of these molecules or compounds may have diagnostic or therapeutic applications.
  • Sequence Listing is a compilation of polynucleotides obtained by sequencing and extension of clone inserts of different cDNAs. Each sequence is identified by a sequence identification number (SEQ ID NO or SEQ ID) and by the clone number (Incyte ID) from which it was obtained.
  • Table 1 lists the fragments and extended polynucleotides by their SEQ ID NO and cDNA respectively, tissue, and by the description associated with at least a fragment of a homologous polynucleotide in GenBank. The descriptions were obtained using the sequences of the Sequence Listing and BLAST analysis.
  • Table 2 lists the source of the RNAs used to produce target polynucleotides for hybridization to the UNIGEM V microarray (Incyte Genomics, Palo Alto CA).
  • the columns present the Source No, Tissue, Age, Ethnicity/Sex, Cause of Death, and Conditions or Diseases, as known for each donor.
  • Table 3 shows the data for each of the clones across each of the tissues used in the experiments.
  • the columns present Clone ID and the tissues (with source number)-heart, skeletal muscle, uterus, stomach, small intestine, lung, liver, kidney, pancreas, spleen and brain. This data was produced using GEMTOOLS software (Incyte Genomics).
  • Table 4 presents the analysis of variance (ANOVA) for the data.
  • the columns present Clone
  • Table 5 shows the cell and tissue specificity of the polynucleotides across tissues (heart, skeletal muscle, uterus, stomach, small intestine, lung, liver, kidney, pancreas, spleen and brain). The cell and tissue specific groupings were produced using mean values [mean (tissue)- mean (entire set)] and grouped using EXCEL98 software (Microsoft).
  • array refers to an ordered arrangement of hybridizable polynucleotides. These are arranged so that there are a "plurality" of polynucleotides, preferably at least one polynucleotide, preferably at least 100 polynucleotides, and more preferably at least 1,000 polynucleotides, and even more preferably at least 10,000 polynucleotides on a 1 cm 2 substrate.
  • the maximum number of polynucleotides is unlimited, but is at least 100,000.
  • the signal from each of the hybridized polynucleotides is individually distinguishable.
  • a “polynucleotide” refers to a chain of nucleotides. Preferably, the chain has from about 15 to 10,000 nucleotides and more preferably from about 400 to 6,000 nucleotides.
  • the term "probe” refers to a probe polynucleotide capable of hybridizing with a target polynucleotide to form a hybridization complex. In most instances, the sequences of the probe and target polynucleotides will be complementary (no mismatches) when aligned. In some instances, there may be up to a 10% mismatch.
  • “Fragment” refers to any part of an Incyte clone or polynucleotide which retains a useful characteristic. Useful fragments may be used in hybridization technologies, to identify or purify ligands, or as a therapeutic to regulate replication, transcription or translation.
  • “Ligand” refers to any agent, molecule, or compound which will bind specifically to a complementary site on a polynucleotide or protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of at least one of the following: inorganic and organic substances including nucleic acids, proteins, carbohydrates, fats, and lipids.
  • “Purified” refers to any molecule or compound that is removed, isolated, or separated from its natural environment and is at least about 60% free, and more preferably about 90% free, from other components with which it is naturally associated.
  • Specific binding refers to a special and precise interaction between two molecules which is dependent upon a particular structure such as molecular side groups. For example, the hydrogen bonding between two single stranded nucleic acids or the binding between an epitope or a protein and an agonist, antagonist, or antibody.
  • sample is used in its broadest sense.
  • a sample containing polynucleotides may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; a finger print, a hair, and the like.
  • Portion refers to any part of a protein used for any purpose, but especially for the screening of molecules or compounds to identify those which specifically bind to that portion and for producing antibodies.
  • polynucleotide encoding a protein refers to nucleic acid sequence that closely aligns with a sequence which encodes a conserved protein motif or domain that were identified by employing analyses well known in the art. These analyses include Hidden Markov Models (HMMs) such as PFAM (Krogh (1994) J Mol Biol 235:1501-1531; Sonnhamer et al. (1988) Nucl Acids Res 26:320-322), BLAST (Basic Local Alignment Search Tool; Altschul (1993) J Mol Evol 36: 290-300; and Altschul et al. (1990) J Mol Biol 215:403-410), or other analytical tools such as BLIMPS (Henikoff et al. (1998) Nucl Acids Res 26:309-12). Additionally, “polynucleotide encoding a protein” may refer to a polynucleotide that is expressed in or associated with specific human metabolic processes, conditions, disorders, or diseases.
  • HMMs Hidden
  • Cell specific refers to those polynucleotides which occur at a statistically significant level in more than one tissue.
  • the commonality between the tissues may be ascribed to the types of cells that are an integral part of or would be expected to be found in a particular tissue, e.g., blood cells, nerve cells, endothelial cells, and the like.
  • the present invention provides a plurality of tissue or cell specific polynucleotides which may be used on an array to produce an expression profile.
  • This profile may define expression of these polynucleotides in normal tissue, during a particular metabolic or developmental process or during the onset, progression, or treatment of a human condition, disease, or disorder.
  • These polynucleotides represent known and novel genes normally expressed in the cells or tissues of the brain, heart, intestine, kidney, liver, lung, smooth muscle, ovary, pancreas, spleen, stomach, or uterus. The expression of these polynucleotides may be compared to the expression of other known or novel genes found on an array.
  • the plurality of polynucleotides comprises SEQ ID NOs:l-416.
  • Tissue or cell-specific reference sets may be selected from SEQ ID NOs:209-218 and 1-10, cell specific polynucleotides of heart and fragments thereof; b) SEQ ID NOs:219-249 and 11-41, cell specific polynucleotides of skeletal muscle and fragments thereof; c) SEQ ID NOs:250-251 and 42-43, cell specific polynucleotides of uterus and fragments thereof; d) SEQ ID NOs: 252-256 and 44-48, cell specific polynucleotides of ovary and fragments thereof; e) SEQ ID NOs:257-263 and 49-55, cell specific polynucleotides of stomach and fragments thereof; f) SEQ ID NOs:264-283 and 56-75, cell specific polynucleotides of intestine and fragments thereof; g) SEQ ID NOs:2
  • the invention also provides a substantially purified polynucleotide selected from SEQ ID NOs:212, 228, 233, 259, 271, 287, 316-319, 324, 370, 379, 380, 383, 410, and 412 or a fragment thereof, SEQ ID NO:4, 20, 25, 51, 63, 79, 108-111, 116, 162, 171, 172, 175, 202, and 204.
  • These polynucleotides may be used in an expression vector transformed into a host cell to produce a protein or a portion thereof by culturing the host cell under conditions for the expression of protein and recovering the protein from
  • the microarray can be used for large scale genetic or gene expression analysis of a large number of novel target polynucleotides.
  • targets are prepared by methods well known in the art and are from mammalian cells or tissues which are in a certain stage of development or differentiation; have been treated with a known molecule or compound, such as a cytokine, growth factor, a drug, and the like; or have been extracted or biopsied from a mammal with a known or unknown condition, disorder, or disease before or after treatment.
  • the plurality of polynuleotides are useful to determine the differentiation of embryonic stem cells toward brain, heart, kidney, liver, lung, muscle or pancreatic tissues or to determine whether a cancer is metastatic or its source by analyzing biopsied tissue from diseased brain, heart, kidney, liver, lung, muscle, ovarian, pancreatic, small intestine, stomach, or uterine tissues.
  • the plurality of polynucleotides may be used during the diagnosis of a cancer, an immunopathology, a neuropathology, and the like.
  • the target polynucleotides are hybridized to the probe polynucleotides for the purpose of defining a novel gene profile associated with that developmental stage, treatment, condition, disorder or disease.
  • the gene profile can be used for diagnosis, prognosis, or monitoring of treatments where altered expression of known and novel genes is associated with a cancer, an immunopathology, a neuropathology, and the like.
  • a gene profile can be used to investigate an individual's predisposition to a condition, disorder or disease such as a cancer, an immunopathology, a neuropathology, and the like.
  • the polynucleotides of the invention are employed as hybridizable polynucleotides on a microarray, the polynucleotides are organized in an ordered fashion so that each polynucleotide is present at a specified location on the substrate. Because the probe polynucleotides are at specified locations on the substrate, their hybridization patterns and intensities can be compared with the hybridization patterns and intensities of other known and novel polynucleotides to create an expression profile. Such a profile, interpreted in terms of expression levels of the cell and tissue specific, known, and novel genes can be correlated with a particular metabolic process, developmental stage, treatment, condition, disorder, disease, or stage of disease.
  • the plurality of polynucleotides can also be used to identify or purify a molecule or compound which specifically binds to at least one of the polynucleotides. These molecules may be identified from a sample or in high throughput mode from a large number of molecules and compounds including mRNAs, cDNAs, genomic fragments, and the like. Typically, the molecules or compounds will be of particular diagnostic or therapeutic interest. If nucleic acid molecules in a sample enhance the hybridization background, it may be advantageous to remove the offending molecules. One method for removing such molecules is by hybridizing the sample with immobilized probe polynucleotides and washing away those molecules that do not form hybridization complexes. At a later point, hybridization complexes can be dissociated, thereby releasing those molecules which specifically bind the probe polynucleotides. Method for Selecting Polynucleotide Probes
  • polynucleotides There are numerous different ways to select polynucleotides. Some of the more common ones include selecting probes from genes which are well known in the literature to have an association with a particular condition, disorder, or disease, which have a common functional characteristic such as the presence of a particular motif or domain or a signal peptide, which are expressed in a particular cell type or tissue such as blood or bone marrow, and the like.
  • the probes are non-redundant; therefore, no more than one probe represents a particular gene. Control sequences, however, may be selected specifically for their redundancy.
  • Polynucleotides of the composition may be manipulated to optimize their performance in ' hybridization technologies. Polynucleotide selection may be optimized by examining the sequences using a computer algorithm to identify fragments lacking potential secondary structure. Computer algorithms such as those employed in Vector NTI software (Informax, N. Bethesda MD) or LASER GENE software (DNASTAR, Madison WI) are well known in the art. These programs search nucleic acid sequences to identify stem loop structures and tandem repeats and to analyze G+C content of the sequence. In mammalian arrays, those sequences with a G+C content greater than 60% may be excluded. Alternatively, polynucleotides can be optimized under experimental conditions to determine whether polynucleotide probes and their complementary targets hybridize optimally.
  • the polynucleotides may be compared with clustered or assembled sequences to assure that each polynucleotide is derived from a different gene.
  • the polynucleotide may be physically extended utilizing the partial nucleotide sequences derived from the Incyte clone and employing the XL-PCR kit (Applied Biosystems, Foster City CA) or other means known in the art.
  • Polynucleotide probes can be genomic DNA or cDNA or mRNA, or any RNA-like or DNA-hke material, such as peptide nucleic acids, branched DNAs and the Uke. They may be the sense or antisense strand. Where targets are double stranded, probes may be either sense or antisense strands. Where targets are single stranded, probes are complementary single strands.
  • polynucleotide probes are cDNAs.
  • the size of the cDNAs may vary and is preferably from 15 to 10,000 nucleotides, more preferably from 60 to 4000 nucleotides, and most preferably from 200-600 nucleotides.
  • probes are plasmids.
  • the cDNA sequence of interest is the insert sequence. Excluding the vector DNA and regulatory sequences, cDNA size may vary preferably from 15 to 10,000 nucleotides, more preferably from 60 to 4000 nucleotides, and most perferably from 200-600 nucleotides.
  • Probes can be prepared by a variety of synthetic or enzymatic methods well known in the art. Probes can be synthesized, in whole or in part, using chemical methods well known in the art (Caruthers et al. (1980) Nucleic Acids Symp Ser (7):215-233). Alternatively, probes can be produced enzymatically or recombinantly, by in vitro or in vivo transcription.
  • Nucleotide analogues can be incorporated into the probes by methods well known in the art. The only requirement is that the incorporated nucleotide analogues of the probe must base pair with target nucleotides. For example, certain guanine nucleotides can be substituted with hypoxanthine which base pairs with cytosine residues. However, these base pairs are less stable than those between guanine and cytosine. Alternatively, adenine nucleotides can be substituted with 2,6-diaminopurine which can form stronger base pairs than those between adenine and thymidine.
  • probes can include nucleotides that have been derivatized chemically or enzymatically. Typical chemical modifications include derivatization with acyl, alkyl, aryl or amino groups.
  • Probes can be synthesized on a substrate. Synthesis on the surface of a substrate may be accomplished using a chemical coupling procedure and a piezoelectric printing apparatus as described by Baldeschweiler et al. (PCT AV095/251116). Alternatively, the probe can be synthesized on a substrate surface using a self-addressable electronic device that controls when reagents are added as described by Heller et al. (USPN 5,605,662).
  • cDNA Complementary DNA
  • Probes can be immobilized by covalent means such as by chemical bonding procedures or UV.
  • a cDNA is bound to a glass surface which has been modified to contain epoxide or aldehyde groups.
  • a cDNA probe is placed on a polylysine coated surface and then UV cross-linked as described by Shalon et al. (PCT/WO95/35505; inco ⁇ orated herein by reference).
  • a DNA is actively transported from a solution to a given position on a substrate by electrical means (Heller et al. supra).
  • probes, clones, plasmids or cells can be arranged on a filter.
  • cells are lysed, proteins and cellular components degraded, and the DNA is coupled to the filter by UV cross-linking.
  • probes do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group.
  • the linker groups are typically about 6 to 50 atoms long to provide exposure of the attached probe.
  • Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like.
  • Reactive groups on the substrate surface react with a terminal group of the linker to bind the linker to the substrate. The other terminus of the linker is then bound to the probe.
  • Probes can be attached to a substrate by sequentially dispensing reagents for probe synthesis on the substrate surface or by dispensing preformed DNA fragments to the substrate surface.
  • Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions efficiently.
  • a sample containing targets is provided.
  • the samples can be any sample containing targets and obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue or forensic preparations.
  • DNA or RNA can be isolated from a sample according to any of a number of methods well known to those of skill in the art. For example, methods of purification of nucleic acids are described in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation. Elsevier Science, New York NY). In one case, total RNA is isolated using TRIZOL reagent (Life Technologies, Gaithersburg MD), and mRNA is isolated using oligo d(T) column chromatography or glass beads.
  • targets when targets are derived from an mRNA, targets can be a DNA reverse transcribed from an mRNA, an RNA transcribed from that DNA, a DNA amplified from that DNA, an RNA transcribed from the amplified DNA, and the like.
  • target when target is derived from DNA, target can be DNA amplified from DNA, or RNA reverse transcribed from DNA.
  • targets are prepared by more than one method.
  • Total mRNA can be amplified by reverse transcription using a reverse transcriptase and a primer consisting of oligo d(T) and a sequence encoding the phage T7 promoter to provide a single stranded DNA template.
  • the second DNA strand is polymerized using a DNA polymerase and an RNAse which assists in breaking up the DNA/RNA hybrid.
  • T7 RNA polymerase can be added, and RNA transcribed from the second DNA strand template as described by Van Gelder et al. (USPN 5,545,522).
  • RNA can be amplified in vitro, in situ or in yiyo (Eberwine, USPN 5,514,545). It is also advantageous to include quantisation controls to assure that amplification and labeling procedures do not change the true abundance of transcripts in a sample.
  • a sample is spiked with a known amount of control nucleic acid, and the probes include control probes which specifically hybridize with the control nucleic acid. After hybridization and processing, the hybridization signals should reflect accurately the amounts of control nucleic acid added to the sample. Prior to hybridization, it may be desirable to fragment the nucleic acids of the sample.
  • Fragmentation improves hybridization by minimizing secondary structure and cross-hybridization among the nucleic acids in the sample or with noncomplementary probes. Fragmentation can be performed by mechanical or chemical means.
  • the nucleic acids may be labeled with one or more labeling moieties to allow for detection and quantitation of hybridization complexes.
  • the labeling moieties can include compositions that can be detected by specfroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means.
  • the labeling moieties include radioisotopes, such as 32 P, 33 P or 35 S; chemiluminescent compounds, labeled binding proteins, heavy metal atoms, specfroscopic markers such as fluorescent markers and dyes; magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the Uke.
  • Exemplary dyes include quinoline dyes, triarylmethane dyes, phthaleins, azo dyes, cyanine dyes, and the Uke.
  • fluorescent markers absorb Ught above about 300 nm, more preferably above 400 nm, and usually emit Ught at wavelengths at least greater than 10 nm above the wavelength of the Ught absorbed.
  • Preferred fluorescent markers include fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5.
  • LabeUng can be carried out during an amplification reaction, such as polymerase chain and in vitro transcription reactions; by nick translation, or by 5' or 3 -end-labeUng reactions.
  • labeled nucleotides are used in an in vitro transcription reaction.
  • the label is inco ⁇ orated after or without an ampUfication step, the label is inco ⁇ orated either by using a terminal transferase or a kinase on the 5 ' end of the target polynucleotide and then incubating overnight with a labeled oUgonucleotide in the presence of T4 RNA Ugase.
  • the labeling moiety can be inco ⁇ orated after hybridization once a probe/target complex has formed.
  • biotin is first incorporated during an ampUfication step as described above. After the hybridization reaction, unbound nucleic acids are rinsed away so that the only biotin remaining bound to the substrate is that attached to targets that are hybridized to probes. Then, an avidin-conjugated fluorophore, such as avidin-phycoerythrin, that binds with high affinity to biotin is added.
  • the labeUng moiety is inco ⁇ orated by intercalation into preformed target/probe complexes. In this case, an intercalating dye such as a psoralen-Unked dye can be employed.
  • Probes or polynucleotides may be used to screen a library of molecules or compounds for specific binding affinity.
  • the Ubraries may be DNA molecules, RNA molecules, PNAs, peptides, proteins such as transcription factors, enhancers, repressors, and other organic or inorganic Ugands which regulate activities such as replication, transcription, or translation of polynucleotides in the biological system.
  • the assay involves combining the probe with the library of molecules or compounds under conditions that allow specific binding, and detecting specific binding to a ligand which specifically binds the probe.
  • a protein or a portion thereof transcribed and translated from a probe may be used to screen Ubraries of molecules or compounds in any of a variety of screening assays.
  • the protein or portion thereof may be free in solution, affixed to an abiotic or biotic substrate, borne on a cell surface, or located intracellularly. Specific binding between the protein and a Ugand may be measured.
  • the assay may be used to identify DNA, RNA, or PNAs, agonists, antagonists, antibodies, immunoglobulins, inhibitors, mimetics, peptides, proteins, drugs, or any other Ugand, that specifically binds the protein.
  • Purification of Ligand Probes may be used to purify a Ugand from a sample. A method for using a probe to purify a
  • Ugand would involve combining the probe with a sample under conditions to allow specific binding, detecting specific binding, recovering the bound protein, and using an appropriate agent to separate the polynucleotide from the purified ligand.
  • the encoded protein or a portion thereof may be used to purify a ligand from a sample.
  • a method for using a protein or a portion thereof to purify a ligand would involve combining the protein or a portion thereof with a sample under conditions to allow specific binding, detecting specific binding between the protein and Ugand, recovering the bound protein, and using an appropriate agent to separate the protein from the purified Ugand.
  • Hybridization and Detection Hybridization causes a denatured polynucleotide probe and a denatured complementary target to form a stable duplex through base pairing. Hybridization methods are well known to those skilled in the art.
  • Conditions can be selected for hybridization where completely complementary probe and target can hybridize, i.e., each base pair must interact with its complementary base pair. Alternatively, conditions can be selected where probe and target have mismatches of up to about 10% but are still able to hybridize. Suitable conditions can be selected by varying the concentrations of salt in the prehybridization, hybridization, and wash solutions or by varying the hybridization and wash temperatures. With some substrates, temperature can be decreased by adding formamide to the prehybridization and hybridization solutions.
  • Hybridization can be performed at low stringency with buffers, such as 5xSSC with 1 % sodium dodecyl sulfate (SDS) at 60°C, which permits hybridization between probe and target sequences that contain some mismatches to form probe/target complexes. Subsequent washes are performed at higher stringency with buffers such as 0.2xSSC with 0.1% SDS at either 45 °C (medium stringency) or 68 °C (high stringency), to maintain hybridization of only those probe/target complexes that contain completely complementary sequences. Background signals can be reduced by the use of detergents such as SDS, Sarcosyl, or TRITON X-100 (Sigma-Aldrich, St. Louis MO) or a blocking agent, such as salmon sperm DNA.
  • buffers such as 5xSSC with 1 % sodium dodecyl sulfate (SDS) at 60°C, which permits hybridization between probe and target sequences that contain some mismatches to form probe/target
  • Hybridization specificity can be evaluated by comparing the hybridization of control probe to target sequences that are added to a sample in a known amount.
  • the control probe may have one or more sequence mismatches compared with the corresponding target. In this manner, it is possible to evaluate whether only complementary probes are hybridizing to the targets or whether mismatched hybrid duplexes are forming.
  • Hybridization reactions can be performed in absolute or differential hybridization formats.
  • absolute hybridization format probes from one sample are hybridized to microarray probes, and signals detected after hybridization complexes form. Signal strength correlates with probe levels in a sample.
  • differential hybridization format differential expression of a set of genes in two biological samples is analyzed. Probes from the two samples are prepared and labeled with different labeUng moieties. A mixture of the two labeled targets is hybridized to the microarray probes, and signals are examined under conditions in which the emissions from the two different labels are individually detectable. Targets in the microarray that are hybridized to substantially equal numbers of probes derived from both biological samples give a distinct combined fluorescence (Shalon,
  • the labels are fluorescent labels with distinguishable emission spectra, such as a lissamine conjugated nucleotide analog and a fluorescein conjugated nucleotide analog.
  • Cy3 and Cy5 fluorophores are employed. After hybridization, the microarray is washed to remove nonhybridized polynucleotides, and complex formation between the hybridizable array probes and the targets is examined. Methods for detecting complex formation are well known to those skilled in the art.
  • the probes are labeled with a fluorescent label, and measurement of levels and patterns of fluorescence indicative of complex formation is accompUshed by fluorescence microscopy, preferably confocal fluorescence microscopy.
  • fluorescence microscopy preferably confocal fluorescence microscopy.
  • An argon ion laser excites the fluorescent label, emissions are directed to a photomultipUer, and the amount of emitted Ught is detected and quantitated.
  • the detected signal should be proportional to the amount of probe/target complexes at each position of the microarray.
  • the fluorescence microscope can be associated with a computer-driven scanner device to generate a quantitative two-dimensional image of hybridization intensity. The scanned image is examined to determine the abundance/expression level of hybridized probe.
  • microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions.
  • individual polynucleotide probe/target complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.
  • This section describes an expression profile using the polynucleotides of this invention.
  • the reference set can be used as part of a expression profile which detects changes in the expression of novel genes whose transcripts are modulated in a particular metabolic response, treatment, condition, disorder, or disease. These genes will include genes whose altered expression is correlated with a cancer, an immunopathology, a neuropathology, and the Uke.
  • the expression profile comprises a pluraUty of detectable hybridization complexes. Each complex is formed by hybridization of one or more probes to one or more complementary targets. At least one of the probes, preferably a pluraUty of probes, is hybridized to a complementary target forming, at least one and preferably, a pluraUty of complexes. A complex is detected by inco ⁇ orating at least one labeUng moiety.
  • the expression profiles provide "snapshots" that can show unique expression patterns that are characteristic of a metaboUc process, treatment, condition, disorder or disease.
  • probes After performing hybridization experiments and detecting signals from a microarray, particular probes can be identified and selected based on their expression patterns. Such probes can be used to clone a full length sequence for the gene, to screen a library for a closely related homolog, to screen for or purify ligands, or to produce a protein.
  • the pluraUty of polynucleotides can be used as hybridizable elements in a microarray.
  • a microarray can be employed in several appUcations including diagnostics, prognostics and treatment regimens, and drug discovery and development for conditions, disorders, and diseases such as cancer, an immunopathology, a neuropathology and the Uke.
  • the microarray is used to monitor the progression of disease.
  • the differences in gene expression between healthy and diseased tissues or cells can be assessed and cataloged.
  • disease can be diagnosed at eariier stages before the patient is symptomatic.
  • the invention can be used to formulate a prognosis and to design a treatment regimen.
  • the invention can also be used to monitor the efficacy of treatment.
  • the microarray is employed to "fine tune" the treatment regimen. A dosage is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment.
  • animal models which mimic a human disease can be used to characterize expression profiles associated with a particular condition, disorder or disease or the freatment of the condition, disorder or disease.
  • Experimental treatment regimens may be tested in these animal models using microarrays to establish and then follow expression profiles over time.
  • microarrays may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects.
  • the invention provides the means to rapidly determine the molecular mode of action of a drug.
  • Embryonic (ES) stem cells isolated from rodent or human embryos retain the potential to form embryonic tissues.
  • ES cells such as the mouse 129/SvJ cell line are placed in a blastocyst from the C57BL/6 mouse strain, they resume normal development and contribute to tissues of the live-born animal.
  • ES cells are preferred for use in the creation of experimental knockout and knockin animals.
  • the method for this process is well known in the art and the steps are: the cDNA is introduced into a vector, the vector is transformed into ES cells, transformed cells are identified and microinjected into mouse cell blastocysts, blastocysts are surgically transferred to pseudopregnant dams.
  • ES cells are also used for the treatment of victims of Parkinson's disease, stroke, and other neuropathologies (The Engineer, 14(18):lff; September 2000). Pharmaceutical companies are also targeting disorders of the liver, kidney, and pancreas, specifically alpha- 1 antifrypsin, polycystic kidney disease, and diabetes, respectively.
  • traumatic damage to the nervous system and internal organs may also be treated by transplantation of cells or organs which are differentiated from embryonic stem cells.
  • the present invention may be used to characterize the developmental pathways of the differentiation processes that give rise to brain, heart, kidney, Uver, lung, muscle, ovarian, pancreatic, small intestine, stomach, or uterine tissues. Knockout Analysis
  • a region of a gene is enzymatically modified to include a non-natural intervening sequence such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292).
  • the modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination.
  • the inserted sequence disrupts transcription and translation of the endogenous gene.
  • ES cells can be used to create knockin humanized animals or transgenic animal models of human diseases.
  • knockin technology a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome.
  • Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on the progression and freatment of the analogous human condition.
  • cDNAs As described herein, the uses of the cDNAs, provided in the Sequence Listing of this appUcation, and their encoded proteins are exemplary of known techniques and are not intended to reflect any limitation on their use in any technique that would be known to the person of average skill in the art.
  • the cDNAs provided in this application may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known to the person of ordinary skill in the art, e.g., the triplet genetic code, specific base pair interactions, and the Uke.
  • reference to a method may include combining more than one method for obtaining, assembling or expressing cDNAs that will be known to those skilled in the art.
  • the BRAINON01 normaUzed cDNA library was constructed from cancerous brain tissue obtained from a 26-year-old Caucasian male during cerebral meningeal excision following diagnosis of grade 4 oUgoastrocytoma localized in the right fronto-parietal part of the brain.
  • the tumor had been irradiated (5800 rads).
  • Patient history included hemiplegia, epilepsy, ptosis of eyelid, and common migraine, and medications included Dilantin® (Parke-Davis, Morris Plains NJ).
  • the frozen tissue was homogenized and lysed using a POLYTRON homogenizer (PT-3000; Brinkmann Instruments, Westbury NY) in guanidinium isothiocyanate solution.
  • the lysate was extracted with acid phenol, pH 4.7, per Stratagene RNA isolation protocol (Stratagene, San Diego CA).
  • the RNA was extracted with an equal volume of acid phenol, reprecipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in DEPC-treated water, and treated with DNase for 25 min at 37°C.
  • the RNA extraction was repeated with phenol, pH 8.7, and precipitated with sodium acetate and ethanol as before.
  • the mRNA was isolated with the OLIGOTEX kit (Qiagen, Chatsworth CA) and used to construct the cDNA library.
  • the mRNA was handled according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies).
  • cDNAs were fractionated on a SEPH AROSE CL4B column (Amersham Pharmacia Biotech), and those cDNAs exceeding 400 bp were Ugated into PSPORT I plasmid (Life Technologies).
  • the plasmid was transformed into DH5 competent cells (Life Technologies) to construct the BRAINOT03 library.
  • the library was normalized in a single round according to the procedure of Soares et al. (1994, Proc Natl Acad Sci 91 :9928-9932) with the following modifications: 1) the primer to template ratio in the primer extension reaction was increased from 2:1 to 10:1, 2) the ddNTP concentration was reduced to 150 ⁇ M to allow generation of longer (400-1000nt) primer extension products, and 3) the reanneaUng hybridization was extended from 13 to 48 hours.
  • Plasmid DNA was released from bacterial cells and purified using the REAL Prep 96 plasmid kit (Qiagen). This kit enabled the simultaneous purification of 96 samples in a 96-well block using multi-channel reagent dispensers. The recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile TERRIFIC BROTH (BD Biosciences, Sparks MD) with carbenicilUn at 25 mg/L and glycerol at 0.4%; 2) the cultures were inoculated, incubated for 19 hours, and then lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water.
  • TERRIFIC BROTH BD Biosciences, Sparks MD
  • the cDNAs were prepared using a MICROLAB 2200 system (Hamilton, Reno NV) in combination with DNA ENGINE thermal cyclers (PTC200; MJ Research, Waltham MA).
  • the cDNAs were sequenced by the method of Sanger and Coulson (1975; J Mol Biol 94:441 f) using ABI PRISM 377 DNA sequencing systems (AppUed Biosystems). Most of the sequences were sequenced using standard ABI protocols and kits (Applied Biosystems) at solution volumes of 0.25x - l.Ox. In the alternative, some of the sequences were sequenced using solutions and dyes from Amersham Pharmacia Biotech.
  • Incyte clones were mapped to non-redundant Unigene clusters (Unigene database (build 46), NCBI; Shuler (1997) J Mol Med 75:694-698), and the 5' clone with the strongest BLAST alignment (at least 90% identity and 100 bp overlap) was chosen, verified, and used in the construction of the microarray.
  • the UNI GEM V microarray (Incyte Genomics) contains 7075 array elements which represent 4610 annotated genes and 2,184 unannotated clusters. Table 1 shows the GenBank 119 annotations for SEQ ID NOs:l-416 of this invention as produced by BLAST analysis.
  • BLAST involves finding similar segments between the query sequence and a database sequence, evaluating the statistical significance of any similarities, and reporting only those matches that satisfy a user-selectable threshold of significance. BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity.
  • HSP High scoring Segment Pair
  • the basis of the search is the product score, which is defined as:
  • the product score takes into account both the degree of identity between two sequences and the length of the sequence match as reflected in the BLAST score.
  • the BLAST score is calculated by scoring +5 for every base that matches in an HSP and -4 for every mismatch. For a product score of
  • the match will be exact within a 1 % to 2% error and for a product score of 70, the match will be exact.
  • Homologous molecules usually show product scores between 15 and 40, although lower scores may identify related molecules.
  • the P- value for any given HSP is a function of its expected frequency of occurrence and the number of HSPs observed against the same database sequence with scores at least as high.
  • Percent sequence identity is found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically using the MEGALIGN program, a component of LASERGENE software (DNASTAR). The percent similarity between two amino acid sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage similarity.
  • Sequences with conserved protein motifs may be searched using the BLOCKS search program.
  • This program analyses sequence information contained in the Swiss-Prot and PROSITE databases and is useful for determining the classification of uncharacterized proteins translated from genomic or cDNA sequences (Bairoch et al. (1997) Nucleic Acids Res 25:217-221; Attwood et al. (1997) J Chem Inf Comput Sci 37:417-424).
  • PROSITE database is a useful source for identifying functional or structural domains that are not detected using motifs due to extreme sequence divergence. Using weight matrices, these domains are calibrated against the SWISS-PROT database to obtain a measure of the chance distribution of the matches.
  • the PRINTS database can be searched using the BLIMPS search program to obtain protein family "finge ⁇ rints".
  • the PRINTS database complements the PROSITE database by exploiting groups of conserved motifs within sequence alignments to build characteristic signatures of different protein famiUes.
  • nucleic acid sequences of the Sequence Listing designed F, R, or T, were produced by extension of an appropriate fragment of the original clone insert using oligonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5' extension of the known sequence, and the other primer, to initiate 3' extension of the known sequence.
  • the initial primers were designed using OLIGO software (Molecular Insights, Cascade CO), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hai ⁇ in structures and primer-primer dimerizations was avoided.
  • Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
  • PCR was performed in 96-well plates using the DNA ENGINE thermal cycler (MJ Research).
  • the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and ⁇ -mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C.
  • the parameters for primer pair T7 and SK+ were as follows: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C.
  • the concentration of DNA in each well was determined by dispensing 100 ⁇ l PICOGREEN reagent (0.25% v/v PICOGREEN (Molecular Probes, Eugene OR) dissolved in lx TE) and 0.5 ⁇ l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent.
  • the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki FI) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 l to 10 ⁇ aUquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose minigel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to religation into pUC 18 vector
  • the digested nucleotides were separated on 0.6% to 0.8% agarose gels, fragments were excised, and agar digested with AGARACE (Promega).
  • Extended clones were reUgated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coU cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2x carbenicilUn tiquid media.
  • the cells were lysed, and DNA was ampUfied using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reampUfied using the conditions described above.
  • mRNA for Target Polynucleotides The mRNAs or tissues for preparing target polynucleotides were obtained from Biochain
  • Probe polynucleotides were amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert and purified using SEPHACRYL-400 beads (Amersham Pharmacia Biotech). Purified polynucleotides were robotically arrayed onto a glass microscope slide (Corning Science Products, Corning NY) previously coated with 0.05% aminopropyl silane (Sigma-Aldrich) and cured at 110°C. The microarray was exposed to UV irradiation in a STRATALINKER UV-crossUnker (Stratagene).
  • mRNA sample shown in Table 2, was reverse transcribed using MMLV reverse transcriptase in the presence of dCTP-Cy3 or dCTP-Cy5 (Amersham Pharmacia Biotech) according to standard protocol. After incubation at 37°C, the reaction was stopped with 0.5 M sodium hydroxide, and RNA was degraded at 85 °C.
  • the target polynucleotides were purified using CHROMASPIN 30 columns (Clontech, Palo Alto CA) and ethanol precipitation.
  • the hybridization mixture containing 0.2 mg of each of the Cy3 and Cy5 labeled target polynucleotides, was heated to 65°C, and dispensed onto the UNIGEM V microarray (Incyte Genomics) surface.
  • the microarray was covered with a coversUp and incubated at 60°C C.
  • the microarrays were sequentially washed at 45°C in moderate stringency buffer (lxSSC and 0.1% SDS) and high stringency buffer (O.lxSSC) and dried.
  • a confocal laser microscope was used to detect the fluorescence-labeled hybridization complexes. Excitation wavelengths were 488 nm for Cy3 and 632 nm for Cy5. Each array was scanned twice, one scan per fluorophore. The emission maxima was 565 nm for Cy3 and 650 nm for Cy5. The emitted light was split into two photomultipUer tube detectors based on wavelength. The output of the photomultipUer tube was digitized and displayed as an image, where the signal intensity was represented using a Unear 20 color transformation, with red representing a high signal and blue a low signal. The fluorescence signal for each probe was integrated to obtain a numerical value corresponding to the signal intensity using GEMTOOLS expression analysis software (Incyte Genomics).
  • Vw Variance within (Vw) categories.
  • F ratio The ratio of Vb divided by Vw (F ratio) was compared to the F distribution for a population of equal degree of freedom (DF) and the probability of the F ratio was returned.
  • Vbetween ⁇ Vwithin Vwithin
  • genes were associated with a primary tissue category according to the highest differential average value.
  • a minimum differential average value of 1.5 was required to associate a gene with a tissue category.
  • genes were associated with a secondary, tertiary, and even quaternary tissue category according to the second, third, and fourth highest differential average values, respectively.
  • the polynucleotide or fragments thereof and the protein or portions thereof are labeled with 32 P-dCTP, Cy3-dCTP, Cy5-dCTP (Amersham Pharmacia Biotech), or BIODIPY or FITC (Molecular Probes), respectively.
  • Candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled nucleic or amino acid. After incubation under conditions for either a polynucleotide or protein, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed. The binding molecule is identified by its arrayed position on the substrate.

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Abstract

L'invention concerne une composition renfermant plusieurs polynucléotides qui sont spécifiques de cellule et/ou de tissu, et peuvent être utilisés dans leur intégralité ou en partie comme références dans la production d'un profil d'expression définissant un processus métabolique ou de développement, un traitement, un état, une maladie ou un trouble.
PCT/US2000/030396 1999-11-04 2000-11-02 Genes specifiques de tissu a signification diagnostique WO2001032927A2 (fr)

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AU1462801A (en) 2001-05-14
WO2001032927A3 (fr) 2002-08-15
EP1255859A2 (fr) 2002-11-13
CA2388511A1 (fr) 2001-05-10

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