US20040067491A1 - Diagnosis of diseases associated with metabolism - Google Patents

Diagnosis of diseases associated with metabolism Download PDF

Info

Publication number
US20040067491A1
US20040067491A1 US10/240,454 US24045403A US2004067491A1 US 20040067491 A1 US20040067491 A1 US 20040067491A1 US 24045403 A US24045403 A US 24045403A US 2004067491 A1 US2004067491 A1 US 2004067491A1
Authority
US
United States
Prior art keywords
dna
recited
sequences
genes
oligomer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/240,454
Other languages
English (en)
Inventor
Alexander Olek
Christian Piepenbrock
Kurt Berlin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Epigenomics AG
Original Assignee
Epigenomics AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE10019058A external-priority patent/DE10019058A1/de
Priority claimed from DE10032529A external-priority patent/DE10032529A1/de
Application filed by Epigenomics AG filed Critical Epigenomics AG
Assigned to EPIGENOMICS AG reassignment EPIGENOMICS AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BERLIN, KURT, OLEK, ALEXANDER, PIEPENBROCK, CHRISTIAN
Publication of US20040067491A1 publication Critical patent/US20040067491A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • 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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/154—Methylation markers
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to nucleic acids, oligonucleotides, PNA-oligomers and to a method for the diagnosis and/or therapy of diseases which have a connection with the genetic and/or epigenetic parameters of genes associated with metabolism and, in particular, with the methylation status thereof.
  • Metabolism is the sum of chemical changes that occur in living organisms. The thousands of different chemical reactions are carried out simultaneously by a cell are closely coordinated. A variety of control mechanisms regulate the activities of key enzymes in response to changing conditions in the cell. One very common form of regulation is a rapidly reversible feedback inhibition exerted on the first enzyme by the final product of that pathway. A longer lasting form of regulation involves the chemical modification of one enzyme by another. Combinations of regulatory mechanisms can produce major and long lasting changes in the metabolism of the cell. Common metabolic diseases are diabetes, cancer, hyperlipidaemia and atherosclerosis. Cancer, for example, is a multistep disease with a multifactorial aetiology.
  • 5-methylcytosine is the most frequent covalent base modification in the DNA of eukaryotic cells. It plays a role, for example, in the regulation of the transcription, in genetic imprinting, and in tumorigenesis. Therefore, the identification of 5-methylcytosine as a component of genetic information is of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behavior as cytosine. Moreover, the epigenetic information carried by 5-methylcytosine is completely lost during PCR amplification.
  • a relatively new and currently the most frequently used method for analyzing DNA for 5-methylcytosine is based upon the specific reaction of bisulfite with cytosine which, upon subsequent alkaline hydrolysis, is converted to uracil which corresponds to thymidine in its base pairing behavior.
  • 5-methylcytosine remains unmodified under these conditions. Consequently, the original DNA is converted in such a manner that methylcytosine, which originally could not be distinguished from cytosine by its hybridization behavior, can now be detected as the only remaining cytosine using “normal” molecular biological techniques, for example, by amplification and hybridization or sequencing. All of these techniques are based on base pairing which can now be fully exploited.
  • the prior art is defined by a method which encloses the DNA to be analyzed in an agarose matrix, thus preventing the diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and which replaces all precipitation and purification steps with fast dialysis (Olek A, Oswald J, Walter J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996 Dec. 15 ;24(24):5064-6). Using this method, it is possible to analyze individual cells, which illustrates the potential of the method.
  • Fluorescently labeled probes are often used for the scanning of immobilized DNA arrays.
  • the simple attachment of Cy3 and Cy5 dyes to the 5′-OH of the specific probe are particularly suitable for fluorescence labels.
  • the detection of the fluorescence of the hybridized probes may be carried out, for example via a confocal microscope. Cy3 and Cy5 dyes, besides many others, are commercially available.
  • Matrix Assisted Laser Desorption Ionization Mass Spectrometry is a very efficient development for the analysis of biomolecules (Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct 15;60(20):2299-301).
  • An analyte is embedded in a light-absorbing matrix. The matrix is evaporated by a short laser pulse thus transporting the analyte molecule into the vapor phase in an unfragmented manner.
  • the analyte is ionized by collisions with matrix molecules.
  • An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, the ions are accelerated at different rates. Smaller ions reach the detector sooner than bigger ones.
  • MALDI-TOF spectrometry is excellently suited to the analysis of peptides and proteins.
  • the analysis of nucleic acids is somewhat more difficult (Gut I G, Beck S. DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Current Innovations and Future Trends. 1995, 1; 147-57).
  • the sensitivity to nucleic acids is approximately 100 times worse than to peptides and decreases disproportionally with increasing fragment size.
  • the ionization process via the matrix is considerably less efficient.
  • the selection of the matrix plays an eminently important role.
  • Genomic DNA is obtained from DNA of cell, tissue or other test samples using standard methods. This standard methodology is found in references such as Fritsch and Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989.
  • the object of the present invention is to provide the chemically modified DNA of genes associated with metabolism, as well as oligonucleotides and/or PNA-oligomers for detecting cytosine methylations, as well as a method which is particularly suitable for the diagnosis and/or therapy of genetic and epigenetic parameters of genes associated with metabolism.
  • the present invention is based on the discovery that genetic and epigenetic parameters and, in particular, the cytosine methylation pattern of genes associated with metabolism are particularly suitable for the diagnosis and/or therapy of diseases associated with metabolism.
  • This objective is achieved according to the present invention using a nucleic acid containing a sequence of at least 18 bases in length of the chemically pretreated DNA of genes associated with metabolism according to one of Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto.
  • the respective data bank numbers accession numbers
  • GenBank was used as the underlying data bank, which is located at the National Institute of Health at the internet address http://www.ncbi.nlm.nih.gov.
  • the object of the present invention is further achieved by an oligonucleotide or oligomer for detecting the cytosine methylation state in chemically pretreated DNA, containing at least one base sequence having a length of at least 13 nucleotides which hybridizes to a chemically pretreated DNA of genes associated with metabolism according to Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto.
  • the oligomer probes according to the present invention constitute important and effective tools which, for the first time, make it possible to ascertain the genetic and epigenetic parameters of genes associated with metabolism.
  • the base sequence of the oligomers preferably contains at least one CpG dinucleotide.
  • the probes may also exist in the form of a PNA (peptide nucleic acid) which has particularly preferred pairing properties.
  • PNA peptide nucleic acid
  • Particularly preferred are oligonucleotides according to the present invention in which the cytosine of the CpG dinucleotide is the 5 th -9 th nucleotide from the 5′-end of the 13-mer; in the case of PNA-oligomers, it is preferred for the cytosine of the CpG dinucleotide to be the 4 th -6 th nucleotide from the 5′-end of the 9-mer.
  • the oligomers according to the present invention are normally used in so called “sets” which contain at least one oligomer for each of the CpG dinucleotides of the sequences of Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto.
  • Preferred is a set which contains at least one oligomer for each of the CpG dinucleotides from one of Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto.
  • the present invention makes available a set of at least two oligonucleotides which can be used as so-called “primer oligonucleotides” for amplifying DNA sequences of one of Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto, or segments thereof.
  • primer oligonucleotides for amplifying DNA sequences of one of Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto, or segments thereof.
  • At least one oligonucleotide is bound to a solid phase.
  • the present invention moreover relates to a set of at least 10 n (oligonucleotides and/or PNA-oligomers) used for detecting the cytosine methylation state in chemically pretreated genomic DNA (Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto).
  • chemically pretreated genomic DNA Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto.
  • the set of oligomers may also be used for detecting single nucleotide polymorphisms (SNPs) in the chemically pretreated DNA of genes associated with metabolism according to one of Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto.
  • SNPs single nucleotide polymorphisms
  • an arrangement of different oligonucleotides and/or PNA-oligomers made available by the present invention is present in a manner that it is likewise bound to a solid phase.
  • This array of different oligonucleotide- and/or PNA-oligomer sequences can be characterized in that it is arranged on the solid phase in the form of a rectangular or hexagonal lattice.
  • the solid phase surface is preferably composed of silicon, glass, polystyrene, aluminium, steel, iron, copper, nickel, silver, or gold.
  • nitrocellulose as well as plastics such as nylon which can exist in the form of pellets or also as resin matrices are possible as well.
  • a further subject matter of the present invention is a method for manufacturing an array fixed to a carrier material for analysis in connection with diseases associated with metabolism in which method at least one oligomer according to the present invention is coupled to a solid phase.
  • Methods for manufacturing such arrays are known, for example, from U.S. Pat. No. 5,744,305 by means of solid-phase chemistry and photolabile protecting groups.
  • a further subject matter of the present invention relates to a DNA chip for the analysis of diseases associated with metabolism which contains at least one nucleic acid according to the present invention.
  • DNA chips are known, for example, for U.S. Pat. No. 5,837,832.
  • kits which may be composed, for example, of a bisulfite-containing reagent, a set of primer oligonucleotides containing at least two oligonucleotides whose sequences in each case correspond or are complementary to an 18 base long segment of the base sequences specified in the appendix (Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto), oligonucleotides and/or PNA-oligomers as well as instructions for carrying out and evaluating the described method.
  • a kit along the lines of the present invention can also contain only part of the aforementioned components.
  • the present invention also makes available a method for ascertaining genetic and/or epigenetic parameters of genes associated with the cycle cell by analyzing cytosine methylations and single nucleotide polymorphisms, including the following steps:
  • a genomic DNA sample is chemically treated in such a manner that cytosine bases which are unmethylated at the 5′-position are converted to uracil, thymine, or another base which is dissimilar to cytosine in terms of hybridization behavior. This will be understood as ‘chemical pretreatment’ hereinafter.
  • the genomic DNA to be analyzed is preferably obtained from usual sources of DNA such as cells or cell components, for example, cell lines, biopsies, blood, sputum, stool, urine, cerebral-spinal fluid, tissue embedded in paraffin such as tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histologic object slides, or combinations thereof.
  • sources of DNA such as cells or cell components, for example, cell lines, biopsies, blood, sputum, stool, urine, cerebral-spinal fluid, tissue embedded in paraffin such as tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histologic object slides, or combinations thereof.
  • genomic DNA is preferably carried out with bisulfite (hydrogen sulfite, disulfite) and subsequent alkaline hydrolysis which results in a conversion of non-methylated cytosine nucleobases to uracil or to another base which is dissimilar to cytosine in terms of base pairing behavior.
  • bisulfite hydrogen sulfite, disulfite
  • Fragments of the chemically pretreated DNA are amplified, using sets of primer oligonucleotides according to the present invention, and a, preferably heat-stable polymerase. Because of statistical and practical considerations, preferably more than ten different fragments having a length of 100-2000 base pairs are amplified.
  • the amplification of several DNA segments can be carried out simultaneously in one and the same reaction vessel. Usually, the amplification is carried out by means of a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the set of primer oligonucleotides includes at least two olignonucleotides whose sequences are each reverse complementary or identical to an at least 18 base-pair long segment of the base sequences specified in the appendix (Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto).
  • the primer oligonucleotides are preferably characterized in that they do not contain any CpG dinucleotides.
  • At least one primer oligonucleotide is bonded to a solid phase during amplification.
  • the different oligonucleotide and/or PNA-oligomer sequences can be arranged on a plane solid phase in the form of a rectangular or hexagonal lattice, the solid phase surface preferably being composed of silicon, glass, polystyrene, aluminium, steel, iron, copper, nickel, silver, or gold, it being possible for other materials such as nitrocellulose or plastics to be used as well.
  • the fragments obtained by means of the amplification can carry a directly or indirectly detectable label.
  • the detection may be carried out and visualized by means of matrix assisted laser desorption/ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).
  • MALDI matrix assisted laser desorption/ionization mass spectrometry
  • ESI electron spray mass spectrometry
  • the amplificates obtained in the second step of the method are subsequently hybridized to an array or a set of oligonucleotides and/or PNA probes.
  • the hybridization takes place in the manner described in the following.
  • the set of probes used during the hybridization is preferably composed of at least 10 oligonucleotides or PNA-oligomers.
  • the amplificates serve as probes which hybridize to oligonucleotides previously bonded to a solid phase. The non-hybridized fragments are subsequently removed.
  • Said oligonucleotides contain at least one base sequence having a length of 13 nucleotides which is reverse complementary or identical to a segment of the base sequences specified in the appendix, the segment containing at least one CpG dinucleotide.
  • the cytosine of the CpG dinucleotide is the 5 th to 9 th nucleotide from the 5′-end of the 13-mer.
  • One oligonucleotide exists for each CpG dinucleotide.
  • Said PNA-oligomers contain at least one base sequence having a length of 9 nucleotides which is reverse complementary or identical to a segment of the base sequences specified in the appendix, the segment containing at least one CpG dinucleotide.
  • the cytosine of the CpG dinucleotide is the 4 th to 6 th nucleotide seen from the 5′-end of the 9-mer.
  • One oligonucleotide exists for each CpG dinucleotide.
  • the non-hybridized amplificates are removed.
  • the hybridized amplificates are detected.
  • labels attached to the amplificates are identifiable at each position of the solid phase at which an oligonucleotide sequence is located.
  • the labels of the amplificates are fluorescence labels, radionuclides, or detachable molecule fragments having a typical mass which can be detected in a mass spectrometer.
  • the mass spectrometer is preferred for the detection of the amplificates, fragments of the amplificates or of probes which are complementary to the amplificates, it being possible for the detection to be carried out and visualized by means of matrix assisted laser desorption/ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).
  • MALDI matrix assisted laser desorption/ionization mass spectrometry
  • ESI electron spray mass spectrometry
  • the produced fragments may have a single positive or negative net charge for better detectability in the mass spectrometer.
  • the aforementioned method is preferably used for ascertaining genetic and/or epigenetic parameters of genes associated with metabolism.
  • the oligomers according to the present invention or arrays thereof as well as a kit according to the present invention are intended to be used for the diagnosis and/or therapy of diseases associated with metabolism by analyzing methylation patterns of genes associated with metabolism.
  • the method is preferably used for the diagnosis and/or therapy of important genetic and/or epigenetic parameters within genes associated with metabolism.
  • the method according to the present invention is used, for example, for the diagnosis and/or therapy of solid tumors and cancer
  • nucleic acids according to the present invention of Seq. ID No.1 through Seq. ID No.64 and sequences complementary thereto and/or oligonucleotide- and/or a chemically pretreated DNA of genes according to the sequences of genes according to table 1 and sequences complementary thereto can be used for the diagnosis and/or therapy of genetic and/or epigenetic parameters of genes associated with metabolism.
  • the present invention moreover relates to a method for manufacturing a diagnostic agent and/or therapeutic agent for the diagnosis and/or therapy of diseases associated with metabolism by analyzing methylation patterns of genes associated with metabolism, the diagnostic agent and/or therapeutic agent being characterized in that at least one nucleic acid according to the present invention is used for manufacturing it, possibly together with suitable additives and auxiliary agents.
  • a further subject matter of the present invention relates to a diagnostic agent and/or therapeutic agent for diseases associated with metabolism by analyzing methylation patterns of genes associated with metabolism, the diagnostic agent and/or therapeutic agent containing at least one nucleic acid according to the present invention, possibly together with suitable additives and auxiliary agents.
  • the present invention moreover relates to the diagnosis and/or prognosis of events which are disadvantageous to patients or individuals in which important genetic and/or epigenetic parameters within genes associated with metabolism said parameters obtained by means of the present invention may be compared to another set of genetic and/or epigenetic parameters, the differences serving as the basis for a diagnosis and/or prognosis of events which are disadvantageous to patients or individuals.
  • hybridization is to be understood as a bond of an oligonucleotide to a completely complementary sequence along the lines of the Watson-Crick base pairings in the sample DNA, forming a duplex structure.
  • stringent hybridization conditions are those conditions in which a hybridization is carried out at 60° C. in 2.5 ⁇ SSC buffer, followed by several washing steps at 37° C. in a low buffer concentration, and remains stable.
  • the term “functional variants” denotes all DNA sequences which are complementary to a DNA sequence, and which hybridize to the reference sequence under stringent conditions and have an activity similar to the corresponding polypeptide according to the present invention.
  • “genetic parameters” are mutations and polymorphisms of genes associated with metabolism and sequences further required for their regulation.
  • mutations are, in particular, insertions, deletions, point mutations, inversions and polymorphisms and, particularly preferred, SNPs (single nucleotide polymorphisms).
  • epigenetic parameters are, in particular, cytosine methylations and further chemical modifications of DNA bases of genes associated with metabolism and sequences further required for their regulation.
  • Further epigenetic parameters include, for example, the acetylation of histones which, however, cannot be directly analyzed using the described method but which, in turn, correlates with the DNA methylation.
  • Sequences having odd sequence numbers exhibit in each case sequences of the chemically pretreated genomic DNAs of different genes associated with metabolism.
  • FIG. 1 A first figure.
  • FIG. 1 shows the hybridisation of fluorescent labelled amplificates to a surface bound olignonucleotide.
  • Sample I being from a pilocytic astrocytoma tumor sample and sample II being from an oligodenrogliome grade II tumor sample.
  • Flourescence at a spot shows hybridisation of the amplificate to the olignonucleotide.
  • Hybridisation to a CG olignonucleotide denotes methylation at the cytosine position being analysed
  • hybridisation to a TG olignonucleotide denotes no methylation at the cytosine position being analysed. It can be seen that Sample I had a higher degree of methylation than Sample II at position 514.
  • Sequence ID Nos. 1 to 64 show sequences of the chemically pretreated genomic DNAs of different genes associated with metabolism.
  • sequences having odd sequence numbers e.g., Seq. ID No. 1, 3, 5, . . .
  • Sequences having even sequence numbers e.g., Seq. ID No. 2, 4, 6, . . .
  • sequences having even sequence numbers exhibit in each case the sequences of the chemically pretreated genomic DNAs of genes associated with metabolism which are complementary to the preceeding sequences (e.g., the complementary sequence to Seq. ID No.1 is Seq. ID No.2, the complementary sequence to Seq. ID No.3 is Seq. ID No.4, etc.).
  • Seq. ID No. 65 to seq. ID No. 68 show specific oligonucleotide sequences as used in Example 1.
  • the following example relates to a fragment of a gene associated with metabolism, in this case, OAT in which a specific CG-position is analyzed for its methylation status.
  • the following example relates to a fragment of the gene OAT in which a specific CG-position is to be analyzed for methylation.
  • a genomic sequence is treated using bisulfite (hydrogen sulfite, disulfite) in such a manner that all cytosines which are not methylated at the 5-position of the base are modified in such a manner that a different base is substituted with regard to the base pairing behavior while the cytosines methylated at the 5-position remain unchanged.
  • bisulfite hydrogen sulfite, disulfite
  • the treated DNA sample is diluted with water or an aqueous solution.
  • the DNA is subsequently desulfonated (10-30 min, 90-100° C.) at an alkaline pH value.
  • the DNA sample is amplified in a polymerase chain reaction, preferably using a heat-resistant DNA polymerase.
  • cytosines of the gene OAT are analyzed.
  • a defined fragment having a length of 572 bp is amplified with the specific primer oligonucleotides TGGAGGTGGATTTAGAGGTA (Sequence ID 65) and AACCAAAACCCCAAAACAAC (Sequence ID No. 66).
  • This amplificate serves as a sample which hybridizes to an oligonucleotide previously bonded to a solid phase, forming a duplex structure, for example GTGTATTCGGTTGTTTTT (Sequence ID No.
  • the cytosine to be detected being located at position 514 of the amplificate.
  • the detection of the hybridization product is based on Cy3 and Cy5 fluorescently labelled primer oligonucleotides which have been used for the amplification.
  • a hybridization reaction of the amplified DNA with the oligonucleotide takes place only if a methylated cytosine was present at this location in the bisulfite-treated DNA.
  • the methylation status of the specific cytosine to be analyzed is inferred from the hybridization product.
  • a sample of the amplificate is further hybridized to another oligonucleotide previously bonded to a solid phase.
  • Said olignonucleotide is identical to the oligonucleotide previously used to analyze the methylation status of the sample, with the exception of the position in question.
  • said oligonucleotide comprises a thymine base as opposed to a cytosine base i.e GTGTATTTGGTTGTTTTT (Sequence ID No. 68). Therefore, the hybridisation reaction only takes place if an unmethylated cytosine was present at the position to be analysed.
  • the procedure was carried out on cell samples from 2 patients, sample I being from a pilocytic astrocytoma tumor sample and sample 11 being from an oligodenrogliome grade II tumor sample.
  • methylation patterns In order to relate the methylation patterns to one of the diseases associated with metabolism, it is initially required to analyze the DNA methylation patterns of a group of diseased and of a group of healthy patients. These analyses are carried out, for example, analogously to Example 1. The results obtained in this manner are stored in a database and the CpG dinucleotides which are methylated differently between the two groups are identified. This can be carried out by determining individual CpG methylation rates as can be done, for example, in a relatively imprecise manner, by sequencing or else, in a very precise manner, by a methylation-sensitive “primer extension reaction”. It is also possible for the entire methylation status to be analyzed simultaneously, and for the patterns to be compared, for example, by clustering analyses which can be carried out, for example, by a computer.
  • Example 2 can be carried out, for example, for metabolic diseases, solid tumours and cancer.
  • Genbank Entry No. Gen http://www.ncbi.nlm.nih.gov
  • DUSP2 NM_004418 EPHX2 NM_001979 QDPR NM_000320 SGSH NM_000199
  • SHMT2 NM_005412 SLC7A2 NM_003046 SLC7A4 NM_004173 TYMS NM_001071
  • FIG. 1 shows the hybridisation of fluorescent labelled amplificates to a surface bound olignonucleotide.
  • Sample I being from a pilocytic astrocytoma tumor sample and sample II being from an oligodenrogliome grade II tumor sample.
  • Flourescence at a spot shows hybridisation of the amplificate to the olignonucleotide.
  • Hybridisation to a CG olignonucleotide denotes methylation at the cytosine position being analysed
  • hybridisation to a TG olignonucleotide denotes no methylation at the cytosine position being analysed. It can be seen that Sample I had a higher degree of methylation than Sample II at position 514.
US10/240,454 2000-04-06 2001-04-06 Diagnosis of diseases associated with metabolism Abandoned US20040067491A1 (en)

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
DE10019058.8 2000-04-06
DE10019058A DE10019058A1 (de) 2000-04-06 2000-04-06 Detektion von Variationen des DNA-Methylierungsprofils
DE10019173.8 2000-04-07
DE10019173 2000-04-07
DE10032529A DE10032529A1 (de) 2000-06-30 2000-06-30 Diagnose von bedeutenden genetischen Parametern innerhalb des Major Histocompatibility Complex (MHC)
DE10032529.7 2000-06-30
DE10043826 2000-09-01
DE10043826.1 2000-09-01
PCT/EP2001/004016 WO2001076451A2 (fr) 2000-04-06 2001-04-06 Diagnostique de maladies associees au metabolisme

Publications (1)

Publication Number Publication Date
US20040067491A1 true US20040067491A1 (en) 2004-04-08

Family

ID=27437807

Family Applications (7)

Application Number Title Priority Date Filing Date
US10/240,454 Abandoned US20040067491A1 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with metabolism
US10/239,676 Expired - Fee Related US7195870B2 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with gene regulation
US10/240,708 Abandoned US20050282157A1 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with dna replication
US10/240,485 Abandoned US20030148327A1 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with metastasis
US10/240,452 Abandoned US20030162194A1 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with apoptosis
US10/240,453 Abandoned US20030148326A1 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with dna transcription
US10/240,589 Abandoned US20040076956A1 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with dna repair

Family Applications After (6)

Application Number Title Priority Date Filing Date
US10/239,676 Expired - Fee Related US7195870B2 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with gene regulation
US10/240,708 Abandoned US20050282157A1 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with dna replication
US10/240,485 Abandoned US20030148327A1 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with metastasis
US10/240,452 Abandoned US20030162194A1 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with apoptosis
US10/240,453 Abandoned US20030148326A1 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with dna transcription
US10/240,589 Abandoned US20040076956A1 (en) 2000-04-06 2001-04-06 Diagnosis of diseases associated with dna repair

Country Status (7)

Country Link
US (7) US20040067491A1 (fr)
EP (9) EP1268857A2 (fr)
JP (3) JP2003534780A (fr)
AT (1) ATE353975T1 (fr)
AU (9) AU2001276331A1 (fr)
DE (1) DE60126593T2 (fr)
WO (8) WO2001077376A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090321626A1 (en) * 2006-05-26 2009-12-31 Akos Vertes Laser desorption ionization and peptide sequencing on laser induced silicon microcolumn arrays
US20100323917A1 (en) * 2009-04-07 2010-12-23 Akos Vertes Tailored nanopost arrays (napa) for laser desorption ionization in mass spectrometry
US9000361B2 (en) 2009-01-17 2015-04-07 The George Washington University Nanophotonic production, modulation and switching of ions by silicon microcolumn arrays

Families Citing this family (76)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6780982B2 (en) * 1996-07-12 2004-08-24 Third Wave Technologies, Inc. Charge tags and the separation of nucleic acid molecules
US6818404B2 (en) 1997-10-23 2004-11-16 Exact Sciences Corporation Methods for detecting hypermethylated nucleic acid in heterogeneous biological samples
US7955794B2 (en) 2000-09-21 2011-06-07 Illumina, Inc. Multiplex nucleic acid reactions
US8076063B2 (en) 2000-02-07 2011-12-13 Illumina, Inc. Multiplexed methylation detection methods
US7582420B2 (en) 2001-07-12 2009-09-01 Illumina, Inc. Multiplex nucleic acid reactions
EP1268857A2 (fr) 2000-04-06 2003-01-02 Epigenomics AG Diagnostic de maladies associees a la regulation genetique
US20040052763A1 (en) * 2000-06-07 2004-03-18 Mond James J. Immunostimulatory RNA/DNA hybrid molecules
WO2002000926A2 (fr) * 2000-06-30 2002-01-03 Epigenomics Ag Diagnostic de maladies associees a une transduction de signal
AUPR142500A0 (en) * 2000-11-13 2000-12-07 Human Genetic Signatures Pty Ltd A peptide nucleic acid-based assay for the detection of specific nucleic acid sequences
DE10128508A1 (de) 2001-06-14 2003-02-06 Epigenomics Ag Verfahren und Nukleinsäuren für die Differenzierung von Prostata-Tumoren
WO2003031932A2 (fr) 2001-10-05 2003-04-17 Case Western Reserve University Procedes et compositions pour detecter les cancers du colon
JP2003144172A (ja) * 2001-11-16 2003-05-20 Nisshinbo Ind Inc メチル化検出用オリゴヌクレオチド固定化基板
US20110151438A9 (en) 2001-11-19 2011-06-23 Affymetrix, Inc. Methods of Analysis of Methylation
DE10161625A1 (de) * 2001-12-14 2003-07-10 Epigenomics Ag Verfahren und Nukleinsäuren für die Analyse einer Lungenzell-Zellteilungsstörung
CA2478592A1 (fr) 2002-03-07 2003-09-18 The Johns Hopkins University School Of Medicine Depistage genomique pour genes lies au cancer rendus epigenetiquement silencieux
EP1344832A1 (fr) * 2002-03-15 2003-09-17 Epigenomics AG Procédé et acides nucléiques pour l'analyse de méthylation dans le gène de melastatin
AU2003225983A1 (en) * 2002-03-25 2003-10-13 St. Jude Children's Research Hospital Cpg retrieval of dna from formalin-fixed pathology specimen for promoter methylation analysis
US20040053880A1 (en) 2002-07-03 2004-03-18 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US7807803B2 (en) 2002-07-03 2010-10-05 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
CA2494508A1 (fr) * 2002-07-03 2004-01-15 Coley Pharmaceutical Group, Inc. Compositions d'acide nucleique destinees a stimuler les reponses immunitaires
EP2157191B1 (fr) 2002-10-01 2013-12-25 Epigenomics AG Utilisation d'acides nucléiques de PITX2 pour le traitement amélioré des troubles cellulaires prolifératifs du sein
WO2004050915A1 (fr) * 2002-12-02 2004-06-17 Solexa Limited Determination de la methylation de sequences d'acide nucleique
DE10304219B3 (de) * 2003-01-30 2004-08-19 Epigenomics Ag Verfahren zum Nachweis von Cytosin-Methylierungsmustern mit hoher Sensitivität
WO2004070062A2 (fr) * 2003-02-04 2004-08-19 Wyeth Compositions et techniques de diagnostic et de traitement de cancers
US20050009059A1 (en) * 2003-05-07 2005-01-13 Affymetrix, Inc. Analysis of methylation status using oligonucleotide arrays
ATE476523T1 (de) 2003-06-17 2010-08-15 Human Genetic Signatures Pty Verfahren zur genomamplifikation
US8415100B2 (en) 2003-08-14 2013-04-09 Case Western Reserve University Methods and compositions for detecting gastrointestinal and other cancers
EP1660683B1 (fr) 2003-08-14 2017-04-19 Case Western Reserve University Procedes et compositions pour le depistage de cancers du colon
DE10338308B4 (de) 2003-08-15 2006-10-19 Epigenomics Ag Verfahren zum Nachweis von Cytosin-Methylierungen in DNA
JP4714148B2 (ja) 2003-09-04 2011-06-29 ヒューマン ジェネティック シグネチャーズ ピーティーワイ リミテッド 核酸検出アッセイ
WO2005038047A1 (fr) * 2003-10-20 2005-04-28 St Vincent's Hospital (Sydney) Limited Evaluation d'un risque de maladie par determination quantitative de l'epimutation dans des tissus normaux
WO2005054517A2 (fr) * 2003-12-01 2005-06-16 Epigenomics Ag Procedes et acides nucleiques pour l'analyse de l'expression genique associee a la survenue de troubles cellulaires proliferants de la prostate
EP1561821B1 (fr) 2003-12-11 2011-02-16 Epigenomics AG Marqueurs pour le pronostic de la réponse à la thérapie et/ou de la survie chez les patients du cancer du sein
CA2496997A1 (fr) * 2004-02-13 2005-08-13 Affymetrix, Inc. Analyse et determination du degre de methylation a l'aide de matrices d'acides nucleiques
US8168777B2 (en) 2004-04-29 2012-05-01 Human Genetic Signatures Pty. Ltd. Bisulphite reagent treatment of nucleic acid
JP4980219B2 (ja) 2004-09-10 2012-07-18 ヒューマン ジェネティック シグネチャーズ ピーティーワイ リミテッド インターカレート型擬似ヌクレオチド（ｉｐｎ）を含有するインターカレーティング核酸（ｉｎａ）を含む増幅ブロッカー
KR20060026595A (ko) 2004-09-21 2006-03-24 (주)지노믹트리 제한효소와 ｄｎａ 칩을 이용한 프로모터의 메틸화 검출방법
KR100617649B1 (ko) * 2004-09-24 2006-09-04 (주)지노믹트리 대장암 특이적 발현감소 유전자의 메틸화된 프로모터를 함유하는 암 진단용 조성물 및 그 용도
WO2006058393A1 (fr) 2004-12-03 2006-06-08 Human Genetic Signatures Pty Ltd Procedes de simplification d’acides nucleiques microbiens par modification chimique de cytosines
US20060134650A1 (en) * 2004-12-21 2006-06-22 Illumina, Inc. Methylation-sensitive restriction enzyme endonuclease method of whole genome methylation analysis
WO2006088978A1 (fr) 2005-02-16 2006-08-24 Epigenomics, Inc. Procede de determination du modele de methylation d'un acide polynucleique
US10731215B2 (en) 2005-04-15 2020-08-04 Epigenomics Ag Method for determining the presence or absence of methylation in a sample
WO2006111586A2 (fr) * 2005-04-20 2006-10-26 Proyecto De Biomedicina Cima, S.L. Procede permettant de determiner in vitro le degre de methylation du promoteur de line-1
EP1883707B1 (fr) 2005-05-26 2014-04-09 Human Genetic Signatures PTY Ltd Amplification par deplacement de brin isotherme utilisant des amorces contenant une base non reguliere
US20060292585A1 (en) * 2005-06-24 2006-12-28 Affymetrix, Inc. Analysis of methylation using nucleic acid arrays
EP1943353B1 (fr) 2005-09-14 2011-11-02 Human Genetic Signatures PTY Ltd Essai pour un etat de sante
US7465544B2 (en) * 2006-01-11 2008-12-16 Wisconsin Alumni Research Foundation Synthetic cofactor analogs of S-adenosylmethionine as ligatable probes of biological methylation and methods for their use
EP1974058B1 (fr) 2006-01-11 2014-06-11 Genomic Health, Inc. Marqueurs d'expression de gène pour pronostic colorectal de cancer
PL1826279T3 (pl) 2006-02-28 2011-11-30 Univ Berlin Charite Wykrywanie i kontrola jakości regulatorowych limfocytów T dzięki analizie metylacji DNA genu FoxP3
US7901882B2 (en) * 2006-03-31 2011-03-08 Affymetrix, Inc. Analysis of methylation using nucleic acid arrays
EP2013364A2 (fr) * 2006-05-02 2009-01-14 University of Southampton Prédiction de phénotypes
WO2008096146A1 (fr) 2007-02-07 2008-08-14 Solexa Limited Préparation de matrices pour l'analyse de méthylation
ES2684219T3 (es) * 2007-10-23 2018-10-01 Clinical Genomics Pty Ltd Un método para el diagnóstico de neoplasias - II
AU2008329549B2 (en) 2007-11-27 2013-08-22 Human Genetic Signatures Pty Ltd Enzymes for amplification and copying bisulphite modified nucleic acids
US9670546B2 (en) 2008-07-15 2017-06-06 Epigenomics Ag Methods and nucleic acids for analyses of cell proliferative disorders
EP2340314B8 (fr) 2008-10-22 2015-02-18 Illumina, Inc. Préservation d'informations liées à une méthylation d'adn génomique
JP2012525159A (ja) 2009-05-01 2012-10-22 ジェノミック ヘルス， インコーポレイテッド 結腸直腸癌の再発および化学療法に対する応答の可能性における遺伝子発現プロファイルアルゴリズムおよび試験
CA2769862A1 (fr) 2009-08-28 2011-03-03 Cellular Dynamics International, Inc. Identification de variation genetique dans les tissus affectes a partir de cellules souches pluripotentes induites
WO2011080314A2 (fr) * 2009-12-31 2011-07-07 Deutsches Krebsforschungszentrum Nouveaux modulateurs de signalisation par trail
EP2702177B1 (fr) * 2011-04-28 2017-04-12 New York University Signatures génomiques d'une métastase dans le cancer de la prostate
US10435743B2 (en) 2011-05-20 2019-10-08 The Regents Of The University Of California Method to estimate age of individual based on epigenetic markers in biological sample
MY167564A (en) 2011-09-07 2018-09-14 Human Genetic Signatures Pty Ltd Molecular detection assay
WO2013129397A1 (fr) * 2012-02-29 2013-09-06 シスメックス株式会社 Procédé pour déterminer la présence ou l'absence de cellule cancéreuse issue d'un carcinome hépatocellulaire, et marqueur de détermination et trousse
US10706957B2 (en) 2012-09-20 2020-07-07 The Chinese University Of Hong Kong Non-invasive determination of methylome of tumor from plasma
US9732390B2 (en) 2012-09-20 2017-08-15 The Chinese University Of Hong Kong Non-invasive determination of methylome of fetus or tumor from plasma
KR101302173B1 (ko) 2012-12-07 2013-08-30 이화여자대학교 산학협력단 Ｈｍｏｘ１ 유전자의 메틸화 변화를 이용한 알츠하이머 질환의 진단용 조성물 및 이를 이용한 알츠하이머 질환의 진단방법
US20140274757A1 (en) 2013-03-14 2014-09-18 Marie K. Kirby Differential Methylation Level of CpG Loci That Are Determinative of a Biochemical Reoccurrence of Prostate Cancer
EP3137633A4 (fr) * 2014-04-28 2017-11-29 Sigma-Aldrich Co. LLC Modification épigénétique de génomes de mammifères à l'aide d'endonucléases ciblées
EP3850083A4 (fr) * 2018-09-14 2022-06-29 Shinozaki, Gen Systèmes et procédés de détection de risque de délire à l'aide de marqueurs épigénétiques
CN111217900A (zh) * 2018-11-27 2020-06-02 上海交通大学 一种血管新生的转录调控因子及其用途
EP3911761A4 (fr) 2019-01-18 2022-10-19 The Regents of the University of California Mesure de méthylation d'adn pour des mammifères sur la base de loci conservés
CN114829631A (zh) 2019-10-14 2022-07-29 建喾立嗣股份公司 通过使用特定基因中的cpg甲基化变化来诊断肝癌的组合物及其用途
KR102637032B1 (ko) 2020-01-28 2024-02-15 주식회사 젠큐릭스 특정 유전자의 CpG 메틸화 변화를 이용한 방광암 진단용 조성물 및 이의 용도
CN115698340A (zh) 2020-04-08 2023-02-03 建喾立嗣股份公司 用glrb基因cpg甲基化变化来诊断结直肠癌、直肠癌或结直肠腺瘤的组合物和其应用
CN111500702B (zh) * 2020-04-26 2021-04-20 江苏大学附属医院 RPN1基因cg00843506位点甲基化在诊断哮喘中的用途
WO2023175019A1 (fr) 2022-03-15 2023-09-21 Genknowme S.A. Procédé pour déterminer la différence entre l'âge biologique et l'âge chronologique d'un sujet

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6017704A (en) * 1996-06-03 2000-01-25 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
US6214556B1 (en) * 1997-11-27 2001-04-10 Epigenomics Ag Method for producing complex DNA methylation fingerprints
US6331393B1 (en) * 1999-05-14 2001-12-18 University Of Southern California Process for high-throughput DNA methylation analysis

Family Cites Families (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5744101A (en) * 1989-06-07 1998-04-28 Affymax Technologies N.V. Photolabile nucleoside protecting groups
US5474796A (en) * 1991-09-04 1995-12-12 Protogene Laboratories, Inc. Method and apparatus for conducting an array of chemical reactions on a support surface
SE501439C2 (sv) 1993-06-22 1995-02-13 Pharmacia Lkb Biotech Sätt och anordning för analys av polynukleotidsekvenser
WO1995011995A1 (fr) * 1993-10-26 1995-05-04 Affymax Technologies N.V. Reseaux de sondes d'acide nucleique sur des microplaquettes biologiques
US5837832A (en) 1993-06-25 1998-11-17 Affymetrix, Inc. Arrays of nucleic acid probes on biological chips
JPH09506253A (ja) 1993-11-30 1997-06-24 マクギル・ユニヴァーシティ Ｄｎａメチルトランスフェラーゼの阻害
US5858661A (en) * 1995-05-16 1999-01-12 Ramot-University Authority For Applied Research And Industrial Development Ataxia-telangiectasia gene and its genomic organization
US5871917A (en) 1996-05-31 1999-02-16 North Shore University Hospital Research Corp. Identification of differentially methylated and mutated nucleic acids
AU7829398A (en) * 1997-06-09 1998-12-30 University Of Southern California A cancer diagnostic method based upon dna methylation differences
DE19750172C1 (de) * 1997-11-12 1998-10-01 Deutsches Krebsforsch DNA mit Promotor-Aktivität für Zellzyklus-Gen
JP2001526381A (ja) * 1997-12-05 2001-12-18 マックス−プランク−ゲゼルシャフト・ツア・フェルデルング・デア・ヴィッセンシャフテン・エー・ファオ マトリックス補助レーザー脱着／イオン化質量分光分析による核酸の同定方法
WO2000005419A1 (fr) * 1998-07-24 2000-02-03 Yeda Research And Development Company Ltd. Prevention d'une metastase au moyen de 5-aza-2'-desoxycytidine
DE19905082C1 (de) * 1999-01-29 2000-05-18 Epigenomics Gmbh Verfahren zur Identifikation von Cytosin-Methylierungsmustern in genomischen DNA-Proben
US6783933B1 (en) * 1999-09-15 2004-08-31 The Johns Hopkins University School Of Medicine CACNA1G polynucleotide, polypeptide and methods of use therefor
US20030190644A1 (en) * 1999-10-13 2003-10-09 Andreas Braun Methods for generating databases and databases for identifying polymorphic genetic markers
WO2001068911A2 (fr) * 2000-03-15 2001-09-20 Epigenomics Ag Diagnostic d'affections associees au cycle cellulaire
EP1268857A2 (fr) * 2000-04-06 2003-01-02 Epigenomics AG Diagnostic de maladies associees a la regulation genetique
WO2002000926A2 (fr) 2000-06-30 2002-01-03 Epigenomics Ag Diagnostic de maladies associees a une transduction de signal
DE10037769A1 (de) 2000-08-03 2002-02-21 Epigenomics Gmbh Diagnose von mit CD24 assoziierten Krankheiten
US6812339B1 (en) * 2000-09-08 2004-11-02 Applera Corporation Polymorphisms in known genes associated with human disease, methods of detection and uses thereof
DE10054974A1 (de) 2000-11-06 2002-06-06 Epigenomics Ag Diagnose von mit Cdk4 assoziierten Krankheiten
DE10054972A1 (de) 2000-11-06 2002-06-06 Epigenomics Ag Diagnose von mit humos assoziierten Krankheiten
DE10128508A1 (de) * 2001-06-14 2003-02-06 Epigenomics Ag Verfahren und Nukleinsäuren für die Differenzierung von Prostata-Tumoren
JP2005516269A (ja) * 2001-07-02 2005-06-02 エピゲノミクス アーゲー エピジェネティックに基づく複合表現型の予測のための分散システム

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6017704A (en) * 1996-06-03 2000-01-25 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
US6214556B1 (en) * 1997-11-27 2001-04-10 Epigenomics Ag Method for producing complex DNA methylation fingerprints
US6331393B1 (en) * 1999-05-14 2001-12-18 University Of Southern California Process for high-throughput DNA methylation analysis

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090321626A1 (en) * 2006-05-26 2009-12-31 Akos Vertes Laser desorption ionization and peptide sequencing on laser induced silicon microcolumn arrays
US8084734B2 (en) 2006-05-26 2011-12-27 The George Washington University Laser desorption ionization and peptide sequencing on laser induced silicon microcolumn arrays
US9000361B2 (en) 2009-01-17 2015-04-07 The George Washington University Nanophotonic production, modulation and switching of ions by silicon microcolumn arrays
US20100323917A1 (en) * 2009-04-07 2010-12-23 Akos Vertes Tailored nanopost arrays (napa) for laser desorption ionization in mass spectrometry
US9490113B2 (en) 2009-04-07 2016-11-08 The George Washington University Tailored nanopost arrays (NAPA) for laser desorption ionization in mass spectrometry

Also Published As

Publication number Publication date
EP1274866A2 (fr) 2003-01-15
US20030082609A1 (en) 2003-05-01
WO2001092565A2 (fr) 2001-12-06
WO2001077164A8 (fr) 2002-03-21
EP1268857A2 (fr) 2003-01-02
AU2001275663A1 (en) 2001-10-23
WO2001081622A3 (fr) 2003-09-04
AU2001276330B2 (en) 2006-08-31
US20030148327A1 (en) 2003-08-07
EP2014776A3 (fr) 2009-04-01
AU2001254794A1 (en) 2001-10-23
JP2003534780A (ja) 2003-11-25
WO2001077377A2 (fr) 2001-10-18
EP1278893A2 (fr) 2003-01-29
WO2001077376A8 (fr) 2002-03-21
AU2001278420A1 (en) 2001-11-07
WO2001077377A8 (fr) 2002-02-28
AU2001277487A1 (en) 2001-10-23
WO2001092565A3 (fr) 2002-09-06
DE60126593D1 (de) 2007-03-29
EP1370685A2 (fr) 2003-12-17
JP2004508807A (ja) 2004-03-25
WO2001076451A3 (fr) 2003-09-04
WO2001081622A8 (fr) 2002-05-16
US20030148326A1 (en) 2003-08-07
WO2001077378A3 (fr) 2002-10-17
AU7633001A (en) 2001-10-23
EP1360319A2 (fr) 2003-11-12
EP1274865B1 (fr) 2007-02-14
WO2001077376A3 (fr) 2002-08-08
WO2001077375A3 (fr) 2002-06-27
US20030162194A1 (en) 2003-08-28
AU2001276331A1 (en) 2001-10-23
WO2001077375A2 (fr) 2001-10-18
AU2001289600A1 (en) 2001-12-11
US20050282157A1 (en) 2005-12-22
WO2001077164A2 (fr) 2001-10-18
WO2001077164A3 (fr) 2002-06-20
WO2001076451A9 (fr) 2002-08-08
EP1272670A2 (fr) 2003-01-08
WO2001077377A3 (fr) 2002-07-11
WO2001081622A2 (fr) 2001-11-01
WO2001077378A2 (fr) 2001-10-18
US20040076956A1 (en) 2004-04-22
EP2014776A2 (fr) 2009-01-14
WO2001077375A8 (fr) 2002-10-24
WO2001092565A8 (fr) 2002-05-30
ATE353975T1 (de) 2007-03-15
JP2003531589A (ja) 2003-10-28
DE60126593T2 (de) 2007-10-31
US7195870B2 (en) 2007-03-27
WO2001077376A2 (fr) 2001-10-18
AU2001254788A1 (en) 2001-10-23
EP1268861A2 (fr) 2003-01-02
EP1274865A2 (fr) 2003-01-15
WO2001076451A2 (fr) 2001-10-18

Similar Documents

Publication Publication Date Title
US20040067491A1 (en) Diagnosis of diseases associated with metabolism
KR102318434B1 (ko) 병태 및 질환 치료용 안티센스 올리고머
KR101708544B1 (ko) 세포 증식 질환을 분석하기 위한 방법 및 핵산
KR102046668B1 (ko) 암 대상자의 예후를 결정하기 위한 방법 및 핵산
KR101778036B1 (ko) 전립선암 마커로서의 포스포디에스테라제 4ｄ7
KR20200103765A (ko) 핵산의 표적화된 통합
WO1998045435A2 (fr) MARQUEURS SECRETES DE SEQUENCE EXPRIMEE (sEST)
KR102110469B1 (ko) 악성의 호르몬 민감성 전립선 암에 대한 마커로서의 포스포디에스테라제 4ｄ7
EP0973896A2 (fr) Marqueurs secretes de sequence exprimee (sest)
CN112195287B (zh) 一种用于人乳头瘤病毒hpv分型和整合检测的探针组及其试剂盒
KR101621273B1 (ko) 카텝신 ｃ의 용도
KR20210107057A (ko) 핵산의 표적화 통합
CN101151371B (zh) 治疗中的逆转录转座子抑制
KR20230038486A (ko) 호흡기 병원체를 진단하는 방법 및 covid-19 관련 결과를 예측하는 방법
KR101691415B1 (ko) 나프록센 노출에 대응하는 히드라 유전자 및 이를 이용한 수생태계 환경오염 진단 방법
KR20220025806A (ko) 핵산의 무작위 구성 표적화 통합
KR101879998B1 (ko) 산화아연 나노입자 노출에 대응하는 히드라 유전자 및 이를 이용한 수생태계 환경오염 진단 방법
KR101474053B1 (ko) 골다공증 또는 골다공성 골절 발생 위험도 예측용 다형성 마커
CN112261951A (zh) 包含合成嵌合痘苗病毒的干细胞及其使用方法
KR101635497B1 (ko) 종자수가 감소한 신품종 수박 및 이의 육종 방법
KR102205790B1 (ko) 이부프로펜 노출에 대응하는 히드라 유전자 및 이를 이용한 수생태계 환경오염과 히드라의 생리 및 대사 변화 진단 방법
MXPA01011882A (es) Productos de expresion de genes implicados en enfermedades relacionadas con el metabolismo del colesterol.
KR20230027043A (ko) 핵산의 표적화 통합
KR20230158917A (ko) 한국인 친족 관계 확인을 위한 482개의 snp 패널 및 이의 용도
US20020142378A1 (en) Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins, and uses thereof

Legal Events

Date Code Title Description
AS Assignment

Owner name: EPIGENOMICS AG, GERMANY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:OLEK, ALEXANDER;PIEPENBROCK, CHRISTIAN;BERLIN, KURT;REEL/FRAME:013829/0839

Effective date: 20021001

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION