WO2001077377A2 - Diagnosis of diseases associated with dna replication by assessing dna methylation - Google Patents
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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 DNA replication and, in particular, with the methylation status thereof.
- the replication of double stranded genomic DNA is a complex activity. It is carried out in three key stages, initiation, elongation and termination. Each stage involves specific protein and enzyme complexes.
- initiation the double helix is temporarily separated and stabilized into two single strands, each of which acts as a template for the replication of the DNA from the replication fork. Separation of the two strands is carried out by a helicase, and stabilisation of the strands is achieved using a single stranded binding protein.
- Replication of the DNA is then carried out by a polymerase after synthesis of a short 'primer' sequence. Replication is carried out in a semi-discontinuous fashion.
- the leading strand is continuously synthe- sised in the 5' to 3' direction.
- replication of the lagging strand in the 3' to 5' direction is made by the synthesis of short fragments in the 5' to 3' direction.
- replication is terminated, and the lagging strand complementary DNA fragments are ligated into a continuous strand.
- a global analysis of the status of DNA replication mechanisms would provide a basis for the development of appropriate and specific therapies for diseases associated with DNA replication.
- the current state of the art is such that the analysis may be carried out in a gene specific manner based on the results of gene expression, e.g. DNA micro array analysis of mRNA expression or proteomic analysis.
- the next step would then be to look at the causal factors involved at earlier stages in the regulatory mechanisms controlling DNA replication.
- DNA methylation provides such a novel level of information at which to analyse the genome.
- 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 behaviour 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 labelled probes are often used for the scanning of immobilised 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 DNA replication, 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 DNA replication.
- the present invention is based on the discovery that genetic and epigenetic parameters and, in particular, the cytosine methylation pattern of genes associated with DNA replication are particularly suitable for the diagnosis and/or therapy of diseases associated with DNA replication.
- nucleic acid containing a sequence of at least 18 bases in length of the chemically pretreated DNA of genes associated with DNA replication according to one of Seq. ID No.l through Seq. ID No.94 and sequences complementary thereto and or a sequence of a chemically pretreated DNA of genes according to table 1 and sequences complementary thereto.
- table 1 after the listed gene designations, the respective data bank numbers (accession numbers) are specified which define the appertaining gene sequences as unique.
- GenBank was used as the underlying data bank, which is located at the National Institute of Health, internet address www.ncbi.nlm.nih.gov.
- the chemically modified nucleic acid could heretofore not be connected with the ascertainment of genetic and epigenetic parameters.
- 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 DNA replication according to Seq. ID No.l through Seq. ID No.94 and sequences complementary thereto and/or a sequence of a chemically pretreated DNA 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 DNA replication.
- 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.
- oligonucleotides according to the present invention in which the cytosine of the CpG dinucleotide is the 5 m - 9 m 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 - 6 m 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.l through Seq. ID No.94 and sequences complementary thereto and/or a sequence of a chemically pretreated DNA of genes according to table 1 and sequences complementary thereto.
- sets which contain at least one oligomer for each of the CpG dinucleotides from one of Seq. ID No.l through Seq. ID No.94 and sequences complementary thereto and/or a sequence of a chemically pretreated DNA 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.l through Seq. ID No.94 and sequences complementary thereto and/or a sequence of a chemically pretreated DNA of genes according to table 1 and sequences complementary thereto, or segments thereof.
- 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.l through Seq. ID No.94 and sequences complementary thereto and/or a sequence of a chemically pretreated DNA of genes according to table 1 and sequences complementary thereto).
- chemically pretreated genomic DNA Seq. ID No.l through Seq. ID No.94 and sequences complementary thereto and/or a sequence of a chemically pretreated DNA 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 DNA replication according to one of Seq. ID No.l through Seq. ID No.94 and sequences complementary thereto and/or a sequence of a chemically pretreated DNA 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 DNA replication 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 US Patent 5,744,305 by means of solid-phase chemistry and photolabile protecting groups.
- 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.l through Seq.
- kits 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:
- the above described treatment of 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 behaviour.
- 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.l through Seq. ID No.94 and sequences complementary thereto and/or a sequence of a chemically pretreated DNA 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 visualised 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 - l i the 5 m to 9 m nucleotide from the 5'-end of the 13-mer.
- One oligonucleotide exists for each
- Said PNA-oligomers contain at least one base sequence having a length of
- the cytosine of the CpG dinucleotide is the 4 m to 6 m 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 detecta- bility in the mass spectrometer.
- the aforementioned method is preferably used for ascertaining genetic and/or epigenetic parameters of genes associated with DNA replication.
- nucleic acids according to the present invention of Seq. ID No.1 through Seq. ID No.94 and sequences complementary thereto and/or a sequence of a chemically pretreated DNA 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 DNA replication.
- 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 DNA replication by analyzing methylation patterns of genes associated with DNA replication, 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 DNA replication by analyzing methylation patterns of genes associated with DNA replication, 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 DNA replication 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 x SSC buffer, followed by several washing steps at 37°C in a low buffer concentration, and remains stable.
- mutations are mutations and polymorphisms of genes associated with DNA replication 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 DNA replication 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.
- Figure 1 shows the hybridisation of fluorescent labelled amplificates to a surface bound olignonucleotide.
- Sample I being from an oligodendroglyome grade II tumour sample and sample II being from astrocytoma grade II cerebrum tissue.
- 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.
- Seq ID Nos. 1 to 94 Sequences having odd sequence numbers (e.g., Seq. ID No. 1, 3, 5, ...) exhibit in each case sequences of the chemically pretreated genomic DNAs of different genes associated with DNA replication. Sequences having even sequence numbers (e.g., Seq. ID No. 2, 4, 6, ...) exhibit in each case the sequences of the chemically pretreated genomic DNAs of genes associated with DNA replication which are complementary to the preceeding sequences (e.g., the complementary sequence to Seq. ID No.l is Seq. ID No.2, the complementary sequence to Seq. ID No.3 is Seq. ID No.4, etc.)
- Seq ID Nos. 95 to 98 show the sequences of oligonucleotides used in Example 1.
- the following example relates to a fragment of a gene associated with DNA replication, in this case, MLH1 in which a specific CG-position is analyzed for its methylation status.
- Example 1 Methylation analysis in the gene MLH1 associated with DNA replication.
- the following example relates to a fragment of the gene MLH1 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 MLH1 are analyzed.
- a defined fragment having a length of 866 bp is amplified with the specific primer oligonucleotides TTTAAGGTAAGAGAATAGGT (Sequence ID No. 95) and
- AAACAACTTAAATACCAATC (Sequence ID No. 96).
- This amplificate serves as a sample which hybridizes to an oligonucleotide previously bonded to a solid phase, forming a duplex structure, for example GGTTTGTACGAGTAGTTT (Sequence ID No. 97), the cytosine to be detected being located at position 135 of the amplificate.
- the detection of the hybridization product is based on Cy3 and Cy5 fluorescently labeled 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 bi- sulfite-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 GGTTTGTATGAGTAGTTT (Sequence ID No. 98). 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 an oligoden- droglyome grade II tumour sample and sample II being from a astrocytoma grade II cerebrum tumor sample.
- methylation patterns In order to relate the methylation patterns to one of the diseases associated with DNA replication, 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 dinu- cleotides 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 methyla- tion-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 the following diseases:
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/240,708 US20050282157A1 (en) | 2000-04-06 | 2001-04-06 | Diagnosis of diseases associated with dna replication |
AU2001275663A AU2001275663A1 (en) | 2000-04-06 | 2001-04-06 | Diagnosis of diseases associated with DNA replication by assessing DNA methylation |
EP01953145A EP1278893A2 (en) | 2000-04-06 | 2001-04-06 | Diagnosis of diseases associated with dna replication |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
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DE10019058A DE10019058A1 (en) | 2000-04-06 | 2000-04-06 | Designing primers and probes for analyzing diseases associated with cytosine methylation state e.g. arthritis, cancer, aging, arteriosclerosis comprising fragments of chemically modified genes associated with cell cycle |
DE10019058.8 | 2000-04-06 | ||
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DE10032529A DE10032529A1 (en) | 2000-06-30 | 2000-06-30 | Diagnosis of major genetic parameters within the Major Histocompatibility Complex (MHC) |
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DE10043826 | 2000-09-01 |
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PCT/EP2001/003969 WO2001077164A2 (en) | 2000-04-06 | 2001-04-06 | Diagnosis of diseases associated with apoptosis by means of assessing the methylation status of genes associated with apoptosis |
PCT/EP2001/003971 WO2001077377A2 (en) | 2000-04-06 | 2001-04-06 | Diagnosis of diseases associated with dna replication by assessing dna methylation |
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PCT/EP2001/003973 WO2001092565A2 (en) | 2000-04-06 | 2001-04-06 | Diagnosis of diseases associated dna transcription by means of assessing the methylation status of genes associated with dna transcription |
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US9850536B2 (en) | 2000-02-07 | 2017-12-26 | Illumina, Inc. | Multiplex nucleic acid reactions |
US9868982B2 (en) | 2007-02-07 | 2018-01-16 | Illumina Cambridge Limited | Preparation of templates for methylation analysis |
US10407717B2 (en) | 2001-11-19 | 2019-09-10 | Affymetrix, Inc. | Methods of analysis of methylation |
Families Citing this family (68)
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 |
US8076063B2 (en) | 2000-02-07 | 2011-12-13 | Illumina, Inc. | Multiplexed methylation detection methods |
US7955794B2 (en) | 2000-09-21 | 2011-06-07 | Illumina, Inc. | Multiplex nucleic acid reactions |
EP1292331A2 (en) * | 2000-06-07 | 2003-03-19 | Biosynexus Incorporated | Immunostimulatory rna/dna hybrid molecules |
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 |
AU2002342004A1 (en) | 2001-10-05 | 2003-04-22 | Case Western Reserve University | Methods and compositions for detecting colon cancers |
JP2003144172A (en) * | 2001-11-16 | 2003-05-20 | Nisshinbo Ind Inc | Oligonucleotide-immobilized board for detecting methylation |
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EP1344832A1 (en) * | 2002-03-15 | 2003-09-17 | Epigenomics AG | Methods and nucleic acids for the analysis of methylation within the gene melastatin |
US7807803B2 (en) | 2002-07-03 | 2010-10-05 | Coley Pharmaceutical Group, Inc. | Nucleic acid compositions for stimulating immune responses |
US20040053880A1 (en) | 2002-07-03 | 2004-03-18 | Coley Pharmaceutical Group, Inc. | Nucleic acid compositions for stimulating immune responses |
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US20050009059A1 (en) * | 2003-05-07 | 2005-01-13 | Affymetrix, Inc. | Analysis of methylation status using oligonucleotide arrays |
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CA2496997A1 (en) * | 2004-02-13 | 2005-08-13 | Affymetrix, Inc. | Analysis of methylation status using nucleic acid arrays |
US8168777B2 (en) | 2004-04-29 | 2012-05-01 | Human Genetic Signatures Pty. Ltd. | Bisulphite reagent treatment of nucleic acid |
WO2006026828A1 (en) | 2004-09-10 | 2006-03-16 | Human Genetic Signatures Pty Ltd | Amplification blocker comprising intercalating nucleic acids (tna) containing intercalating pseudonucleotides (ipn) |
KR20060026595A (en) | 2004-09-21 | 2006-03-24 | (주)지노믹트리 | Method for detecting methylaion of promoter using restriction enzyme and dna chip |
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US20060134650A1 (en) * | 2004-12-21 | 2006-06-22 | Illumina, Inc. | Methylation-sensitive restriction enzyme endonuclease method of whole genome methylation analysis |
WO2006088978A1 (en) | 2005-02-16 | 2006-08-24 | Epigenomics, Inc. | Method for determining the methylation pattern of a polynucleic acid |
DK1871912T3 (en) | 2005-04-15 | 2012-05-14 | Epigenomics Ag | Method for determining DNA methylation in blood or urine samples |
WO2006111586A2 (en) * | 2005-04-20 | 2006-10-26 | Proyecto De Biomedicina Cima, S.L. | Method for the in vitro determination of the degree of methylation of the line-1 promoter |
AU2006251866B2 (en) | 2005-05-26 | 2007-11-29 | Human Genetic Signatures Pty Ltd | Isothermal strand displacement amplification using primers containing a non-regular base |
US20060292585A1 (en) * | 2005-06-24 | 2006-12-28 | Affymetrix, Inc. | Analysis of methylation using nucleic acid arrays |
US8343738B2 (en) | 2005-09-14 | 2013-01-01 | Human Genetic Signatures Pty. Ltd. | Assay for screening for potential cervical cancer |
BRPI0706511A2 (en) | 2006-01-11 | 2011-03-29 | Genomic Health Inc | gene expression markers for colorectal cancer prognosis |
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 |
US20070243161A1 (en) * | 2006-02-28 | 2007-10-18 | Sven Olek | Epigenetic modification of the loci for CAMTA1 and/or FOXP3 as a marker for cancer treatment |
US20090104615A1 (en) * | 2006-05-02 | 2009-04-23 | Keith Malcolm Godfrey | Phenotype prediction |
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WO2010127322A1 (en) | 2009-05-01 | 2010-11-04 | Genomic Health Inc. | Gene expression profile algorithm and test for likelihood of recurrence of colorectal cancer and response to chemotherapy |
WO2011025852A1 (en) | 2009-08-28 | 2011-03-03 | Cellular Dynamics International, Inc. | Identifying genetic variation in affected tissues |
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996036691A1 (en) * | 1995-05-16 | 1996-11-21 | Ramot-Univ. Authority For Applied Research And Industrial Development Ltd. | Ataxia-telangiectasia gene and its genomic organization |
US5744305A (en) * | 1989-06-07 | 1998-04-28 | Affymetrix, Inc. | Arrays of materials attached to a substrate |
WO1999024605A2 (en) * | 1997-11-12 | 1999-05-20 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Dna with promoter activity for cell cycle genes |
WO1999028498A2 (en) * | 1997-11-27 | 1999-06-10 | Epigenomics Gmbh | Method for producing complex dna methylation fingerprints |
WO1999029898A2 (en) * | 1997-12-05 | 1999-06-17 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Method for identifying nucleic acids by means of matrix-assisted laser desorption/ionisation mass spectrometry |
WO2001092565A2 (en) * | 2000-04-06 | 2001-12-06 | Epigenomics Ag | Diagnosis of diseases associated dna transcription by means of assessing the methylation status of genes associated with dna transcription |
Family Cites Families (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 (en) | 1993-06-22 | 1995-02-13 | Pharmacia Lkb Biotech | Method and apparatus for analyzing polynucleotide sequences |
US5837832A (en) | 1993-06-25 | 1998-11-17 | Affymetrix, Inc. | Arrays of nucleic acid probes on biological chips |
JPH09507121A (en) * | 1993-10-26 | 1997-07-22 | アフィマックス テクノロジーズ ナームロゼ ベノートスハップ | Nucleic acid probe array on biological chip |
US6184211B1 (en) | 1993-11-30 | 2001-02-06 | Methylgene Inc. | Inhibition of DNA methyltransferase |
US5871917A (en) | 1996-05-31 | 1999-02-16 | North Shore University Hospital Research Corp. | Identification of differentially methylated and mutated nucleic acids |
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 |
US6251594B1 (en) * | 1997-06-09 | 2001-06-26 | Usc/Norris Comprehensive Cancer Ctr. | Cancer diagnostic method based upon DNA methylation differences |
AU5128999A (en) * | 1998-07-24 | 2000-02-14 | Yeda Research And Development Co. Ltd. | Prevention of metastasis with 5-aza-2'-deoxycytidine |
DE19905082C1 (en) * | 1999-01-29 | 2000-05-18 | Epigenomics Gmbh | Identification of methylation patterns of cytosine in genome DNA comprises chemical treatment to produce different base pairing behavior between cytosine and 5-methylcytosine |
US6331393B1 (en) * | 1999-05-14 | 2001-12-18 | University Of Southern California | Process for high-throughput DNA methylation analysis |
US6783933B1 (en) * | 1999-09-15 | 2004-08-31 | The Johns Hopkins University School Of Medicine | CACNA1G polynucleotide, polypeptide and methods of use therefor |
US7332275B2 (en) * | 1999-10-13 | 2008-02-19 | Sequenom, Inc. | Methods for detecting methylated nucleotides |
EP1268855A2 (en) | 2000-03-15 | 2003-01-02 | Epigenomics AG | Diagnosis of diseases associated with tumor suppressor genes and oncogenes |
AU2001289617A1 (en) | 2000-06-30 | 2002-01-08 | Epigenomics Ag | Diagnosis of diseases associated with signal transduction |
DE10037769A1 (en) | 2000-08-03 | 2002-02-21 | Epigenomics Gmbh | Diagnosis of diseases associated with CD24 |
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 (en) | 2000-11-06 | 2002-06-06 | Epigenomics Ag | Diagnosis of diseases associated with Cdk4 |
DE10054972A1 (en) | 2000-11-06 | 2002-06-06 | Epigenomics Ag | Diagnosis of diseases associated with humus |
DE10128508A1 (en) * | 2001-06-14 | 2003-02-06 | Epigenomics Ag | Methods and nucleic acids for the differentiation of prostate tumors |
EP1502222A2 (en) * | 2001-07-02 | 2005-02-02 | Epigenomics AG | A distributed system for epigenetic based prediction of complex phenotypes |
-
2001
- 2001-04-06 EP EP01956429A patent/EP1370685A2/en not_active Withdrawn
- 2001-04-06 AU AU2001276331A patent/AU2001276331A1/en not_active Abandoned
- 2001-04-06 US US10/240,454 patent/US20040067491A1/en not_active Abandoned
- 2001-04-06 US US10/240,452 patent/US20030162194A1/en not_active Abandoned
- 2001-04-06 EP EP01953145A patent/EP1278893A2/en not_active Withdrawn
- 2001-04-06 AU AU2001254788A patent/AU2001254788A1/en not_active Abandoned
- 2001-04-06 AU AU2001254794A patent/AU2001254794A1/en not_active Abandoned
- 2001-04-06 JP JP2001575229A patent/JP2004508807A/en not_active Withdrawn
- 2001-04-06 AU AU2001277487A patent/AU2001277487A1/en not_active Abandoned
- 2001-04-06 US US10/240,485 patent/US20030148327A1/en not_active Abandoned
- 2001-04-06 AT AT01953936T patent/ATE353975T1/en not_active IP Right Cessation
- 2001-04-06 US US10/240,589 patent/US20040076956A1/en not_active Abandoned
- 2001-04-06 WO PCT/EP2001/003970 patent/WO2001077376A2/en active Application Filing
- 2001-04-06 EP EP01953937A patent/EP1274866A2/en not_active Ceased
- 2001-04-06 AU AU2001275663A patent/AU2001275663A1/en not_active Abandoned
- 2001-04-06 US US10/239,676 patent/US7195870B2/en not_active Expired - Fee Related
- 2001-04-06 WO PCT/EP2001/003969 patent/WO2001077164A2/en active IP Right Grant
- 2001-04-06 WO PCT/EP2001/003971 patent/WO2001077377A2/en not_active Application Discontinuation
- 2001-04-06 EP EP01927895A patent/EP1272670A2/en not_active Withdrawn
- 2001-04-06 WO PCT/EP2001/004016 patent/WO2001076451A2/en not_active Application Discontinuation
- 2001-04-06 EP EP08012765A patent/EP2014776A3/en not_active Ceased
- 2001-04-06 JP JP2001575230A patent/JP2003534780A/en not_active Withdrawn
- 2001-04-06 EP EP01955278A patent/EP1360319A2/en not_active Withdrawn
- 2001-04-06 DE DE60126593T patent/DE60126593T2/en not_active Expired - Fee Related
- 2001-04-06 AU AU2001278420A patent/AU2001278420A1/en not_active Abandoned
- 2001-04-06 JP JP2001575634A patent/JP2003531589A/en not_active Withdrawn
- 2001-04-06 WO PCT/EP2001/003972 patent/WO2001081622A2/en active Application Filing
- 2001-04-06 AU AU7633001A patent/AU7633001A/en active Pending
- 2001-04-06 WO PCT/EP2001/003968 patent/WO2001077375A2/en not_active Application Discontinuation
- 2001-04-06 US US10/240,708 patent/US20050282157A1/en not_active Abandoned
- 2001-04-06 EP EP01953936A patent/EP1274865B1/en not_active Expired - Lifetime
- 2001-04-06 US US10/240,453 patent/US20030148326A1/en not_active Abandoned
- 2001-04-06 AU AU2001276330A patent/AU2001276330B2/en not_active Ceased
- 2001-04-06 WO PCT/EP2001/004015 patent/WO2001077378A2/en not_active Application Discontinuation
- 2001-04-06 EP EP01927887A patent/EP1268857A2/en not_active Withdrawn
- 2001-04-06 AU AU2001289600A patent/AU2001289600A1/en not_active Abandoned
- 2001-04-06 EP EP01969303A patent/EP1268861A2/en not_active Withdrawn
- 2001-04-06 WO PCT/EP2001/003973 patent/WO2001092565A2/en active Application Filing
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5744305A (en) * | 1989-06-07 | 1998-04-28 | Affymetrix, Inc. | Arrays of materials attached to a substrate |
WO1996036691A1 (en) * | 1995-05-16 | 1996-11-21 | Ramot-Univ. Authority For Applied Research And Industrial Development Ltd. | Ataxia-telangiectasia gene and its genomic organization |
WO1999024605A2 (en) * | 1997-11-12 | 1999-05-20 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Dna with promoter activity for cell cycle genes |
WO1999028498A2 (en) * | 1997-11-27 | 1999-06-10 | Epigenomics Gmbh | Method for producing complex dna methylation fingerprints |
WO1999029898A2 (en) * | 1997-12-05 | 1999-06-17 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Method for identifying nucleic acids by means of matrix-assisted laser desorption/ionisation mass spectrometry |
WO2001092565A2 (en) * | 2000-04-06 | 2001-12-06 | Epigenomics Ag | Diagnosis of diseases associated dna transcription by means of assessing the methylation status of genes associated with dna transcription |
Non-Patent Citations (18)
Title |
---|
BACHOO S, GIBBONS R J: "Germline and gonosomal mosaicism in the ATR-X syndrome" EUROPEAN JOURNAL OF HUMAN GENETICS, vol. 7, no. 8, December 1999 (1999-12), pages 933-936, XP002190211 * |
BAYLIN S B ET AL: "DNA hypermethylation in tumorigenesis: epigenetics joins genetics" TRENDS IN GENETICS, ELSEVIER, AMSTERDAM, NL, vol. 16, no. 4, April 2000 (2000-04), pages 168-174, XP004194021 ISSN: 0168-9525 * |
DATABASE EMBL [Online] EBI; 10 October 1995 (1995-10-10) OGURA T.:: "NAD+ ADP-ribosyltransferase; PADPRP-I gene, promoter" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. x16674 XP002190213 -& OGURA T ET AL.: "Characterization of a putative promoter region of the human poly(ADP-ribose) polymerase beta gene." BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 167, no. 2, 16 March 1990 (1990-03-16), pages 701-710, XP001026656 * |
DATABASE EMBL [Online] EBI; 11 October 1999 (1999-10-11) BIRREN B ET AL.: "Homo sapiens clone RP11-15H13" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. AC011651 XP002190215 * |
DATABASE EMBL [Online] EBI; 12 January 1996 (1996-01-12) BYRD P J: "H. sapiens mRNA for E14 and A-T proteins" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. X91196 XP002190218 -& BYRD P J ET AL.: "Mutations revealed by sequencing the 5' half of the gene for ataxia telangiectasia" HUMAN MOLECULAR GENETICS, vol. 5, no. 1, 1996, pages 145-149, XP002190210 * |
DATABASE EMBL [Online] EBI; 14 March 2000 (2000-03-14) BIRREN, B. ET AL.: "Homo sapiens chromosome 1 clone RP11-274N19 map 1" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. AC025385 XP002180960 * |
DATABASE EMBL [Online] EBI; 15 August 1997 (1997-08-15) PLATZER M ET AL.: "Homo sapiens ataxia telangiectasia (ATM) gene, complete cds." retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. U82828 XP002190216 * |
DATABASE EMBL [Online] EBI; 2 July 1999 (1999-07-02) HERZOG ET AL.: "Human poly(ADP-ribose) polymerase gene, 5' end" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. M60436 XP002190214 * |
DATABASE EMBL [Online] EBI; 21 February 2000 (2000-02-21) BIRREN B ET AL.: "Homo sapiens chromosome 4 clone RP11-631I16 map 4" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. AC023984 XP002190223 * |
DATABASE EMBL [Online] EBI; 22 August 1997 (1997-08-22) VILLARD L ET AL.: "Homo sapiens X-linked nuclear protein (ATRX) gene, exon 1" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. AF000153 XP002190220 * |
DATABASE EMBL [Online] EBI; 22 January 1997 (1997-01-22) BIRD C: "Human DNA sequence" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. Z84487 XP002190219 * |
DATABASE EMBL [Online] EBI; 26 April 1999 (1999-04-26) LIU, Y. ET AL.: "Homo sapiens RNA-specific adenosine deaminase ADAR1 (Adar1) gene, promoter" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. AF084517 XP002180959 -& LIU Y ET AL.: "Functionally distinct double-stranded RNA-binding domains associated with alternative splice site variants of the interferon-inducible double-stranded RNA-specific adenosine deaminase." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 7, 14 February 1997 (1997-02-14), pages 4419-4428, XP002180957 * |
DATABASE EMBL [Online] EBI; 4 April 1997 (1997-04-04) EVANS G A ET AL.: "Human chromosome 15 pac pDJ24m8, complete sequence" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. AC000379 XP002190221 * |
DATABASE EMBL [Online] EBI; 9 March 1996 (1996-03-09) IMAI T: "Homo sapiens ATM gene exon 1 and NPAT gene exon 1" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. D83244 XP002190217 -& LUO L ET AL.: "Ataxia-telangiectasia an T-cell leukemias: No evidence for somatic ATM mutation in sporadic T-ALL or hypermethylatio of the ATM-NPAT/E14 bidirectional promoter in T-PLL" CANCER RESEARCH, vol. 58, no. 11, 1 June 1998 (1998-06-01), pages 2293-2297, XP001056079 -& IMAI T ET AL.: "The structure and organisation of the human NPAT gene" GENOMICS, vol. 42, 1997, pages 388-392, XP002190209 * |
DATABASE GENBANK [Online] NCBI; 10 January 1995 (1995-01-10) PURANAM, K.L., ET AL.: "Homo sapiens (clone 1311) DNA helicase (REQL) mRNA, complete cds." retrieved from HTTP://WWW.NCBI.NLM.NIH.GOV Database accession no. L36140 XP002180958 -& PURNAM K L ET AL.: "Cloning and characterization of RECQL, a potential human homologue of the escherichia coli DNA helicase RECQ." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 47, 25 November 1994 (1994-11-25), pages 29838-29845, XP002032093 * |
DATABASE GENBANK [Online] NCBI; 16 February 1996 (1996-02-16) ELLIS N A ET AL: "Human Bloom's syndrome protein (BLM) mRNA, complete cds" retrieved from HTTP://WWW.NCBI.NLM.GOV Database accession no. U39817 XP002190222 -& ELLIS N A ET AL.: "The Bloom's syndrome gene product is homologous to RecQ helicase" CELL, vol. 83, 17 November 1995 (1995-11-17), pages 655-666, XP002113488 * |
HERMAN J G ET AL: "INCIDENCE AND FUNCTIONAL CONSEQUENCES OF HMLH1 PROMOTOR HYPERMETHYLATION IN COLORECTAL CARCINOMA" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 95, June 1998 (1998-06), pages 6870-6875, XP002944676 ISSN: 0027-8424 * |
POPPE M ET AL.: "Use of PCR to screen for promoter elements in genomic DNA library clones" BIOTECHNIQUES, vol. 26, no. 4, April 1999 (1999-04), pages 718-726, XP001026489 * |
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