US20040067491A1 - Diagnosis of diseases associated with metabolism - Google Patents

Diagnosis of diseases associated with metabolism Download PDF

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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
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dna
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Alexander Olek
Christian Piepenbrock
Kurt Berlin
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Epigenomics AG
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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 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.
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DE10032529A DE10032529A1 (de) 2000-06-30 2000-06-30 Diagnose von bedeutenden genetischen Parametern innerhalb des Major Histocompatibility Complex (MHC)
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US7195870B2 (en) 2007-03-27
WO2001077378A2 (en) 2001-10-18
AU2001276331A1 (en) 2001-10-23
DE60126593T2 (de) 2007-10-31
US20030148327A1 (en) 2003-08-07
EP1278893A2 (de) 2003-01-29
US20030162194A1 (en) 2003-08-28
DE60126593D1 (de) 2007-03-29
AU2001275663A1 (en) 2001-10-23
EP1360319A2 (de) 2003-11-12
EP1370685A2 (de) 2003-12-17
EP1274865A2 (de) 2003-01-15
WO2001092565A3 (en) 2002-09-06
WO2001077375A2 (en) 2001-10-18

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