Abstract: A method is described for the analysis of cytosine methylation patterns in genomic DNA samples. In the first method step, the genomic DNA is isolated from cells or other accompanying materials and bound essentially irreversibly to a surface. Then the DNA bound to the surface is treated, preferably with a bisulfite, in such a way that cytosine is converted into a base that is different in its base pairing behavior in the DNA duplex, while 5-methylcytosine remains unchanged. Then the reagents that were used are removed in a washing step. Finally, selected segments of the immobilized DNA are amplified in a polymerase reaction and the amplified products are investigated with respect to their sequence.

Abstract: Described is a method for detecting 5-methylcytosine in genomic DNA samples. First, a genomic DNA from a DNA sample is chemically converted with a reagent, 5-methylcytosine and cytosine reacting differently, and the pretreated DNA is subsequently amplified using a polymerase and at least one primer. In the next step, the amplified genomic DNA is hybridized to at least two oligonucleotides, the latter being joined by inserting at least one oligonucleotide. In the ligation product, one nucleotide carries a detectable label, and the elongation depends on the methylation status of the specific cytosine in the genomic DNA sample. In the next step, the elongated oligonucleotides are analyzed for the presence of the label.

Abstract: Chemically modified nucleic acid sequences of genomic DNA, oligonucleotides and/or PNA-oligomers for detecting the cytosine methylation state of genomic DNA. In addition, a method for ascertaining genetic and/or epigenetic parameters of genes for the characterization, classification, differentiation, grading, staging, treatment and diagnosis of oligodendrogliomas, astrocytomas and oligoastrocytomas.

Abstract: The present invention relates to chemically modified genomic sequences, to oligonucleotides and/or PNA-oligomers for detecting the cytosine methylation state of genomic DNA, as well as to a method for ascertaining genetic and/or epigenetic parameters of genes for use in characterisation, classification, differentiation, diagnosis and therapy of prostate lesions.

Abstract: The present invention provides methods for identification of methylated, and/or potentially methylatable CpG dinucleotides in genomic DNA sequences, and methods for isolating genomic DNA sequences comprising methylated CpG dinucleotide sequences. The present invention further provides methods for comparison of the methylation status of specific CpG dinucleotides, and patterns thereof between normal and diseased genomic DNA sequences, along with methods for determining all potentially methylatable CpG dinucleotides in a genomic DNA sample. Specifically, the present invention discloses a novel use of 5-methylcytosine DNA glycosylase (5-MCDG) in combination with art-recognized DNA base excision repair (BER) enzymes, and in particular embodiments, in combination with DNA methyltransferase to specifically label methylated CpG dinucleotide sequences in genomic DNA sequences.

Abstract: The present invention concerns a method for the detection of cytosine methylation in DNA samples, wherein the following steps are conducted: (a) a genomic DNA sample, which comprises the DNA to be investigated and background DNA, is chemically treated in such a way that all of the unmethylated cytosine bases are converted to uracil, whereas the 5-methylcytosine bases remain unchanged; (b) the chemically treated DNA sample is amplified with the use of at least 1 primer oligonucleotide as well as a polymerase, whereby the DNA to be investigated is preferred as the template over the background DNA, and (c) the amplified products are analyzed and the methylation status in the DNA to be investigated is concluded from the presence of an amplified product and/or from the analysis of additional positions.

Abstract: The present invention relates to modified and genomic sequences, to oligonucleotides and/or PNA-oligomers for detecting the cytosine methylation state of genomic DNA, as well as to a method for ascertaining genetic and/or epigenetic parameters of genes for use in the differentiation, diagnosis, treatment and/or monitoring of lung cell proliferative disorders, or the predisposition to lung cell proliferative disorders.

Abstract: Chemically modified genomic sequences of genes associated with gene regulation, to oligonucleotides and/or PNA-oligomers for detecting the cytosine methylation state of genes associated with gene regulation which are directed against the sequence are disclosed. In addition, a method for ascertaining genetic and/or epigenetic parameters of genes associated with gene regulation is disclosed.

Abstract: A method is described for distinguishing 5-position methylation changes of cytosine bases and cytosine-to-thymine mutations and for the detection of single nucleotide polymorphisms (SNPs) or point mutations in genomic DNA, in which: a) a genomic DNA sample is treated with sulfite or disulfite in such a way that all of the cytosine bases that are not methylated in the 5-position of the base are changed in such a way that a base is formed that is different in its base-pairing behavior, whereas the cytosines methylated at the 5-position remain unchanged, and b) an aliquot of the same genomic DNA sample is quantitatively methylated with Sss1 or another methyltransferase prior to the chemical treatment according to a) and c) both of the DNA samples treated in this way are investigated for the presence of cytosine by means of the same analytical method, and d) the cytosine positions that are determined are matched with a reference DNA sequence.

Abstract: The present invention relates to modified and genomic sequences, to oligonucleotides and/or PNA-oligomers for detecting the cytosine methylation state of genomic DNA, as well as to a method for ascertaining genetic and/or epigenetic parameters of genes for use in the differentiation, diagnosis, treatment and/or monitoring of breast cell proliferative disorders, or the predisposition to breast cell proliferative disorders.

Abstract: A method is described for the relative quantification of the methylation of cytosine bases in DNA samples, wherein the following method steps are conducted: a) a genomic DNA sample is chemically converted with a reagent, wherein 5-methylcytosine and cytosine react differently and show a different base pairing behavior in the DNA duplex after the reaction; b) the DNA sample is amplified, whereby a fluorescently labeled dCTP or dGTP derivative is added; c) the amplified products are separated spatially from each other; and d) the fluorescence of the separated amplified products is measured quantitatively.

Abstract: The invention provides methods, nucleic acids and kits for detecting, or for detecting and distinguishing between or among liver cell proliferative disorders or for detecting, or for detecting and distinguishing between or among colorectal cell proliferative disorders. The invention discloses genomic sequences the methylation patterns of which have utility for the improved detection of and differentiation between said class of disorders, thereby enabling the improved diagnosis and treatment of patients.

Abstract: The present invention relates to modified and genomic sequences, to oligonucleotides and/or PNA-oligomers for detecting the cytosine methylation state of genomic DNA, as well as to a method for ascertaining genetic and/or epigenetic parameters of genes for use in the differentiation, diagnosis, treatment and/or monitoring of colon cell proliferative disorders, or the predisposition to colon cell proliferative disorders.

Abstract: A method is described for the detection of 5-methylcytosine in genomic DNA samples. First, a genomic DNA from a DNA sample is chemically converted with a reagent, whereby 5-methylcytosine and cytosine react differently. Then the pretreated DNA is amplified with the use of a polymerase with primers of different sequence. In the next step, the amplified genomic DNA is hybridized to an oligonucleotide array and PCR products are obtained, which must be provided with a label. Alternatively, the PCR products can be extended in a primer extension reaction, wherein the extension products are also provided with a label. In the last step, the extended oligonucleotides are investigated for the presence of the label.

Abstract: There is disclosed an improved high-throughput and quantitative process for determining methylation patterns in genomic DNA samples based on amplifying modified nucleic acid, and detecting methylated nucleic acid based on amplification-dependent displacement of specifically annealed hybridization probes. Specifically, the inventive process provides for treating genomic DNA samples with sodium bisulfite to create methylation-dependent sequence differences, followed by detection with fluorescence-based quantitative PCR techniques. The process is particularly well suited for the rapid analysis of a large number of nucleic acid samples, such as those from collections of tumor tissues.

Abstract: Particular aspects relate to a method for determining the methylation pattern of a polynucleic acid, comprising: a) preparing a solution comprising a mixture of fragments of the polynucleic acid; b) coupling the fragments with a substance being detectable with a detection method; c) contacting a solution comprising the fragments of b) with a DNA microarray having a plurality of different immobilized oligonucleotides, each comprising at least one methylation site, at respectively assigned different locations thereon, the contacing under conditions affording hybridization of fragments with correlated immobilized oligonucleotides under defined stringency, and wherein the immobilized oligonucleotides have a length of less than 200 bases; d) optionally performing a a washing step; and e) detecting, using the physical detection method, such immobilized nucleic acids to which solution fragments are hybridized and/or to which solution fragments are not hybridized.

Abstract: The present invention provides, inter alia, a method for generating a genome-wide epigenomic map, comprising a correlation between methylation variable CpG positions (MVP) and genomic DNA sample types. MVP are those CpG positions that show a variable quantitative level of methylation between sample types. Particular genomic regions of interest (ROI) provide preferred marker sequences that comprise multiple, and preferably proximate MVP, and that have novel utility for distinguishing sample types. The epigenic maps have broad utility, for example, in identifying sample types, or for distinguishing between and among sample types. In a preferred embodiment the epigenomic map is based on methylation variable regions (MVP) within the major histocompatibility complex (MHC), and has utility, for example, in identifying the cell or tissue source of a genomic DNA sample, or for distinguishing one or more particular cell or tissue types among other cell or tissue types.

Abstract: Particular aspects provide methods for specific amplification of template DNA in the presence of potentially contaminating PCR products from previous amplification experiments. Particular embodiments comprise, in a first step, contacting DNA with a bisulfite solution, which sulfonates unmethylated (but not methylated) cytosines, resulting in cytosine deamination and generation of sulfonated uracil. Such sulfonation protects the template nucleic acid from being a target for the enzyme uracil-DNA-glycosylase (UNG), whereas any contaminating DNA, which contains unprotected unsulfonated or desulfonated uracils, is degraded enzymatically while the UNG is active. After UNG treatment and inactivation thereof, the sulfonated uracil bases are converted into uracil by desulfonation. Such aspects have substantial utility for decontamination of nucleic acid samples; e.g., for avoiding amplification of ‘carry over products’ in the context of DNA methylation analysis.

Abstract: A method is described for controllably conducting complex PCR amplifications, wherein at least the following steps are conducted: a) PCR amplification with at least 50 primers of a first type (type 1) of different sequence, which are complementary to one of the strands of a random DNA sample, and also with a primer or a library of primers of a second type (type 2), which is complementary to the other strand of the DNA sample used, wherein the type 2 primers contain a first label (label 1); b) hybridizing of the amplified products to an oligomer array, which comprises oligonucleotides that hybridize to the primers utilized in the PCR reaction or to oligonucleotides that are complementary to these; or hybridizing of the amplified products to an oligomer array, which contains oligomers complementary to the primers utilized in the PCR reaction; c) length determination of the amplified products bound to the array by a second label (label 2) which can be correlated with the length of the respective DNA fragment,

Abstract: Particular aspects of the present invention provide a method for quantification of two different variations of a DNA sequence. Particularly, the invention relates to a quantification of methylated DNA, and for this purpose, the test DNA is converted so that cytosine is converted to uracil, while 5-methylcytosine remains unchanged. The converted DNA is amplified by means of a real-time PCR, wherein two labeled real-time probe types are utilized: one specific for methylated DNA; and one for unmethylated DNA. Preferably, the degree of methylation of the test DNA is calculated from the ratio of the signal intensities of the probes or from the Ct values. The inventive methods have substantial utility for diagnosis and prognosis of cancer and other disorders associated with altered or characteristic DNA methylation status, as well as having substantial utility for analysis of SNPs, allelic expression, and prediction of drug response, drug interactions, among other uses.