Epigenomics Ag

Christian Piepenbrock

Kurt Berlin

Alexander Olek

Ralf Lesche

Juergen Distler

Jörn Lewin

Peter Adorjan

Thomas Koenig

Armin Schmitt

Anne Fassbender

Tamas Rujan

David Guetig

A method is described for the detection of the degree of methylation of a specific cytosine in the sequence context 5'-CpG-3&#x2032; of a genomic DNA sample. In the first step, the genomic DNA is chemically treated in such a way that the cytosine bases are converted to uracil, but not the 5-methylcytosine bases. Then segments of the genomic DNA which contain the said specific cytosine are amplified, whereby the amplified products are given a detectable label and in the following steps the extent of hybridization of the amplified products on two classes of oligonucleotides is determined by detection of the label of the amplified products, and a conclusion is made on the extent of methylation of said specific cytosine in the genomic DNA sample from the ratio of the labels detected on the two classes of oligonucleotides as a consequence of the hybridization.

Method for determining the degree of methylation of defined cytosines in genomic DNA in the sequence context of 5'-CpG-3&#x2032;

The invention describes a method for the quantitative sequencing of nucleic acids treated with bisulfite. The invention particularly describes a method for the determination of the degree of methylation of a cytosine in the base sequence 5'-CG-3&#x2032;, thus in a so-called CpG position, if the genomic sequence of the investigated DNA region is known. The invention makes possible the validation of sequence information in the sequencing of DNA treated with bisulfite. It is particularly described how the data obtained with conventional sequencing methods are first standardized with respect to their signal intensity, how the conversion rate of cytosine to uracil after bisulfite treatment is determined from these data, and how a correction factor can be determined with the help of this conversion rate, and this factor produces an essentially improved estimation of the degree of methylation actually present.

Method for quantitative determination of the degree of methylation of cytosines in CpG positions

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 contacting 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.

Method for determining the methylation pattern of a polynucleic acid

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.

Diagnosis of diseases associated with gene regulation

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Berlin, Germany

Christian Piepenbrock