Epigenomics Ag

Theo De Vos

Joe Lograsso

Fabian Model

Volker Liebenberg

Cathy Lofton-Day

Jennifer Maas

Andrew Sledziewski

Matthias Ballhause

Kurt Berlin

Matthias Schuster

Dimo Dietrich

Reimo Tetzner

Antje Kluth Lukas

Ute Wagner

Reinhold Wasserkort

Heike Ziebarth

Ina Fuhrmann

Karen Cardon

David Gütig

Catherine E Lofton-Day

Andrew Z Sledziewski

Aspects of the present invention relate to compositions and methods for providing DNA fragments from a remote sample. In particular aspects a remote sample comprising DNA is provided, DNA is isolated from the remote sample, and the isolated DNA is treated in a way which allows differentiation of methylated and unmethylated cytosine. Additional, particular embodiments provide compositions and methods for methylation analysis of DNA derived from a remote sample. Other aspects provide for compositions and methods of whole genome amplification of bisulfite treated DNA. Other aspects provide methods for determining the presence or absence of methylation of at least one cytosine, or a series of cytosines in cis, in human DNA of a blood sample, a plasma sample, a serum sample or a urine sample from a human individual.

Method for determining the presence or absence of methylation in a sample

Aspects of the present invention relate to compositions and methods for providing DNA fragments from an archived sample (e.g., paraffin-embedded and/or fixed-tissue biopsies, etc.). Particular aspects provide methods whereby high yields of DNA are isolated as well as a substantial portion of the DNA consists of long DNA fragments, and where the isolated genomic DNA is free of associated or cross-linked contaminants like proteins, peptides, amino acids or RNA. The methods are facile, cost-effective, and are characterized by high reproducibility and reliability. Particular aspects provide methods for providing DNA fragments derived from an archived sample, wherein the yield of DNA before, for example, an amplification step is at least 20%, and amplicons up to a length of about 1,000 base pairs are amplifiable.

Method for providing DNA fragments derived from an archived sample

Aspects of the present invention relate to compositions and methods for providing DNA fragments from an archived sample (e.g., paraffin-embedded and/or fixed-tissue biopsies, etc). Particular aspects provide methods whereby high yields of DNA are isolated as well as a substantial portion of the DNA consists of long DNA fragments, and where the isolated genomic DNA is free of associated or cross-linked contaminants like proteins, peptides, amino acids or RNA. The methods are facile, cost-effective, and are characterized by high reproducibility and reliability. Particular aspects provide methods for providing DNA fragments derived from an archived sample, wherein the yield of DNA before, for example, an amplification step is at least 20%, and amplicons up to a length of about 1,000 base pairs are amplifiable.

The present invention relates to an improved method for the bisulfite conversion of DNA. In certain time-temperature ranges the efficacy of the bisulfite conversion is clearly improved. By combination with denaturating solvents, new reaction conditions and new purification methods the efficacy can be further increased The converted DNA can subsequently be analysed by different methods. The present invention facilitates the analysis of cytosine methylation.

Bisulfite conversion of DNA

The present invention provides an improved method for the bisulfite conversion of DNA, and facilitates the analysis of cytosine methylation of genomic DNA. Novel combinations of denaturing solvents, new reaction conditions and new purification methods provide surprisingly efficacious methods for bisulfite conversion of DNA relative to prior art methods. The converted DNA may subsequently be analyzed by many different methods.

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.

Method for the detection of cytosine methylations in immobilized DNA samples

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Berlin, Germany

Matthias Ballhause