1Jörg D. Hoheisel, 1Ole Brandt, 1Verena Beier, 2Markus Beier, 3Hans-Josef Gröne, 4Heinrich Arlinghaus, 5Frank Lyko, 1Anette Jacob
1Functional Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, 2febit AG, Käfertalerstr. 190, 68167 Mannheim, Germany, 3Cellular and Molecular Pathology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, 4Physikalisches Institut, Universität Münster, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany, 5Epigenetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
In the human genome, about 4% of the cytosine residues are modified by methylation that regulates the expression of many genes. This regulation involves a variety of factors, like DNA methyltransferases, methyl-DNA binding proteins and chromatin proteins. The synergistic action of all factors results in the establishment and maintenance of a specific DNA methylation pattern. Epigenetic programming of the genome dictates the interpretation of the genetic information. Different cell types can be distinguished by their specific epigenetic programmes, for example. Also, it is generally assumed that epigenetic mis-regulation of cancer-related genes plays a major role in cellular transformation.
Progress in the field has been hampered by the unavailability of methods for genome-wide epigenetic profiling. However, recent developments in array- technologies provide an opportunity for a comprehensive analysis. Based on the bisulphite conversion of unmethylated cytosine to uracil, methylation patterns can be detected in the same way as single nucleotide polymorphisms (SNPs).
We pursue different approaches. At low complexity, samples are hybridised directly to in situ synthesised oligomer microarrays of flexible design. At higher complexity, we employ the superior discrimination power of polymerases. Due to appropriately modified nucleotide monomers, oligonucleotides can be synthesised in 5'-3' direction, thus acting as substrates for polymerases.
Last, we take advantage of the unique features of peptide nucleic acid (PNA) in order to enable an immediate analysis of genomic DNA without prior amplification or labelling. Since PNA does not contain any phosphates, the phosphates of hybridising nucleic acids can act as an inherent label, detection being performed by secondary ion mass spectrometry (SIMS). Thereby, an unlimited number of methylation sites could be studied in a single experiment, the detection process being restricted solely by the reaction kinetics rather than procedural factors.
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