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The Epigenetics Group of the Centre National de Génotypage is lead by Dr. Jörg Tost and is part of the Technology Development Department headed by Dr. Ivo Gut. The Epigenetics Group is involved in the development and application of technologies to analyze DNA methylation at high resolution of both target loci and genome-wide. Aberrant DNA methylation patterns have been found to underlie many complex diseases and might contribute to pathogenesis as much as genetic changes do. In the last few years the laboratory has devised several methods for the analysis of DNA methylation patterns based on matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and pyrosequencing. The mass spectrometry-based epigenotyping assay was first used in a pilot project for the Human Epigenome Project in which our laboratory participated. Established technological platforms are applied to various in-house and collaborative projects. One research axis is the identification of epigenetic markers that could serve for the early detection of disease and/or might be used for prognosis, optimal treatment strategy, etc. of disease outcome. Current projects include breast, colon, lung, pancreatic cancer, and type 2 diabetes. The second interest of research comprises interindividual epigenetic variations, especially in correlation to imprinting and development and how changes in DNA methylation influence phenotypes in nondisease situations.
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The Technique
Pyrosequencing has evolved in the last years as a powerful tool for the analysis of DNA methylation patterns after bisulfite treatment. Based on the enzymatic conversion of pyrophosphate that is released upon nucleotide incorporation into a luminometric signal, the technology unites simplicity, reproducibility, robustness, and most importantly, quantitative accuracy with a resolution of better than 5%. Pyrosequencing has the potential to supersede the laborious and time-consuming procedure of cloning and subsequent sequencing, which is still considered the gold standard for high-resolution quantitative DNA methylation analysis. However, the short read-length of a pyrosequencing primer required multiple PCR amplifications to scan an entire product, which used up precious DNA resources. In our article we present a modification of the pyrosequencing protocol that permits recovery of the sequenced template strand and repeated annealing of new sequencing primers. This improved protocol has significantly speeded up the workflow in our laboratory, contributes to notable cost savings, and conserves the precious DNA resources for the analysis of multiple genes instead of only a few loci.
Serial pyrosequencing for quantitative DNA methylation analysis, p. 721.
