DNA Methylation Analysis of ChIP Products at Single Nucleotide Resolution by Pyrosequencing®

International audienceInteraction and co-occurrence of protein and DNA-based epigenetic modifi cations have become a topic of interest for many fundamental and biomedical questions. We describe within this chapter a protocol that combines two techniques in order to determine the methylation status of the DNA specifi cally associated with a protein of interest. First, DNA that directly interacts with the selected protein (such as a specifi c histone modifi cation, a transcription factor, or any other DNA-associated protein) is purifi ed by standard chromatin immunoprecipitation (ChIP). Second, the level of DNA methylation of this immunoprecipitated DNA is measured by bisulfi te conversion and Pyrosequencing, a quantitative sequencing-by-synthesis method. This procedure allows determining the methylation status of genomic DNA associated to a specifi c protein at single nucleotide resolution

Microarrays as research tools and diagnostic devices

Molecular diagnostics comprises a main analytical division in clinical laboratory diagnostics. The analysis of RNA or DNA helps to diagnose infectious diseases and identify genetic determined disorders or even cancer. Starting from mono-parametric tests within the last years, technologies have evolved that allow for the detection of many parameters in parallel, e.g., by using multiplex nucleic acid amplification techniques, microarrays, or next-generation sequencing technologies. The introduction of closed-tube systems as well as lab-on-a-chip devices further resulted in a higher automation degree with a reduced contamination risk. These applications complement or even stepwise replace classical methods in clinical microbiology like virus cultures, resistance determination, microscopic and metabolic analyses, as well as biochemical or immunohistochemical assays. In addition, novel diagnostic markers appear, like noncoding RNAs and miRNAs providing additional room for novel biomarkers. This article provides an overview of microarrays as diagnostics devices and research tools. Introduced in 1995 for transcription analysis, microarrays are used today to detect several different biomolecules like DNA, RNA, miRNA, and proteins among others. Mainly used in research, some microarrays also found their way to clinical dia- gnostics. Further, closed lab-on-a-chip devices that use DNA microarrays as detection tools are discussed, and additionally, an outlook toward applications of next-generation sequencing tools in diagnostics will be given

Forest tree genomics: 10 achievements from the past 10 years and future prospects

This review highlights some of the discoveries and applications made possible by “omics” technologies over the last 10 years and provides perspectives for pioneering research to increase our understanding of tree biology.ContextA decade after the first forest tree genome sequence was released into the public domain, the rapidly evolving genomics and bioinformatics toolbox has advanced our understanding of the structure, functioning, and evolution of forest tree genomes.Aims and methodsThis review highlights some of the discoveries and applications that “omics” technologies have made possible for forest trees over the past 10 years.ResultsIn this review, we start by our current understanding of genome evolution and intricacies of gene regulation for reproduction, development, and responses to biotic and abiotic stresses. We then skim over advances in interactome analysis and epigenomics, the knowledge of the extent of genetic variation within and between species, revealing micro- and macro-evolutionary processes and species history, together with the complex architecture of quantitative traits. We finally end with applications in genetic resource conservation and breeding.ConclusionThe knowledge gained through the use of these technologies has a huge potential impact for adapting forests to the main challenges they will have to face: changing demand from ecosystem services with potentially conflicting strategies in terms of conservation and use, as well as climate changes and associated threats. Genomics will undoubtedly play a major role over the next decade and beyond, not only to further understand the mechanisms underlying adaptation and evolution but also to develop and implement innovative management and policy actions to preserve the adaptability of natural forests and intensively managed plantations