Group1principalofmethlatedDNAimmunoprecipitation.pptVIP

Principles of Methylated DNA immunoprecipitation Group1： Chen Zong Cuiling Li Jingyun Guan Xiaofei Han 2012.12.18 Background DNA Methylation, referring to the reversible methylation of the 5 position of cytosine by methyltransferases, is a major epigenetic modification in multicellular organisms. In mammals, this modification primarily occurs at CpG sites, which in turn tend to cluster in regions called CpG islands. The function of DNA methylation is silencing transcription. There is a small fraction of CpG islands that can overlap or be in close to promoter regions of transcription start sites. Methylation at either of these sites can repress gene expression by either interfering with the binding of transcription factors or modifying chromatin structure to a repressive state. MeDIP: Methylated DNA immunoprecipitation MeDIP or mDIP is a chromosome-or genome-wide purification technique in molecular biology that is used to enrich for methylated DNA sequences. It consists of isolating methylated DNA fragments via an antibody raised against 5-methylcytosine 5mC . Coimmunoprecipitation principles The procedure of MeDIP 1.Extraction of Nucleic Acids and DNA Purification 2.DNA Shearing 3.Denaturation 4.Incubation with 5mc antibodies 5.Magnetic beads conjugated to anti-mouse-IgG 6.DNA Elution and Purification 7.qPCR Analysis of Immunoprecipitated DNA Workflow overview of the MeDIP procedure A.MeDIP and array-based hybridization MeDIP-chip Downstream bioinformatics analysis Studies using MeDIP-seq or MeDIP-chip are both genome-wide approaches that have the common aim of obtaining the functional mapping of the DNA methylation. Once regions of DNA methylation are identified, a number of bioinformatics analyses can be applied to answer certain biological questions. One obvious step is to investigate genes contained in these regions and investigate the functional significance of their repression. Limitations of MeDIP Limitations to take note when using MeDIP