Project description:We demonstrate that mammalian cells are able to salvage uracil analogs, yielding high background incorporation into cellular RNA. We also discover the enzymatic pathways responsible for this background. To overcome these limitations, we have developed a novel small-molecule/enzyme pair consisting of uridine/cytidine kinase 2 (UCK2) and 2'-azidouridine (2'AzUd).
Project description:We report the setup of a new method to map 5-hydroxymethylcytosine (5hmC) genome-wide at CpG resolution. The method combines selective chemical labeling by 5hmC b-glucosyltransferase and exonuclease digestion of the DNA molecules bound to streptavidin beads after biotinylation of the 5-glucosylmethylcytosines. Associated with a straightforward bioinformatic analysis, this new procedure provides a cost-effective and fast method for mapping 5hmC at high resolution.
Project description:Conventional prokaryotic RNA labeling method usually requires large amounts of starting materials and tends to generate high background signals. Recently, two novel methods based on amplification systems were introduced. Here, we compared three alternative strategies: direct labeling method, ployadenylation-involved oligo-dT priming amplification method and random priming amplification method (hereafter referred to as DL, PAOD and RPA method in this article) for prokaryotic RNA labeling employing the expression profiling investigation in Escherichia coli (E. coli) heat shock model.
Project description:Conventional prokaryotic RNA labeling method usually requires large amounts of starting materials and tends to generate high background signals. Recently, two novel methods based on amplification systems were introduced. Here, we compared three alternative strategies: direct labeling method, ployadenylation-involved oligo-dT priming amplification method and random priming amplification method (hereafter referred to as DL, PAOD and RPA method in this article) for prokaryotic RNA labeling employing the expression profiling investigation in Escherichia coli (E. coli) heat shock model. To identify an optimal RNA labeling method for prokaryotic microarray analysis, experiments were performed with starting RNA obtained from E.coli growing at control (37°C) or heat shock (43°C) condition. We employed 0.5 μg of total RNA for a single round of amplification using PAOD and RPA methods coupling to IVT to generate cDNA targets. In addition, an approach using DL method was also performed. Three types of cDNA mixtures containing Cy5-labeled (or Cy3-labeled) control DNA and Cy3-labeled (or Cy5-labeled) DNA targets were hybridized with E.coli microarrays in a dye-swap strategy. Replicate experiments were conducted from the same batch of total RNA to assess technique reproducibility of each approach.
Project description:5-Methylcytosine (5-mC) is an important DNA modification found in eukaryotes that impacts gene regulation and disease pathogenesis. Recently, 5-hydroxymethylcytosine (5-hmC), another form of DNA modification, has been identified in substantial amounts in certain mammalian cell types; however, its roles as well as its distribution in mammalian genomes are unknown. Here we present a selective chemical labeling method for 5-hmC by utilizing T4 bacteriophage BGT-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC, which in turn can chemically incorporate a biotin group for detection, affinity enrichment, and sequencing of 5-hmC in mammalian genomes. Using this highly effective method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized. We also find a gene expression level-dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders
Project description:5-Methylcytosine (5-mC) is an important DNA modification found in eukaryotes that impacts gene regulation and disease pathogenesis. Recently, 5-hydroxymethylcytosine (5-hmC), another form of DNA modification, has been identified in substantial amounts in certain mammalian cell types; however, its roles as well as its distribution in mammalian genomes are unknown. Here we present a selective chemical labeling method for 5-hmC by utilizing T4 bacteriophage BGT-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC, which in turn can chemically incorporate a biotin group for detection, affinity enrichment, and sequencing of 5-hmC in mammalian genomes. Using this highly effective method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized. We also find a gene expression level-dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders Identification of 5hmC enriched genmoic regions in mouse cerebellum