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Full Description
This book provides an overview of methods and experimental protocols that are currently used to analyze the presence and abundance of non-canonical DNA nucleotides in different biological systems. Focusing particularly on the newly discovered and less studied DNA modifications that are enzymatically produced and are likely to play specific roles in various biological processes, the volume explores chromatography- and mass spectrometry-based techniques for the detection and quantification of DNA modifications, antibody-based approaches to study their spatial distribution in different cells and tissues, and methods to analyze their genomic distribution with the help of bioinformatics tools that interrogate the corresponding datasets. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and comprehensive, DNA Modifications: Methods and Protocols serves as an ideal guide to research scientists and PhD students in this rapidly developing discipline, and, thus, will ultimately contribute to deciphering the roles of non-canonical DNA nucleotides in different biological systems.
Contents
Modified Forms of Cytosine in Eukaryotes: DNA (De)Methylation and Beyond.- Evidence for Non-Cytosine Epigenetic DNA Modifications in Multicellular Eukaryotes: An Overview.- Mass Spectrometry-Based Analysis of DNA Modifications: Potential Applications in Basic Research and Clinic.- Mapping DNA Methylation in Mammals: The State of the Art.- Detection of Modified Bases in Bacteriophage Genomic DNA.- Liquid Chromatography-Mass Spectrometry Analysis of Cytosine Modifications.- Detection of DNA Methylation in Genomic DNA by UHPLC-MS/MS.- Quantification of DNA Modifications Using Two-Dimensional Ultra-Performance Liquid Chromatography Tandem Mass-Spectrometry (2D-UPLC-MS/MS).- MS Analysis of DNA Modifications in Urinary/Body Fluids.- Preparation of Internal Standards for 2D-UPLC-MS/MS Quantification of Non-Canonical DNA Bases.- Detection of TET-Oxidized 5-Methylcytosine Bases by Capillary Gel Electrophoresis.- Immunochemical Detection of Modified Cytosine Species in Mammalian PreimplantationEmbryos.- Immunochemical Detection of Modified Cytosine Species in Lampbrush Chromatin.- Detection of Low-Abundance DNA Modifications Using Signal Amplification-Based Immunocytochemistry.- Immunohistochemical Detection of Modified Cytosine Bases in Rodent Brain.- Immunohistochemical Detection of 5-Hydroxymethylcytosine and 5-Carboxylcytosine in Sections of Zebrafish Embryos.- Immunochemical Detection of Modified Species of Cytosine in Plant Tissues.- Antigen Retrieval for Immunostaining of Modified Cytosine Species.- Computational Analysis of DNA Modifications in Confocal Images.- Three-Dimensional Confocal Analysis of Chromosome Positioning Coupled with Immunofluorescence in Mouse Sperm Nuclei.- Analysis of 5-Hydroxymethyluracil Levels Using Flow Cytometry.- Hairpin-Bisulfite PCR.- Genome-Wide Mapping of DNA Methylation 5mC by Methylated DNA Immunoprecipitation (MeDIP)-Sequencing.- Analysis of 5-Carboxylcytosine Distribution Using DNA Immunoprecipitation.- High Resolution Analysis of5-Hydroxymethylcytosine by TET-Assisted Bisulfite Sequencing.- Oxidative Bisulfite Sequencing: An Experimental and Computational Protocol.- Bisulfite-Free Sequencing of 5-Hydroxymethylcytosine with APOBEC-Coupled Epigenetic Sequencing (ACE-Seq).- Single-Nucleotide-Resolution Sequencing of N6-Methyldeoxyadenosine.- Design and Application of DNA Modification-Specific Transcription-Activator-Like Effectors.- Notes on Functional Modules in the Assembly of CRISPR/Cas9-Mediated Epigenetic Modifiers.- Analyzing DNA-Immunoprecipitation Sequencing Data.- Bioinformatics Analysis of DNA Methylation through Bisulfite Sequencing Data.- Unified Analysis of Multiple ChIP-Seq Datasets.- Integrated Analysis of DNA Methylation, Hydroxymethylation, and Gene Expression Data Using ME-Class2.
