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DNA Methylation Protocols

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Cover of 'DNA Methylation Protocols'

Table of Contents

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    Book Overview
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    Chapter 1 A Summary of the Biological Processes, Disease-Associated Changes, and Clinical Applications of DNA Methylation
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    Chapter 2 Considerations for Design and Analysis of DNA Methylation Studies
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    Chapter 3 Quantification of Global DNA Methylation Levels by Mass Spectrometry
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    Chapter 4 Antibody-Based Detection of Global Nuclear DNA Methylation in Cells, Tissue Sections, and Mammalian Embryos
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    Chapter 5 Whole-Genome Bisulfite Sequencing Using the Ovation® Ultralow Methyl-Seq Protocol
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    Chapter 6 Tagmentation-Based Library Preparation for Low DNA Input Whole Genome Bisulfite Sequencing
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    Chapter 7 Post-Bisulfite Adaptor Tagging for PCR-Free Whole-Genome Bisulfite Sequencing
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    Chapter 8 Multiplexed Reduced Representation Bisulfite Sequencing with Magnetic Bead Fragment Size Selection
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    Chapter 9 Low Input Whole-Genome Bisulfite Sequencing Using a Post-Bisulfite Adapter Tagging Approach
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    Chapter 10 Methyl-CpG-Binding Domain Sequencing: MBD-seq
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    Chapter 11 The HELP-Based DNA Methylation Assays
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    Chapter 12 Comprehensive Whole DNA Methylome Analysis by Integrating MeDIP-seq and MRE-seq
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    Chapter 13 Digital Restriction Enzyme Analysis of Methylation (DREAM)
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    Chapter 14 Nucleosome Occupancy and Methylome Sequencing (NOMe-seq)
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    Chapter 15 Bisulphite Sequencing of Chromatin Immunoprecipitated DNA (BisChIP-seq)
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    Chapter 16 A Guide to Illumina BeadChip Data Analysis
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    Chapter 17 Microdroplet PCR for Highly Multiplexed Targeted Bisulfite Sequencing
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    Chapter 18 Multiplexed DNA Methylation Analysis of Target Regions Using Microfluidics (Fluidigm)
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    Chapter 19 Large-Scale Targeted DNA Methylation Analysis Using Bisulfite Padlock Probes
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    Chapter 20 Targeted Bisulfite Sequencing Using the SeqCap Epi Enrichment System
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    Chapter 21 Multiplexed and Sensitive DNA Methylation Testing Using Methylation-Sensitive Restriction Enzymes “MSRE-qPCR”
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    Chapter 22 Quantitative DNA Methylation Analysis at Single-Nucleotide Resolution by Pyrosequencing®
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    Chapter 23 Methylation-Specific PCR
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    Chapter 24 Quantitation of DNA Methylation by Quantitative Multiplex Methylation-Specific PCR (QM-MSP) Assay
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    Chapter 25 MethyLight and Digital MethyLight
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    Chapter 26 Quantitative Region-Specific DNA Methylation Analysis by the EpiTYPER™ Technology
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    Chapter 27 Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA)
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    Chapter 28 Methylation-Sensitive High Resolution Melting (MS-HRM)
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    Chapter 29 Hairpin Bisulfite Sequencing: Synchronous Methylation Analysis on Complementary DNA Strands of Individual Chromosomes
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    Chapter 30 Helper-Dependent Chain Reaction (HDCR) for Selective Amplification of Methylated DNA Sequences
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    Chapter 31 DNA Methylation Analysis from Blood Spots: Increasing Yield and Quality for Genome-Wide and Locus-Specific Methylation Analysis
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    Chapter 32 DNA Methylation Analysis of Free-Circulating DNA in Body Fluids
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    Chapter 33 Tet-Assisted Bisulfite Sequencing (TAB-seq)
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    Chapter 34 Multiplexing for Oxidative Bisulfite Sequencing (oxBS-seq)
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    Chapter 35 Affinity-Based Enrichment Techniques for the Genome-Wide Analysis of 5-Hydroxymethylcytosine
Attention for Chapter 25: MethyLight and Digital MethyLight
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Chapter title
MethyLight and Digital MethyLight
Chapter number 25
Book title
DNA Methylation Protocols
Published in
Methods in molecular biology, January 2018
DOI 10.1007/978-1-4939-7481-8_25
Pubmed ID
Book ISBNs
978-1-4939-7479-5, 978-1-4939-7481-8
Authors

Mihaela Campan, Daniel J. Weisenberger, Binh Trinh, Peter W. Laird

Abstract

MethyLight is a quantitative, fluorescence-based, real-time PCR method to sensitively detect and quantify DNA methylation of candidate regions of the genome. MethyLight is uniquely suited for detecting low-frequency methylated DNA regions against a high background of unmethylated DNA, as it combines methylation-specific priming with methylation-specific fluorescent probing. The quantitative accuracy of real-time PCR and the ability to design bisulfite-dependent, DNA methylation-independent control reactions together allow for a quantitative assessment of these low frequency methylation events. Here we describe the experimental steps of MethyLight analysis in detail. Furthermore, we present principles and design examples for three types of quality control reactions. QC-1 reactions are methylation-independent reactions to monitor sample quantity and integrity. QC-2 reactions are bisulfite-independent reactions to monitor recovery efficiencies of the bisulfite conversion methodology used. QC-3 reactions are bisulfite-independently primed reactions with variable bisulfite-dependent probing to monitor completeness of the sodium bisulfite treatment. We show that these control reactions perform as expected in a time course experiment interrupting sodium bisulfite conversion at various timepoints. Finally, we describe Digital MethyLight, in which MethyLight is combined with Digital PCR, for the highly sensitive detection of individual methylated molecules, with use in disease detection and screening.

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Mendeley readers

The data shown below were compiled from readership statistics for 19 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 19 100%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 3 16%
Researcher 3 16%
Student > Master 3 16%
Student > Postgraduate 2 11%
Lecturer 1 5%
Other 2 11%
Unknown 5 26%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 5 26%
Agricultural and Biological Sciences 3 16%
Medicine and Dentistry 2 11%
Nursing and Health Professions 1 5%
Psychology 1 5%
Other 1 5%
Unknown 6 32%