DNA Modification Detection Methods (Springer Protocols Handbooks)

✍ Scribed by Bi-Feng Yuan (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 189
• Category: Library

✦ Synopsis

Understanding the functional roles of DNA modifications relies on the accurate detection, quantification, and mapping of DNA modifications. Methods for deciphering DNA modifications have substantially improved over the last several years, which greatly revolutionize the field of DNA modifications. In addition to DNA cytosine methylation (5-methylcytosine, 5mC), the best-characterized epigenetic modification, many new modifications have been discovered to present in DNA in recent years.

This book provides a comprehensive overview of available techniques and methods together with detailed step-by-step protocols for experimental procedures required to successfully perform analysis on various types of DNA modifications, including 5mC, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxycytosine (5caC), 5-hydroxymethyluracil (5hmU), 5-formyluracil (5fU), N6-methyladenine (6mA), β-D-glucosyl-5-hydroxymethyluracil (base J) and 8-oxo-7,8-dihydroguanine (OG). This laboratory manual is a valuable source for biochemists and molecular biologists from different fields who wish to investigate DNA modifications.

✦ Table of Contents

Contents
Part I: Detection of 5mC, 5hmC, 5fC, and 5caC
Chapter 1: Quantitative Assessment of the Oxidation Products of 5-Methylcytosine in DNA by Liquid Chromatography-Tandem Mass S...
1 Introduction
2 Materials
2.1 DNA Extraction
2.1.1 Enzymes
2.1.2 Chemicals and Solutions
2.1.3 Equipment
2.2 Enzymatic Digestion
2.2.1 Enzymes
2.2.2 Chemicals and Solutions
2.3 HPLC Enrichment
2.3.1 Chemicals and Solutions
2.3.2 Equipment
2.4 Quantification with LC-MS/MS/MS
2.4.1 Chemicals and Solutions
2.4.2 Equipment
3 Method
3.1 DNA Extraction
3.2 Enzymatic Digestion
3.3 HPLC Enrichment
3.4 Quantification with LC-MS/MS/MS
3.5 Data Analysis
4 Notes
References
Chapter 2: Determination of Cytosine Modifications in DNA by Chemical Labeling-Mass Spectrometry Analysis
1 Introduction
2 Materials
2.1 Enzymes
2.2 Stock Solution of Standards
2.3 Chemicals and Reagents
2.4 Equipment
3 Methods
3.1 DNA Extraction
3.2 Enzymatic Digestion of DNA
3.3 Remove Salts in Digestion Mixture with Graphitized Carbon Black
3.4 Chemical Labeling
3.5 Determination of the Derivatives of 5-mdC, 5-hmdC, 5-fdC, and 5-cadC by LC-ESI-MS/MS
3.6 Construction of Calibration Curve
3.7 Simultaneous Determination of 5-mdC, 5-hmdC, 5-fdC, and 5-cadC in Genomic DNA of Human CRC Tissues
4 Notes
References
Chapter 3: Analysis of 5-Methylcytosine and 5-Hydroxymethylcytosine in Genomic DNA by Capillary Electrophoresis-Mass Spectrome...
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Equipment
2.3 Software
2.4 Enzymes and Kit
3 Methods
3.1 Extraction and Purification of DNA
3.2 DNA Digestion
3.3 CESI-MS/MS Analysis
4 Notes
References
Chapter 4: Simple Quantification of Epigenetic DNA Modifications and DNA Damage on Multi-Well Slides
1 Introduction
2 Materials
2.1 General Reagents
2.2 Kits for DNA Extraction and Purification
2.3 Reagents for Slides Preparation
2.4 Reagents for Unmodified CpGs Labeling
2.5 Reagents for 5-hmC Labeling
2.6 Reagents for Labeling Oxidation Damage and UV-Induced Damage
3 Methods
3.1 Summary of the Workflow
3.2 Preparation of Multi-Well Slides
3.3 Labeling of Unmodified CpGs
3.4 Labeling of 5-hmC
3.5 Labeling of Oxidation Damage
3.6 Labeling of UV-Induced DNA Damage
3.7 Preparation of Negative Control and Calibration Samples
3.7.1 Calibration Samples
3.7.2 Control Samples
3.8 Loading of Labeled DNA on Slides
3.9 Imaging of Fluorescent Labels
3.10 Total DNA Quantification
3.11 Imaging of Total DNA
3.12 Analysis
4 Notes
References
Chapter 5: A Label-Free Electrochemical Biosensor for Sensitive Detection of 5-Hydroxymethylcytosine
1 Introduction
2 Materials
2.1 Solutions
2.2 Equipment
2.3 Enzymes, Kit, and Other Materials
3 Methods
3.1 Preparation of Biotinylated 5-hmC DNA
3.2 Preparation of MBs Linking 5-hmC DNA
3.3 Electrochemical Measurements
3.4 Isolation of DNA from Cell Lines
3.5 Real Sample Analysis
4 Notes
References
Chapter 6: Electrochemical Assay for Continuous Monitoring of Dynamic DNA Methylation Process
1 Introduction
2 Materials
2.1 Reagents
2.2 Solutions
2.3 Equipment
3 Methods
3.1 Electrochemical Label Preparation
3.2 Electrochemical Sensor Construction
3.3 Pause Point: Pretreatment of the Gold Electrode
3.3.1 Pause Point: Store at -20 C for Further Use or Use Immediately
3.4 DNA Methylation and Bromine Derivatization
3.5 Continuous Measurements
4 Notes
References
Chapter 7: Electrogenerated Chemiluminescence Method for Determination of 5-Hydroxymethylcytosine in DNA
1 Introduction
2 Materials
2.1 Solutions
2.2 Equipment
2.3 DNA Sequence
3 Methods
3.1 Oxidation of DNA by KRuO4
3.2 ABEI Labeling of 5-fC DNA
3.3 Characteristic of ABEI-Labeled 5-fC-Containing DNA
3.4 Polyacrylamide Gel Electrophoresis (PAGE)
3.5 Fabrication of ss-DNA-Modified Gold Electrode
3.6 Electrochemical Measurement
3.7 ECL Measurement
4 Notes
References
Chapter 8: Quantification of Site-Specific 5-Formylcytosine by Integrating Peptide Nucleic Acid-Clamped Ligation with Loop-Med...
1 Introduction
2 Materials
2.1 Specific Chemical Conversion of 5fC to 5fC-M
2.1.1 Sequence Information
2.1.2 Reagents and Solution
2.1.3 Equipment
2.2 Distinguish 5fC from Its Analogues by PNA-Clamped Ligation Reaction
2.2.1 Sequence Information
2.2.2 Reagents and Solution
2.2.3 Equipment and Consumables
2.3 Signal Amplification by LAMP
2.3.1 Reagents and Solution
2.3.2 Equipment and Consumables
2.4 Polyacrylamide Gel Electrophoresis (PAGE) Analysis
2.4.1 Reagents and Equipment
3 Methods
3.1 Malononitrile-Based 5fC-Specific Conversion Reaction
3.2 PNA-Clamped Ligation Reaction
3.3 LAMP Reaction
3.4 PAGE Characterization of the Amplification Products of ODN-5fC and ODN-X
3.5 Quantitative Analysis of 5fC
3.5.1 Quantitative Analysis of 5fC in ODN-5fC and dsDNA-5fC
3.5.2 Quantitative Analysis 5fC in Mouse Brain Tissue Genomic DNA
4 Note
References
Chapter 9: Global DNA Methylation Analysis Using Methylcytosine Dioxygenase
1 Introduction
2 Materials
2.1 Protein Expression
2.1.1 Equipment
2.1.2 Reagents and Consumables
2.2 Quantification of Succinate
2.2.1 Equipment
2.2.2 Reagents and Consumables
3 Methods
3.1 TET2 Expression Vector
3.2 Preparation of TET2
3.2.1 Protein Expression
3.2.2 Protein Purification
3.3 Global DNA Methylation Analysis by TET2
4 Notes
References
Part II: Detection of 6mA
Chapter 10: Metabolically Generated Stable Isotope for Identification of DNA N6-Methyladenine Origin in Cultured Mammalian Cel...
1 Introduction
2 Materials
2.1 Cell Culture and Cell Proliferation Detection
2.2 Mycoplasma Detection
2.3 DNA Extraction
2.4 DNA Digestion and Ultrafiltration
2.5 UHPLC-MS/MS Analysis
3 Methods
3.1 Cell Culture of HEK293T Cells
3.2 Detection of Mycoplasma in Cell Culture
3.3 The Toxicity of Deoxyadenine for Cultured Cells
3.4 Cell Treatment Using Appropriate Concentration of 15N5-dA
3.5 Genomic DNA Preparation
3.6 DNA Digestion and Sample Handling for UHPLC-MS/MS Analysis
3.7 UHPLC-MS/MS Analysis of 15N5-6mA, 15N4-6mA, Unlabled 6mA, 15N5-dA, 15N4-dA, and Unlabled dA
4 Notes
References
Chapter 11: N6-\Methyladenine in DNA of Mammals and Plants by?>Determination of N6-Methyladenine in DNA of Mammals and Plants ...
1 Introduction
2 Materials
2.1 Enzymes
2.2 Stock Solutions of Standards
2.3 Chemicals and Reagents
2.4 Biological Samples
2.5 Equipment
3 Methods
3.1 DNA Extraction
3.2 Dpn I Digestion and Size-Exclusion Ultrafiltration
3.3 Enzymatic Digestion of DNA
3.4 Analysis of Nucleosides by LC-MS/MS
3.5 Construction of Calibration Curve
3.6 Determination of 6mA in Genomic DNA of Mammals and Plants
4 Notes
References
Part III: Detection of 5hmU and 5fU
Chapter 12: Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry for Detection of 5-Hydroxymethyluracil and 5-Formy...
1 Introduction
2 Materials
2.1 Enzymes
2.2 Buffers and Other Reagents
2.3 Liquid Chromatography
2.4 Nucleoside Standards
2.5 Equipment
3 Methods
3.1 Extraction of Genomic DNA
3.2 Enzymatic Digestion of Genomic DNA
3.3 Off-line HPLC Enrichment
3.4 LC-MS3 Analysis
4 Notes
References
Chapter 13: Detection of 5-Formylcytosine and 5-Formyluracil Based on Photo-Assisted Domino Reaction
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Equipment
3 Methods
3.1 Optimization of Derivatization Conditions for 5fC by YC-CN (see Note 1)
3.1.1 Optimization of Wittig Olefination Conditions
3.1.2 Optimization of Photocatalytic Reaction Time
3.2 Verify the Specificity of YC-CN for 5fC
3.3 Analyze 5fC Content in DNA Samples
4 Notes
References
Chapter 14: Detection of 5-Formyluracil and 5-Formylcytosine in DNA by Fluorescence Labeling
1 Introduction
2 Materials
3 Methods
3.1 Fluorescence Labeling Method for Detection of 5-Formylcytosine
3.2 Fluorescence Labeling Method for Detection of 5-Formyluracil
4 Notes
References
Part IV: Detection of Base J and 8-oxo-7,8-dihydroguanine
Chapter 15: Mass Spectrometry-Based Quantification of β-d-Glucosyl-5-Hydroxymethyluracil in Genomic DNA
1 Introduction
2 Materials
2.1 Solutions
2.2 DNA Oligomers and Enzymes
2.3 Columns
2.4 Equipments
2.5 Software
3 Methods
3.1 Preparation of Surrogate Internal Standard
3.1.1 Synthesis and Purification
3.1.2 Verify the Identity of the Purified ODNs by LC-MS/MS
3.2 DNA Extraction
3.3 Enzymatic Digestion of Genomic DNA
3.4 Off-Line HPLC Enrichment
3.5 Quantitative Analysis of Base J
3.5.1 LC-MS/MS Analysis of dJ
3.5.2 Calibration Curve Construction for dJ Quantification
3.6 Quantitative Analysis of 5-HmdU
4 Notes
References
Chapter 16: Determination of 8-Oxo-7,8-Dihydroguanine in DNA at Single-Base Resolution by Polymerase-Mediated Differential Cod...
1 Introduction
2 Materials
2.1 Oligonucleotides
2.2 Chemicals and Reagents
2.3 Equipment
3 Methods
3.1 Quantitative Analysis of 8OG in DNA
3.1.1 Steady-State Kinetics Study
3.1.2 Quantitative Evaluation of OG in DNA via Primer Extension
3.1.3 Analysis of 8OG in Telomeric DNA from HeLa Cells
3.2 Analysis of OG by Sequencing
3.2.1 Analysis of OG in Synthesized DNA by Sanger Sequencing
3.2.2 Sanger Sequencing Analysis of OG in Genomic DNA
4 Notes
References