Chromosome Analysis: Methods and Protocols (Methods in Molecular Biology, 2519)

✍ Scribed by Eisuke Gotoh (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 195
• Edition: 1st ed. 2023
• Category: Library

✦ Synopsis

This volume provides essential and fundamental protocols on manipulation chromosome. Chapters details methods on the preparation of mitotic chromosome, chromosome aberration, micronucleus (MN), comet assay, karyotyping, Fluorescent in situ hybridization (FISH), premature chromosome condensation (PCC), immunohistochemistry (IHC) staining, new generation sequencing technology and new chromosome concepts, such as epigenetic and its cause of cancer are presented. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

 

Authoritative and cutting-edge, Chromosome Analysis: Methods and Protocols aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge. 

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: A Basic and Simple Chromosome Preparation Protocol
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 2: Metaphase Chromosome Preparation and Classification of Chromosomal Aberrations
1 Introduction
2 Materials and Equipment
2.1 Materials
2.2 Equipment
3 Methods
3.1 Metaphase Chromosome Preparation
3.2 Analysis of Chromosomal Aberrations
4 Notes
References
Chapter 3: Mitotic Index Analysis
1 Introduction
2 Materials
2.1 Cell Culture Solutions, Cell Treatment Buffer
2.2 Equipment
3 Methods
3.1 Mitotic Index Analysis with Simple Metaphase Spreads
3.2 pHH3 Immunocytochemistry Analysis with Fluorescence Microscope
3.3 pHH3 Imaging or Flow Cytometer Analysis for High-Throughput Analysis
3.4 Time Lapse Image Analysis for Mitosis Event at Single Cell Level
4 Note
References
Chapter 4: Preparation of Mitotic Cells for Fluorescence Microscopy
1 Introduction
2 Materials
2.1 Solutions
2.2 Tools for Immunofluorescence Staining
2.3 Reagent for Synchronization of Cell Cycle
3 Methods
3.1 Immunostaining of Cells Fixed on Coverslip
3.2 Cell Extraction Before the Fixation
3.2.1 Extraction of the Cells During the Fixation
3.2.2 Extraction of the Cells Before the Fixation
3.2.3 Hypotonic Treatment of Cells and Cytocentrifugation
3.3 Coating of Coverslip with Reagents to Assist the Attachment of Cells
3.3.1 Coating Coverslip with Fibronectin
3.3.2 Coating of Coverslip with Cell-Tak
3.4 Synchronization of Cell Cycle and Accumulation of Mitotic Cells
3.4.1 Thymidine Block and Release
3.4.2 Prometaphase Arrest with Nocodazole or Taxol
3.4.3 Metaphase Arrest with MG132
4 Notes
References
Chapter 5: Chemical-Induced Premature Chromosome Condensation Protocol
1 Introduction
2 Materials
3 Methods
4 Notes
References
6: Apoptosis Detection Assays
1 Introduction
1.1 Apoptosis and Cell Death
1.2 Annexin V
1.3 DNA Condensation
1.4 TUNEL Assay
2 Materials and Equipment
2.1 Materials
2.2 Equipment
3 Methods
3.1 Annexin PI Method
3.2 Slide Preparation for DNA Condensation Analysis and TUNEL Stain
3.3 DNA Condensation Analysis
3.4 TUNEL Assay
4 Note
References
Chapter 7: Alkaline Comet Assay to Detect DNA Damage
1 Introduction
2 Materials and Equipment
2.1 Materials
2.2 Equipment
3 Methods
3.1 Slide Preparation Pre-Etching and Coating
3.2 Alkaline Lysis and Electrophoresis
3.3 Data Analysis
4 Notes
References
Chapter 8: Analysis for Sister Chromatid Exchange
1 Introduction
2 Materials and Equipment
2.1 Materials
2.2 Equipment
3 Methods
3.1 Cell Culture
3.2 Staining
3.2.1 Fluorescence Plus Giemsa (FPG) Staining
3.3 BrdU or EdU Uptake for Fluorescent Images
3.4 BrdU Antibody Fluorescent Immunostaining
3.5 Click Reaction Staining for EdU
3.6 Analysis of SCE
4 Note
References
Chapter 9: Cytokinesis Blocked Micronuclei Aberration Analysis
1 Introduction
2 Materials and Equipment
2.1 Materials
2.2 Equipment
3 Methods
3.1 The Standard Micronuclei Formation and Giemsa Staining
3.2 The Micronuclei Staining with Centromere Staining
3.3 Micronuclei Staining with Immunocytochemistry
4 Notes
References
Chapter 10: DNA Damage Foci on Metaphase Chromosomes
1 Introduction
2 Materials and Equipment
2.1 Materials
2.2 Equipment
3 Methods
3.1 DNA Damage-Induced Foci Metaphase by Cytocentrifugation and Fixation
3.2 Staining and Analysis
4 Notes
References
Chapter 11: FISH with Whole Chromosome Painting Probes
1 Introduction
2 Materials and Equipment
2.1 Materials
2.2 Equipment
3 Methods
3.1 The Standard Metaphase Chromosome Spread Preparation
3.2 Whole Chromosome Painting
4 Notes
References
Chapter 12: Telomere Aberration Detection by PNA FISH Probe
1 Introduction
2 Materials and Equipment
2.1 Materials
2.2 Equipment
3 Methods
3.1 The Standard Metaphase Chromosome Spread Preparation
3.2 Telomere Staining
4 Notes
References
Chapter 13: Nontraditional Method for Telomere Staining by PNA Probes
1 Introduction
2 Materials and Equipment
2.1 Materials
2.2 Equipment
3 Methods
3.1 The Standard Metaphase Chromosome Spread Preparation
3.2 Telomere Staining
4 Notes
References
Chapter 14: Visualizing Active Replication Regions in S-Phase Chromosomes
1 Introduction
2 Materials
3 Methods
3.1 Cell Culture and Cy3-dUTP Labeling by a Bead Loading Method
3.2 Premature Chromosome Condensation
3.3 Laser Scanning Microscopy
3.4 Brief Result
4 Notes
References
Chapter 15: Hi-C Analysis to Identify Genome-Wide Chromatin Structural Aberration in Cancer
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Crosslinking
3.2 Digestion
3.3 Marking of DNA Ends and Proximity Ligation
3.4 Reverse Crosslink, DNA Purification, DNA Shearing, and Size Selection
3.5 Biotin Pull-Down and Preparation for Illumina Sequencing
3.6 Final Amplification and Purification
3.7 Data Analysis
4 Notes
References
Chapter 16: CUT
1 Introduction
2 Materials
2.1 Preparation of Con A-Conjugated Dynabeads
2.2 Preparation of Tn5 Adapter Complex
2.3 CUT&Tag
3 Methods
3.1 Preparation of Con A-Conjugated Dynabeads
3.2 pA-Tn5 Transposase Purification
3.3 pA-Tn5 Adapter Transposome Preparation
3.4 CUT&Tag
3.5 PCR Amplification
4 Notes
References
Chapter 17: Live Cell Synthetic Histone Acetylation by Chemical Catalyst
1 Introduction
2 Materials
2.1 Synthetic Histone Acetylation Reaction in Living Cells
2.2 Purification of Histones After Histone Acetylation Reaction
2.3 Quantification of Acetylation Yield Using LC-MS/MS
3 Methods
3.1 Synthetic Histone Acetylation Reaction in Living Cells
3.2 Purification of Histones After Histone Acetylation Reaction
3.3 Quantification of Acetylation Yield Using LC-MS/MS
4 Notes
References
Chapter 18: Histone Modification Analysis of Low-Mappability Regions
1 Introduction
2 Materials
2.1 Design of ChIP Primers
2.2 Cell Culture and Preparation of Single Cell Suspension from Specimens
2.3 Stock Solutions
2.4 ChIP (Day 1)
2.5 ChIP (Day 2)
2.6 Quantitative Real-Time PCR (qPCR)
3 Methods
3.1 Design of ChIP Primers
3.1.1 Retrieving Genomic Sequences
3.1.2 Multiple Alignments, Homology Search and Selecting a Prototype of Family Genes
3.1.3 Selection of Primer Target Sites
3.1.4 Validation of ChIP Primers
3.2 Cell Culture and Preparation of Single Cell Suspension from Specimens
3.3 ChIP (Day 1)
3.4 ChIP (Day 2)
3.5 Real-Time PCR
4 Notes
References
Index