Full Description
This detailed book contains techniques to explore the unusual properties of the peculiar microsatellites known as trinucleotide repeats. Beginning with technical challenges raised by secondary structure-forming repeats and their propensity to contract and expand over time, the book continues with genetic screens in Saccharomyces cerevisiae, experimental systems to study trinucleotide repeat instability in human cells, dedicated experimental systems in appropriate cell types, as well as gene therapy approaches using the CRISPR-Cas family of endonucleases. 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 practical, Trinucleotide Repeats: Methods and Protocols serves as avaluable aid to experts and newcomers alike who seek to investigate this fascinating and ever-expanding field of study.
Contents
Experimenting with Trinucleotide Repeats: Facts and Technical Issues.- Fast Assays to Detect Interruptions in CTG.CAG Repeat Expansions.- Tracking Expansions of Stable and Threshold Length Trinucleotide Repeat Tracts In Vivo and In Vitro Using Saccharomyces cerevisiae.- Quantifying Replication Fork Progression at CTG Repeats by 2D Gel Electrophoresis.- Genetic Assays to Study Repeat Fragility in Saccharomyces cerevisiae.- Genetic Screens to Study GAA/TTC and Inverted Repeat Instability in Saccharomyces cerevisiae.- Monitoring Double-Strand Break Repair of Trinucleotide Repeats Using a Yeast Fluorescent Reporter Assay.- Analysis of Trinucleotide Repeat Stability by Integration at a Chromosomal Ectopic Site.- Experimental System to Study Instability of (CGG)n Repeats in Cultured Mammalian Cells.- Assessing Triplet Repeat Expansions in Human SVG-A Cell Culture.- The Isolation and Analysis of the CGG-Repeat Size in Male and Female Gametes from a Fragile X Mouse Model.- In Vitro Synthesis and RNA Structure Probing of CUG Triplet Repeat RNA.- FISH Protocol for Myotonic Dystrophy Type 1 Cells.- Real Time Videomicroscopy and Semi-Automated Analysis of Brain Cell Culture Models of Trinucleotide Repeat Expansion Diseases.- Primary Cultures of Pure Embryonic Dorsal Root Ganglia Sensory Neurons as a New Cellular Model for Friedreich's Ataxia.- GFP Reporters to Monitor Instability and Expression of Expanded CAG/CTG Repeats.- Gene Therapy for Huntington's Disease Using Targeted Endonucleases.
