Trinucleotide Repeat Protocols

Trinucleotide Repeat Protocols

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Trinucleotide repeats are relatively common in the human genome. These simple repeats have received much attention since epoch-making discoveries were made that particular trinucleotide repeats are expanded in the causal genes of human hereditary neurological disorders. For example, the CGG repeat is expanded in fragile X syndrome at the 5' untranslated region (UTR) of its causal gene. In myotonic dystrophy, it is the CTG repeat that is expanded at the 3' UTR of its causal gene. The CAG repeat was also found expanded in coding regions of the genes responsible for X-linked spinal and bulbar muscular atrophy, Huntington's disease, spinocerebellar ataxia, and other disorders. On the other hand, expansion of the GAA repeat was identified in the intron of the gene responsible for the Friedreich's ataxia. For these trinucleotide repeat diseases, the longer the trinucleotide expansion, the earlier the age of onset and the more severe the syndrome. Thus, these findings that showed the intriguing link between a particular trinucleotide expansion and its associated neurological disorders have led to a new field of intensive study. Active research addressing the underlying mechanisms for trinucleotide repeat diseases has employed various approaches ranging from DNA biochemistry to animal models for the diseases. In particular, animal models for the triplet repeat diseases have provided excellent resources not only for understanding the mechanisms but also for exploring therapeutic interventions.
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Product details

  • Hardback | 342 pages
  • 152.4 x 233.7 x 25.4mm | 703.08g
  • Totowa, NJ, United States
  • English
  • 2004 ed.
  • XII, 342 p.
  • 1588292436
  • 9781588292438

Back cover copy

The discovery that trinucleotide repeats significantly influence the age of onset and severity of a variety of hereditary neurological disorders has opened the door to a deeper understanding of the disease mechanisms involved, as well as to a more productive search for novel therapeutic interventions. In Trinucleotide Repeat Protocols, established leaders in trinucleotide repeat disease describe in step-by-step detail their best techniques for studying trinucleotide pathology at the molecular level. The protocols cover a variety of targets, ranging from DNA and RNA to proteins and whole animals, and focus not only on causal genes, but also on their consequent products, such as transcription factors, neurotransmitter receptors, proteasomes, and mitochondria/oxidation damage. Experimental systems employed include E. coli, yeast, C. elegans, mouse, and generally take a clinical point of view. The authors utilize a wide range of techniques, including gel electrophoresis, quantitative RT-PCR, immunological analysis, antibody usage and its applications, receptor assays using radioisotope handling, gene delivery by virus, brain cell and organotypic cultures, gender dependency, and neuron structure analysis. Each protocol follows the successful Methods in Molecular Biology(TM) series format, offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Cutting-edge and highly practical, Trinucleotide Repeat Protocols offers neuroscientists powerful tools to elucidate both normal brain function and the mechanisms of hereditary neurological disease, as well as to develop the next generation of therapies for neuronal genetic diseases.
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Table of contents

Part I. Introduction

Mouse Models of Triplet Repeat Diseases
Gillian P. Bates and David G. Hay

Part II. Analysis of Triplet Repeat DNAs and RNAs

Analysis of Triplet Repeat Replication by Two-Dimensional Gel Electrophoresis
Maria M. Krasilnikova and Sergei M. Mirkin

Genetic Analysis for Triplet Repeat Instability in Yeast
Michael J. Dixon, Saumitri Bhattacharyya, and Robert S. Lahue

Detection and Isolation of Trinucleotide Repeat Expansions Using the RED Method
Qiu-Ping Yuan and Martin Schalling

Analysis of Unstable Triplet Repeats Using Small-Pool Polymerase Chain Reaction
Mario Gomes-Pereira, Sanjay I. Bidichandani, and Darren G. Monckton

Real-Time RT-PCR for CTG Repeat-Containing Genes
Maria Eriksson

Part III. Detection and Analysis of Polyglutamine-Containing Proteins and Their Aggregates

Antibodies Against Huntingtin: Production and Screening of Monoclonals and Single-Chain Recombinant Forms
Ali Khoshnan, Susan Ou, Jan Ko, and Paul H. Patterson

Using Antibodies to Analyze Polyglutamine Stretches
Elizabeth Brooks, Montserrat Arrasate, Kenneth Cheung, and Steven M. Finkbeiner

Solubilization of Aggregates Formed by Expanded Polyglutamine Tract Expression in Cultured Cells
Noriko Hazeki and Ichiro Kanazawa

Part IV. Establishment of Animal and Cultured Cell Models for Trinucleotide Repeat Diseases

Caenorhabditis elegans as a Model System for Triplet Repeat Diseases
Cindy Voisine and Anne C. Hart

Monitoring Aggregate Formation in Organotypic Slice Cultures From Transgenic Mice
Donna L. Smith and Gillian P. Bates

The CGG Repeat and the FMR1 Gene
Violeta Stoyanova and Ben A. Oostra

Analysis of CTG Repeats Using DM1 Model Mice
Cedric Savouret, Claudine Junien, and Genevieve Gourdon

Lentiviral-Mediated Gene Transfer toModel Triplet Repeat Disorders
Etienne Regulier, Diana Zala, Patrick Aebischer, and Nicole Deglon

Mouse Tissue Culture Models of Unstable Triplet Repeats
Mario Gomes-Pereira and Darren G. Monckton

Part V. In Vivo Analysis of Trinucleotide Repeat Diseases

Neurotransmitter Receptor Analysis in Transgenic Mouse Models
Caroline L. Benn, Laurie A. Farrell, and Jang-Ho J. Cha

Chromatin Immunoprecipitation Technique for Study of Transcriptional Dysregulation in Intact Mouse Brain
Melissa W. Braveman, Alice S. Chen-Plotkin, George J. Yohrling, and Jang-Ho J. Cha

Techniques for Thick-Section Golgi Impregnation of Formalin-Fixed Brain Tissue
Tracie L. Moss and William O. Whetsell, Jr.

Assessment of Impaired Proteasomal Function in a Cellular Model of Polyglutamine Diseases
Nihar Ranjan Jana and Nobuyuki Nukina

Assessment of In Vitro and In Vivo Mitochondrial Function in Friedreich's Ataxia and Huntington's Disease
Anthony Schapira and Raffaele Lodi

Triplet Repeats and DNA Repair: Germ Cell and Somatic Cell Instability in Transgenic Mice
Irina V. Kovtun, Craig Spiro, and Cynthia T. McMurray

Oxidative Damage in Huntington's Disease
Jose Segovia and Francisca Perez-Severiano

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