Trinucleotide Repeat Protocols

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.

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 a valuable aid to experts and newcomers alike who seek to investigate this fascinating and ever-expanding field of study.

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.

PCR Cloning Protocols, Second Edition, updates and expands Bruce White's best-selling PCR Cloning Protocols (1997) with the newest procedures for DNA cloning and mutagenesis. Here the researcher will find readily reproducible methods for all the major aspects of PCR use, including PCR optimization, computer programs for PCR primer design and analysis, and novel variations for cloning genes of special characteristics or origin, with emphasis on long distance PCR and GC-rich template amplification. Also included are both conventional and novel enzyme-free and restriction site-free procedures to clone PCR products into a range of vectors, as well as state-of-the-art protocols to facilitate DNA mutagenesis and recombination, and to clone the challenging uncharacterized DNA flanking a known DNA fragment.

The rapid identification and characterization of genes of neurological relevance holds great potential for offering insight into the diagnosis, management, and und- standing of the pathophysiologic mechanisms of neurological diseases. This volume in the Methods in Molecular BiologyTM series was conceived to highlight many of the contemporary methodological approaches utilized for the characterization of neu- logically relevant gene mutations and their protein products. Although an emphasis has been placed upon descriptions of methodologies with a defined clinical utility, it is hoped that Neurogenetics: Methods and Protocols will appeal not only to clinical laboratory diagnosticians, but also to clinicians, and to biomedical researchers with an interest in advances in disease diagnosis and the functional consequences of neu- logically relevant gene mutations. To meet this challenge, more than 60 authors graciously accepted my invitation to contribute to the 32 chapters of this book. Through their collective commitment and diligence, what has emerged is a comprehensive and timely treatise that covers many methodological aspects of mutation detection and screening, including disc- sions on quantitative PCR, trinucleotide repeat detection, sequence-based mutation detection, molecular detection of imprinted genes, fluorescence in situ hybridization (FISH), in vitro protein expression systems, and studies of protein expression and function. I would like to take this opportunity to formally thank my colleagues for their effort and dedication to this work.

The field of epigenetics has grown exponentially in the past decade, and a steady flow of exciting discoveries in this area has served to move it to the forefront of molecular biology. Although epigenetics may previously have been considered a peripheral science, recent advances have shown considerable progress in unraveling the many mysteries of nontraditional genetic processes. Given the fast pace of epigenetic discoveries and the groundbreaking nature of these developments, a thorough treatment of the methods in the area seems timely and appropriate and is the goal of Epigenetics Protocols. The scope of epigenetics is vast, and an exhaustive analysis of all of the techniques employed by investigators would be unrealistic. However, this TM volume of Methods in Molecular Biology covers three main areas that should be of greatest interest to epigenetics investigators: (1) techniques related to analysis of chromatin remodeling, such as histone acetylation and methylation; (2) methods in newly developed and especially promising areas of epigenetics such as telomere position effects, quantitative epigenetics, and ADP ribosylation; and (3) an updated analysis of techniques involving DNA methylation and its role in the modification, as well as the maintenance, of chromatin structure.

There are a number of outstanding volumes that provide a comprehensive overview of bioconjugation techniques. However, many of the conventional approaches to the synthesis of chemically modified protein conjugates lack efficient means to control the stoichiometry of conjugation, as well as the s- cific site of attachment of the conjugated moiety. Moreover, the recent dev- opments in microarray technologies as well as in nanobiotechnology—a novel field of research rapidly evolving at the crossroads of physics, chemistry, b- technology, and materials science—call for a summary of modern bioconjugation strategies to overcome the limitations of the classical approaches. Bioconjugation Protocols: Methods and Strategies is intended to provide an update of many of the classic techniques and also to introduce and summarize newer approaches that go beyond the pure biomedical applications of bioconjugation. The purpose of Bioconjugation Protocols: Methods and Str- egies is therefore to provide instruction and inspiration for all those scientists confronting the challenges of semisynthesizing functional biomolecular reagents for a wide variety of applications ranging from novel biomedical diagnostics, to therapeutics, to biomaterials. Part I contains seven protocols for the preparation of protein conjugates.

In 1995, Signal Transduction Protocols, edited by David A. Kendall and Stephen J. Hill, was published in the Methods in Molecular Biology series. This second edition represents an update to that previous work with an emp- sis on new methodologies that have developed in the last few years. The goal, then and now, is to provide procedures written by experts with first-hand ex- rience in a detail that goes far beyond what is generally encountered in the “methods” section of most journals and thus actually permits a particular p- cedure to be replicated. In addition, we have had as a secondary goal the id- tification of protocols for the assay of general classes of signal transduction components that, ideally, can be adapted to the assay of any member of that class. The ability to do this has resulted in large part from the use of affini- based assays, the ease with which specific proteins can be specifically tagged, and an explosion in the availability of highly specific antibodies from comm- cial sources, especially antibodies raised against signaling proteins of human origin. The number of available approaches is, fortunately for those working in signaling research, far too great to fit within the confines of this volume, so hard choices as to what to include had to be made.