Pyrosequencing Protocols

This book presents detailed protocols for the multidisciplinary application of Pyrosequencing® technology, all written by world-renowned experts. This comprehensive volume enables quick reference by collecting the primary applications for Pyrosequencing®, and supplementing each protocol with troubleshooting tips specific to that method. This volume both highlights the versatility of and provides detailed protocols for the application of Pyrosequencing®.

The primary purpose of this volume is to demonstrate the range of applications of the Pyrosequencing technology in research and diagnostics and to provide detailed protocols. Beginning with an up-to-date overview of the biochemistry, the volume continues with quantitative analysis of genetic variation, ratio of expressed alleles at the RNA level, analysis of DNA methylation, global DNA methylation assays, specialized applications for DNA methylation analysis including loss of imprinting, single blastocyst analysis, allele-specific DNA methylation patterns, DNA methylation patterns associated with specific histone modifications. The volume further details tools and protocols for the detection of viruses and bacteria, and genetic and epigenetic analyses for forensics using Pyrosequencing. As a volume in the highly successful Methods in Molecular Biology series, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols and tips on troubleshooting and avoiding known pitfalls. Comprehensive and adaptable, Pyrosequencing: Methods and Protocols, Second Edition will greatly aid doctorial students, postdoctoral investigators and research scientists studying different aspects of genetics and cellular and molecular biology.

Expert researchers and physician/clinicians describe in detail the newest and most commonly used technologies today in this rapidly advancing field. The authors provide readily reproducible methods for assessing the functional consequences of a certain polymorphism, evaluate the variety of genotyping platforms currently available, and discuss the management of pharmacogenomic information. Highlights include techniques for making a snapshot of the allele-specific variation in human gene expression, genome wide analysis of allele-specific expression using oligo microarrays, in vivo assays with HaploChIP, SNP genotyping in DNA pools, and PharmGKB, the pharmacogenetics and pharmacogenomics knowledge base. The methodologies for genotyping include denaturing high-performance liquid chromarography, pyrosequencing, kinetic-fluorescence detection, mass spectrometry, and TaqMan assay for insertion/deletions.

The aim of this book is to improve pyrosequencing protocols as well as instrumentation for better clinical use by describing improvements and novel applications of pyrosequencing technology. Divided into five parts, the book's thirty chapters explore advances in pyrosequencing template preparation, pyrosequencing technology innovations, multiplex pyrosequencing based on barcodes, the miniaturization of pyrosequencing equipment, as well as various applications. As part of the Springer Protocols program, chapters contain the kind of detail and practical implementation advice to guarantee successful results in the lab. Comprehensive and thorough, Advances and Clinical Practice in Pyrosequencing serves as a valuable reference for researchers who are engaged in personalized medicine, disease control, and DNA diagnosis in numerous other fields.

This detailed volume provides a comprehensive collection of protocols for epigenomic research, powering our ability to analyze epigenetic modifications across the entire genome. Beginning with methods used to investigate epigenomic modifications such as DNA methylation, histone modifications, and chromatin structure, the book continues with methods for manipulating the epigenome, including platforms for epigenome editing, inducible systems for epigenome editing, and epigenetically modified animals. Written for the highly successful Methods in Molecular Biology series, chapters feature introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Epigenomics: Methods and Protocols serves as an ideal resource for researchers looking to further expand the utility and scope of epigenomics research.

The ability to successfully clone genes underlies the majority of our knowledge in molecular and cellular biology. Gene Cloning introduces the diverse array of techniques available to clone genes and how they can be used effectively both in the research laboratory, to gain knowledge about the gene, and for use in biotechnology, medicine, the pharmaceutical industry, and agriculture. It shows how cloning genes is an integral part of genomics and underlines its relevance in the post-genomic age, as a tool required to test predictions of gene regulation and function made through bioinformatics. Applications of gene cloning in medicine, both for diagnosis and treatment, and in the pharmaceutical industry and agriculture, are also covered in the book. Gene Cloning takes a fresh approach to teaching molecular and cellular biology and will be a valuable resource to both undergraduates and lecturers of biological and biomedical science courses.

The 2e of Molecular Diagnostics, the only book dealing with diagnosis on a molecular level, discusses current molecular biological techniques used to identify the underlying molecular defects in inherited disease. The book delves further into the principle and brief description of the technique, followed by examples from the authors' own expertise. Contributors to the 2e are well-known experts in their field, and derive from a variety of disciplines, to ensure breadth and depth of coverage. Molecular Diagnostics, 2e , is a needed resource for graduate students, researchers, physicians and practicing scientists in molecular genetics and professionals from similar backgrounds working in diagnostic laboratories in academia or industry, as well as academic institutions and hospital libraries. Deals exclusively with the currently used molecular biology techniques to identify the underlying molecular defect of inherited diseases Includes pharmacogenetics and pharmacogenomics relating to new cancer therapies Provies a comprehensive guide through emerging concepts and demonstrates how the available mutation screening technology can be implemented in diagnostic laboratories and provide better healthcare

In recent years, the field of epigenetics has grown significantly, driving new understanding of human developmental processes and disease expression, as well as advances in diagnostics and therapeutics. As the field of epigenetics continues to grow, methods and technologies have multiplied, resulting in a wide range of approaches and tools researchers might employ. Epigenetics Methods offers comprehensive instruction in methods, protocols, and experimental approaches applied in field of epigenetics. Here, across thirty-five chapters, specialists offer step-by-step overviews of methods used to study various epigenetic mechanisms, as employed in basic and translational research. Leading the reader from fundamental to more advanced methods, the book begins with thorough instruction in DNA methylation techniques and gene or locus-specific methylation analyses, followed by histone modification methods, chromatin evaluation, enzyme analyses of histone methylation, and studies of non-coding RNAs as epigenetic modulators. Recently developed techniques and technologies discussed include single-cell epigenomics, epigenetic editing, computational epigenetics, systems biology epigenetic methods, and forensic epigenetic approaches. Epigenetics methods currently in-development, and their implication for future research, are also considered in-depth. In addition, as with the wider life sciences, reproducibility across experiments, labs, and subdisciplines is a growing issue for epigenetics researchers. This volume provides consensus-driven methods instruction and overviews. Tollefsbol and contributing authors survey the range of existing methods; identify best practices, common themes, and challenges; and bring unity of approach to a diverse and ever-evolving field. Includes contributions by leading international investigators involved in epigenetic research and clinical and therapeutic application Integrates technology and translation with fundamental chapters on epigenetics methods, as well as chapters on more novel and advanced epigenetics methods Written at verbal and technical levels that can be understood by scientists and students alike Includes chapters on state-of-the-art techniques such as single-cell epigenomics, use of CRISPR/Cas9 for epigenetic editing, and epigenetics methods applied to forensics