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RNA Methodologies: A Laboratory Guide for Isolation and Characterization, Sixth Edition provides the most up-to-date ribonucleic acid lab techniques for seasoned scientists and graduate students alike. This edition features new material on RNA sequencing, RNA in Situ Hybridization, non-coding RNAs, computational RNA biology, transcriptomes and bioinformatics, along with the latest advances in methods and protocols across the field of RNA investigation. As a leader in the field, Dr. Farrell provides a wealth of knowledge on the topic of RNA biology while also giving readers helpful hints and troubleshooting techniques from his own personal experience in this subject area. This book presents the essential knowledge and techniques to use when working with RNA for the experienced practitioner, while also aiding the beginner in fully understanding this important branch of molecular biology. Presents the latest information covering all aspects of working with RNA, delivering a holistic understanding of this leading field in molecular biology Builds from basic information on RNA techniques to in-depth protocols for specific applications Features new chapters on RNA sequencing and RNA in situ hybridization Includes new material on RNA clinical applications and innovations, including RNA therapeutics and RNA vaccines, with particular relevance to coronavirus Comprises the latest developments in transcriptomes and bioinformatics, with new material on computational RNA biology, RNA CHiP analysis, aptamer biology and RNA epigenetics

This volume focuses on newly emerging technologies that facilitate the isolation and characterization of genes. The detailed protocols will be useful to the seasoned professional and easily understood by the novice. The vast majority of methods are applic

This textbook helps you to prepare for your next exams and practical courses by combining theory with virtual lab simulations. The “Labster Virtual Lab Experiments” series gives you a unique opportunity to apply your newly acquired knowledge in a learning game that simulates exciting laboratory experiments. Try out different techniques and work with machines that you otherwise wouldn't have access to.In this book, you'll learn the fundamental concepts of basic biochemistry focusing on:Ionic and Covalent BondsIntroduction to Biological MacromoleculesCarbohydratesEnzyme KineticsIn each chapter, you'll be introduced to one virtual lab simulation and a true-to-life challenge. Following a theory section, you'll be able to play the relevant simulation that includes quiz questions to reinforce your understanding of the covered topics. 3D animations will show you molecular processes not otherwise visible to the human eye.If you have purchased a printed copy of this book, you get free access to five simulations for the duration of six months. If you're using the e-book version, you can sign up and buy access to the simulations at www.labster.com/springer.If you like this book, try out other topics in this series, including “Basic Biology”, “Basic Genetics”, and “Genetics of Human Diseases”.Please note that the simulations in the book are not virtual reality (VR) but 2D virtual experiments.

Cytogenetics is the study of chromosome morphology, structure, pathology, function, and behavior. The field has evolved to embrace molecular cytogenetic changes, now termed cytogenomics. Cytogeneticists utilize an assortment of procedures to investigate the full complement of chromosomes and/or a targeted region within a specific chromosome in metaphase or interphase. Tools include routine analysis of G-banded chromosomes, specialized stains that address specific chromosomal structures, and molecular probes, such as fluorescence in situ hybridization (FISH) and chromosome microarray analysis, which employ a variety of methods to highlight a region as small as a single, specific genetic sequence under investigation. The AGT Cytogenetics Laboratory Manual, Fourth Edition offers a comprehensive description of the diagnostic tests offered by the clinical laboratory and explains the science behind them. One of the most valuable assets is its rich compilation of laboratory-tested protocols currently being used in leading laboratories, along with practical advice for nearly every area of interest to cytogeneticists. In addition to covering essential topics that have been the backbone of cytogenetics for over 60 years, such as the basic components of a cell, use of a microscope, human tissue processing for cytogenetic analysis (prenatal, constitutional, and neoplastic), laboratory safety, and the mechanisms behind chromosome rearrangement and aneuploidy, this edition introduces new and expanded chapters by experts in the field. Some of these new topics include a unique collection of chromosome heteromorphisms; clinical examples of genomic imprinting; an example-driven overview of chromosomal microarray; mathematics specifically geared for the cytogeneticist; usage of ISCN's cytogenetic language to describe chromosome changes; tips for laboratory management; examples of laboratory information systems; a collection of internet and library resources; and a special chapter on animal chromosomes for the research and zoo cytogeneticist. The range of topics is thus broad yet comprehensive, offering the student a resource that teaches the procedures performed in the cytogenetics laboratory environment, and the laboratory professional with a peer-reviewed reference that explores the basis of each of these procedures. This makes it a useful resource for researchers, clinicians, and lab professionals, as well as students in a university or medical school setting.

This fifth edition of the successful, long-selling classic has been completely revised and expanded, omitting some topics on obsolete DNA electrophoresis, but now with a completely new section on electrophoretic micro-methods and on-the-chip electrophoresis.The text is geared towards advanced students and professionals and contains extended background sections, protocols and a trouble-shooting section. It is now also backed by a supplementary website providing all the figures for teaching purposes, as well as a selection of animated figures tested in many workshops to explain the underlying principles of the different electrophoretic methods.

Today's synthetic biologists are in the early stages of engineering living cells to help treat diseases, sense toxic compounds in the environment, and produce valuable drugs. With this manual, you can be part of it. Based on the BioBuilder curriculum, this valuable book provides open-access, modular, hands-on lessons in synthetic biology for secondary and post-secondary classrooms and laboratories. It also serves as an introduction to the field for science and engineering enthusiasts.Developed at MIT in collaboration with award-winning high school teachers, BioBuilder teaches the foundational ideas of the emerging synthetic biology field, as well as key aspects of biological engineering that researchers are exploring in labs throughout the world. These lessons will empower teachers and students to explore and be part of solving persistent real-world challenges.Learn the fundamentals of biodesign and DNA engineeringExplore important ethical issues raised by examples of synthetic biologyInvestigate the BioBuilder labs that probe the design-build-test cycleTest synthetic living systems designed and built by engineersMeasure several variants of an enzyme-generating genetic circuitModel'bacterial photography'that changes a strain's light sensitivityBuild living systems to produce purple or green pigmentOptimize baker's yeast to produce?-carotene

Microorganisms play an important role in the maintenance of the ecosystem structure and function. Bacteria constitute the major part of the microorganisms and possess tremendous potential in many important applications from environmental clean up to the drug discovery. Much advancement has been taken place in the field of research on bacterial systems. This book summarizes the experimental setups required for applied microbiological studies. Important background information, representative results, step by step protocol in this book will be of great use to the students, early career researchers as well as the academicians. The book describes many experiments covering the basic microbiological experiments to the applications of microbial systems for advanced research. Researchers in any field who utilize bacterial systems will find this book very useful. In addition to microbiology and bacteriology, this book will also find useful in molecular biology, genetics, and pathology and the volume should prove to be a valuable laboratory resource in clinical and environmental microbiology, microbial genetics and agricultural research. Unique features • Easy to follow by the users as the experiments have been written in simple language and step-wise manner. • Role of each reagents to be used in each experiment have been described which will help the beginners to understand quickly and design their own experiment. • Each experiment has been equipped with the coloured illustrations for proper understanding of the concept. • Trouble-shootings at the end of each experiment will be helpful in overcoming the problems faced by the users. • Flow-chart of each experiment will quickly guide the users in performing the experiments.

Gene therapy as a treatment for cancer is at a critical point in its evolution. Exciting new developments in gene targeting and vector technology, coupled with results from the first generation of preclinical and clinical studies have led to the design and testing of new therapeutic approaches. The Third Edition of Gene Therapy of Cancer provides crucial updates on the basic and applied sciences of gene therapy. It offers a comprehensive assessment of the field including the areas of suicide gene therapy, oncogene and suppressor gene targeting, immunotherapy, drug resistance gene therapy, and the genetic modification of stem cells. Researchers at all levels of development, from basic laboratory investigators to clinical practitioners, will find this book to be instructive. Cancer gene therapy, like cancer therapy in general, is evolving rapidly, testing new concepts, targets and pathways, evoking new technologies, and passing new regulatory hurdles. Its essence, however, has not changed: the hope and challenges of returning altered genes to normal, using targeted gene expression to alter the function of both tumor and microenvironment, and in some cases normal cells, and delivering functionally important genes to specific cell types to increase sensitivity to killing or to protect normal cells from cancer therapies. In some instances, gene therapy for cancer forms a continuum from gene repair through the use of molecularly modified cells; the use of viral and non-viral vector based gene delivery to both tumor and tumor microenvironment; the use of viral and gene based vaccines; and development of new gene-based therapeutics. The unique mechanistically chosen vector platforms are at the heart of this technology because they allow for direct and selective cell death and transient to sustained delivery of vaccine molecules or molecules that affect the microenvironment, vasculature, or the immune response. Explains the underlying cancer biology necessary for understanding proposed therapeutic approaches Presents in-depth description of targeting systems and treatment strategies Covers the breadth of gene therapy approaches including immunotherapeutic, drug resistance,oncolytic viruses, as well as regulatory perspectives from both the NCI and FDA

The book Protocols in Livestock Genome Analysis Presents the Fundamental principle and detailed protocols of laboratory techniques with an adequate amount of thought been applied towards making it simplistic and effective. The book has been structured into seventeen chapters. It begins with Chapter 1, givingreaders some basic safety and precautions while working in molecular genetics laboratory that is exposed to hazardous agents. Chapter 2, 3 and 4 explains the most basic and fundamental procedures in molecular genetics laboratory i.e. isolation of genomic DNA, plasmid DNA, total RNA which are prerequisite for researchers conducting downstream genetics research like gene amplification or expression. Chapter 5 deals with quantification of DNA and RNA with specific examples. Chapter 6 deals with technique of polymerase chain reaction for amplification of a specific DNA sequences while Chapter 7 focuses on various variants of polymerase chain reaction that have been developed over the years for specific applications. Chapter 8 discusses the reverse transcriptionpolymerase chain reaction which is important prerequisite for gene expression studies and genetic characterization. Chapter 9 describes at length, agarose gel electrophoresis for the separation of nucleic acids and their analysis. Chapter 10describes single strand conformation polymorphism, a technique that is capable of identifying most sequence variations in a single strand of DNA. Chapter 11 details the restriction digestion of DNA molecules by restriction endonucleases. Chapter 12 describes real-time PCR that can detect their accumulation and quantify the number of substrate DNA molecules present in starting sample. Chapter 13 discusses the method of chromatin immunoprecipitation, a very important technique for elucidating the transcriptional regulation of gene expression. Chapter 14 deals with detailed analysis of microsatellite repeat sequences along with statistical tools for data analysis. Chapter 15 explains the very important procedures of DNA ligation, competent cells preparation and transformation involved in cloning of any desired DNA sequence. Chapter 16 describes the set of steps for retrieving QTL & SNP information from online Animal QTL databases. Chapter 17 provides a simple understanding of DNA microarrays. Chapter 18 details the in situ hybridization method for localization of genetic material. Chapter 19 provides the recipe for preparation of buffers and solutions required for conducting various procedures outlined in previous chapters.

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers recent research and methods development for changing the DNA sequence within the genomes of cells and organisms. Focusing on enzymes that generate double-strand breaks in DNA, the chapters describe use of molecular tools to introduce or delete genetic information at specific sites in the genomes of animal, plant and bacterial cells. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods in biomineralization science Contains sections on such topics as genome editing, genome engineering, CRISPR, Cas9, TALEN and zinc finger nuclease

Plant tissue culture has a long history, dating back to the work of Gottlieb Haberlandt and others at the end of the 19th century, but the associated concepts and techniques have reached a level of usefulness and application which has never been greater. The technical innovations have given new insights into fundamental aspects of plant differentiation and development, and have paved the way to the identification of strategies for the genetic manipula­ tion of plants. It is the aim of this manual to deliver a broad range of these techniques in a form which is accessible to students and research scientists of diverse backgrounds, including those with little or no previous experience. The themes of the manual aim to reflect those research areas which have been advanced by tissue culture technology. As was the case for the sister volume Plant Molecular Biology Manual, the objective has been from the start to produce a manual which is at home on the laboratory bench. The plastic-covered, ring-bound format has proved to be most popular and is retained here. Equally, the emphasis has been on producing a collection of detailed step-by-step protocols, each supplemented with an introductory text and practical footnotes, to provide the next best thing to a supervisor at one's shoulder.

Covering state-of-the-art technologies and a broad range of practical applications, the Third Edition of Gene Biotechnology presents tools that researchers and students need to understand and apply today's biotechnology techniques. Many of the currently available books in molecular biology contain only protocol recipes, failing to explain the princ

This is the fourth edition of the successful laboratory guide which has translated the rich story of riboneucleic acid for over fifteen years. RNA Methodologies 4e presents the latest collection of tested laboratory protocols for the isolation and characterization of eukaryotic and prokaryotic RNA with greater emphasis on transcript profiling, including quantification issues and elucidation of alternative transcription start sites. Collectively the chapters work together providing analysis with clear take-home lessons to assist researchers to understand RNA and to optimize time at the bench. The abundant use of flow charts, tables and graphs are especially helpful in the planning and implementation phases of a project and facilitate learning. 30% new material in this edition includes the addition of RNA isolation protocols including RNA isolation from tissue, expansion of PCR optimization analysis and RNA interference sections, the introduction of a new chapter dealing with the molecular biology of plants, and an expanded glossary. 30% new material with the addition of RNA isolation protocols including RNA isolation from tissue, expansion of PCR optimization analysis and RNA interference sections, the introduction of a new chapter dealing with the molecular biology of plants, and an expanded glossary Author is a well-recognized expert in the field of RNA experimentation and founded Exon-Intron, a well-known biotechnology educational workshop center Includes classic and contemporary techniques useful for all labs

Due to its prolific reproduction and the external development of the transparent embryo, the zebrafish is the prime model for genetic and developmental studies, as well as research in genomics. While genetically distant from humans, nonetheless the vertebrate zebrafish has comparable organs and tissues that make it the model organism for study of vertebrate development.This book, one of two new volumes in the Reliable Lab Solutions series dealing with zebrafish, brings together a robust and up-to-date collection of time-tested methods presented by the world's leading scientists. Culled from previously published chapters in Methods in Cell Biology and updated by the original authors where relevant, it provides a comprehensive collection of protocols describing the most widely used techniques relevant to the study of zebrafish genetics and genomics. The methods in this volume were hand-selected by the editors, whose goal was to a provide a handy and cost-effective collection of fail-safe methods, tips, and'tricks of the trade'to both experienced researchers and more junior members in the lab. Provides busy researchers a quick reference for time-tested methods and protocols that really work, updated where possible by the original authors Gives pragmatic wisdom to the non-specialist from experts in the field with years of experience with trial and error

The combined power of genetic analysis and recombinant DNA technology to analyse entire genomes has moved biomedical research into a new and revolutionary phase. The complete sequencing and mapping of the human genome, as well as the genomes of other model organisms, will be the basis for our future understanding of human disease, and will allow us to answer fundamental questions about development and evolution. T The new ICRF Handbook of Genome Analysis is the essential guide to the enormous range of techniques available to the researcher for both the genetic and physical mapping of the genome, as well as the sequencing and analysis of DNA. It is both a protocol manual and a comprehensive information resource. Written by international experts, each chapter presents a state-of-the-art review of a methodology. Methods are fully described and evaluated; their advantages and disadvantages discussed; and their suitability for different investigations considered. Step-by-step protocols, including computer analyses, are given for 123 essential experimental procedures.'Troubleshooting'sections discuss possible reasons for failure and offer remedies. The primary focus is on human genetics and the benefits of an understanding of the genome for the diagnosis and treatment of human disease. The book also considers the current state of progress in the analysis of genomes of many model organisms, including plants. A major part of the work provides detail on Internet resources as well as basic data on human and other genomes, including mapped disease genes and mouse knockouts. Covers not only the human genome in relation to cancers and other human diseases, but also the genomes of all important model organisms Contains 123 easy-to-follow protocols for essential experimental procedures Reviews a vast range of other information resources, including journals and the Internet • provides an invaluable listing of suppliers of laboratory materials Has been written by international experts from their own practical experience Is mandated by the Imperial Cancer Research Fund - a leader in research in this field Has a sturdy spiral binding within a hardback case for ease of use in the lab

Stem cells are self-replicating and undifferentiated, meaning their function is not yet cell, tissue, or organ-specific. Due to the unique nature of these cells, research into their biology and function holds great promise for therapeutic applications through replacement or repair of diseased and damaged cells. This reader-friendly manual provides a practical'hands on'guide to the culture of human embryonic and somatic stem cells. By presenting methods for embryonic and adult lines side-by-side, the authors lay out an elegant and unique path to understanding the science of stem cell practice. The authors begin with a broad-based introduction to the field, and also review legal and regulatory issues and patents. Each experimental strategy is presented with an historical introduction, detailed method, discussion of alternative methods, and common pitfalls. This lab guide for researchers also serves as a textbook for undergraduate and graduate students in laboratory courses.

With the advent of high-throughput technologies, the number of genes of interest has expanded and the traditional methods for gene function analysis cannot achieve the throughput necessary for large-scale exploration. In Gene Function Analysis, a select team of experts bring together a number of recently developed techniques for studying gene function. The volume begins with a variety of computational techniques, which provide an excellent point of departure for the cutting-edge experimental methods that follow. Written in the highly successful Methods in Molecular Biology series format, each chapter surveys its subject with readily reproducible laboratory protocols, a list of the necessary materials, and the popular Notes section, which contains tips for troubleshooting and avoiding known pitfalls. Comprehensive and timely, Gene Function Analysis offers researchers a clear guide and the important tools they need to further study the intricate human genome.

Protein expression is an increasingly important tool for research on gene function. What is needed is not just a lab manual providing established methods as well as the latest state-of-the-art protocols, but also clear advice on what expression system to choose when. Expression Systems: Methods Express uniquely fills this need. It covers expression across a broad range of systems, including the following. •Baculovirus expression vectors •CHO cells • E. coli •HEK293-EBNA1 cells • Lactococcus lactis and other gram positive bacteria • S. cerevisiae •transfected insect cells • Pichia pastoris •mammalian cells using BacMam viruses •lentiviral vectors •wheat germ cell-free system The book takes the reader through how to make an informed choice of appropriate system, taking into account the protein target, the time involved, the ultimate use of the expressed protein, and the laboratory equipment required. It also provides step-by-step methods for each system. In addition, the book describes the optimisation of expression strategies, expression engineering using ribosome display, and how to select protein variants with improved expression. Every chapter discusses the merits and limitations of the approaches available, describes the key techniques in full practical detail, and provides sensible advice for immediate use at the bench. In summary, Expression Systems: Methods Express is a comprehensive laboratory manual and information resource for researchers at all levels, from postgraduate student to principal investigator.

Dictyostelium discoideum Protocols presents a comprehensive collection of cutting-edge molecular biological protocols for studying D. discoideum, with emphasis on its utility in the study of fundamental cellular processes including signal transduction, chemotaxis, cell motility, cytokinesis, phagocytosis, and aspects of development such as cell sorting, pattern formation, and cell type differentiation. Written by experts in the field, this important guide provides easy access to current methods and techniques for the study of D. discoideum. For the newcomer to the field, it offers a complete and inclusive introduction to this important model organism. The book is divided into four main parts, which include an introduction to the organism that provides integral community resources and genome-wide approaches; basic methods and available molecular genetic techniques for study of the organism; imaging and localization methods; and a discussion of D. discoideum, with emphasis on its unique advantages as a model system. This in-depth guide provides a complete introduction to and thorough handbook for the study of D. discoideum, including a discussion of the implications of the recently completed Dictyostelium genome project in strengthening the position of D. discoideum as a model organism for studying fundamental cell processes and aspects of development. Researchers at all levels of experience will benefit from the presentations throughout of the most current, useful, and innovative techniques for the study of D. discoideum from leading Dictyostelium scientists.

Integrated Genomics: A Discovery-Based Laboratory Course introduces the excitement of discovery to the basic molecular biology laboratory. Utilizing up-to-date molecular biology protocols and a basic experimental design, this text offers experience with three different model systems. Students will become familiar with the simplicity and power of single-celled organisms, Escherichia coli and Saccharomyces cerevisiae, as they search for genes that interact and function within the nematode Caenorhabditis elegans. Incorporated throughout the course are exercises designed to offer students familiarity with the wealth of bioinformatics data that can be accessed on the World Wide Web. Following completion of interaction studies within the yeast, the course is designed to allow students to examine the functional consequences of reducing a gene's function within the multicellular worm that is both simple and inexpensive to maintain within a laboratory. The inclusion of alternative experiments allow for flexibility in determining the ending date or goal of the laboratory, as well as working within the available budget and resources of most any classroom environment. Further striking features of this title are: An accompanying Web site providing PowerPoint slides, plus links to the internet, and regular updates as bioinformatics databases evolve and methods improve. www.wiley.com/go/caldwell Inclusion of modern genomic/proteomic technologies such as the yeast two-hybrid system and RNAi Detailed experimental protocols and easy access to instructional materials This discovery-based laboratory course provides excellent practical training for those pursuing career paths in biomedicine, pharmacy, and biotechnology.

This four-volume laboratory manual contains comprehensive state-of-the-art protocols essential for research in the life sciences. Techniques are presented in a friendly step-by-step fashion, providing useful tips and potential pitfalls. The important steps and results are beautifully illustrated for further ease of use. This collection enables researchers at all stages of their careers to embark on basic biological problems using a variety of technologies and model systems. This thoroughly updated third edition contains 165 new articles in classical as well as rapidly emerging technologies. Topics covered include: Cell and Tissue Culture: Associated Techniques, Viruses, Antibodies, Immunocytochemistry (Volume 1) Organelle and Cellular Structures, Assays (Volume 2) Imaging Techniques, Electron Microscopy, Scanning Probe and Scanning Electron Microscopy, Microdissection, Tissue Arrays, Cytogenetics and In Situ Hybridization, Genomics and Transgenic Knockouts and Knock-down Methods (Volume 3) Transfer of Macromolecules, Expression Systems, Gene Expression Profiling (Volume 4) Indispensable bench companion for every life science laboratory Provides the latest information on the plethora of technologies needed to tackle complex biological problems Includes numerous illustrations, some in full color, supporting steps and results

Numerous emerging technologies have propelled the growth of proteomics into a wide-ranging discipline with great potential to improve human health through the discovery of new protein biomarkers and therapeutics. In New and Emerging Proteomic Techniques, a panel of leading researchers and innovators describe in step-by-step detail the latest techniques that promise to significantly impact the practice of proteomics, as well as its success in developing novel clinical agents. The methods described here span the entire spectrum of top-down and bottom-up approaches, including microarrays, gels, chromatography, and affinity separations, and address every aspect of the human proteome, both quantitatively and qualitatively. The techniques of protein detection utilized are diverse and range from fluorescence and resonance light scattering to surface plasmon resonance and mass spectrometry. Where necessary, two or more distinct techniques are provided for a given problem, and advanced bioinformatics approaches are presented for the analysis of complex proteomic data. The protocols follow the successful Methods in Molecular Biology series format, each offering step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and easy to use, New and Emerging Proteomic Techniques offers expert and novice investigators alike today's most promising proteomics techniques for probing the biological processes and understanding the diseases of the human body.

This laboratory guide represents a growing collection of tried, tested and optimized laboratory protocols for the isolation and characterization of eukaryotic RNA, with lesser emphasis on the characterization of prokaryotic transcripts. Collectively the chapters work together to embellish the RNA story, each presenting clear take-home lessons, liberally incorporating flow charts, tables and graphs to facilitate learning and assist in the planning and implementation phases of a project.RNA Methodologies, 3rd edition includes approximately 30% new material, including chapters on the more recent technologies of RNA interference including: RNAi; Microarrays; Bioinformatics. It also includes new sections on: new and improved RT-PCR techniques; innovative 5'and 3'RACE techniques; subtractive PCR methods; methods for improving cDNA synthesis.• Author is a well-recognized expert in the field of RNA experimentation and founded Exon-Intron, a well-known biotechnology educational workshop center • Includes classic and contemporary techniques • Incorporates flow charts, tables, and graphs to facilitate learning and assist in the planning phases of projects

The past decade has witnessed a true revolution in our understanding of how RNA molecules can reduce or silence gene expression, a process that holds great promise in basic research and for antiviral and anticancer applications. In RNA Silencing: Methods and Protocols, many of the most notable and distinguished researchers in the field describe in step-by-step detail their best methods for the design, preparation, and use of RNAs to reduce gene expression in cells and organisms. The techniques range widely and include methods addressing the biochemical aspects of the silencing machinery, RNA silencing in non-mammalian organisms, and the in vivo delivery of siRNAs and silencing vectors. There are also techniques for designing, preparing, and using RNAs to silence gene expression, for fine-tuning regulation by targeting specific isoforms of a given gene, and for the study and use of microRNAs. The protocols follow the successful Methods in Molecular Biology series format, each 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. Comprehensive and state-of-the-art, RNA Silencing: Methods and Protocols offers today's laboratory investigators a host of optimized and validated techniques to study RNA silencing and translate its concepts into successful practical applications.

Maximizing the potential of RNA interference in functional genomics - as well as in the development of therapeutics - continues to be at the forefront of biomedical research. Unlike journal articles, Gene Silencing by RNA Interference: Technology and Application combines essential background to the RNAi field with practical techniques designed by r

Many scientists find themselves working in the laboratory without sufficient background in current biotechnology methods. Others want to keep up with the revolution in biotechnology and the flood of new methodologies. This book provides a solution for both: a multidisciplinary approach to the methods essential to biotechnical development. C

Recombinant vaccinia viruses have become important in recent years, both as protein expression vectors, and-in the age of bioterrorism-as powerful tools for developing new smallpox vaccines, diagnostics, and understanding of poxvirus pathogenesis. In Vaccinia Virus and Poxvirology: Methods and Protocols, internationally recognized experts and leaders in poxvirology describe in step-by-step detail the successful techniques they have perfected to study and work with vaccinia virus and other poxviruses. They offer cutting-edge techniques for the construction and characterization of recombinant viruses; for the study of poxvirus gene transcription and DNA replication; and for the use of the virus in immunologic and cell-cell fusion assays. Other methods cover poxvirus bioinformatics, aspects of viral pathogenesis at both a protein and an animal model level, and the study of immune responses to poxvirus-the latter critical to the important role of vaccinia virus in smallpox vaccination and its potential role as a vaccine vector directed against infectious agents and cancer. The protocols presented follow the successful Methods in Molecular Biology'!series format, each one offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and timely, Vaccinia Virus and Poxvirology: Methods and Protocols offers virologists and vaccinologists an unequaled source of proven techniques for understanding poxviruses and developing defenses against their use as bioterrorist agents

Assembly of the basic units of mammalian chromosomes into artificial constructs, and the reduction of their components to more manageable dimensions, has now become a reality. In Mammalian Artificial Chromosomes: Methods and Protocols, leading scientists in cell biology, genetics, cell culturing, and gene transfer present their most productive methods for the preparation, characterization, and use of artificial chromosomes. The authors cover all relevant areas of artificial chromosome research-from basic genetics to daring attempts at building new gene therapy tools-and describe possible applications in genetic manipulation requiring large portions of the genome. Each fully tested protocol is described in step-by-step detail and includes a background introduction outlining the principle behind the technique, reagent and equipment lists, and tips on troubleshooting and how to avoid pitfalls. Cutting-edge and much-needed, Mammalian Artificial Chromosomes: Methods and Protocols offers a timely collection of meticulous techniques that will help elucidate the mechanisms of nuclear duplication, allow the introduction of changes into the human genome, and revolutionize our understanding of how chromosomes work.

In this completely updated and expanded edition of a classic bench manual, hands-on experts take advantage of the latest advances in ribozyme, DNAzyme, hammerhead ribozymes and derivatives, and RNA interference technologies to describe in detail the exciting and successful methods now available for gene inactivation in vitro and in vivo. Their optimized techniques employ hairpin ribozymes, DNAzymes, hammerhead ribozymes and derivatives, group I intron ribozymes, RNase P ribozymes, and siRNAs, as well as general methods for RNA structure analysis, delivery of oligonucleotides, and gene therapy. Also provided are novel methods for identifying accessible cellular mRNA sites; group I intron and RNAse P ribozyme protocols for effective design, selection, and therapeutic applications; and the latest RNAi methods for sequence-specific gene silencing in a wide variety of organisms. Additional techniques cover the analysis of ribozyme structures and conformational transitions using nucleotide analog interference mapping and fluorescence resonance energy transfer, the use of ribozymes in clinical and gene therapy, and the use of ribozymes and DNAzymes in rodent models of human disease. Each proven protocol includes a background introduction outlining the principle behind the technique, step-by-step instructions, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and up-to-date, Ribozymes and siRNA Protocols details for experienced and novice investigators alike the many exciting advances in our understanding of nucleic acid enzymes, as well as demonstrating how they may be used to analyze gene function and target validation, and to productively develop novel therapeutics for human diseases.

Probably no single experimental organism has contributed as much to our understanding of the structure, function, and mechanism of transmission of eukaryotic chromosomes as has the fruit fly, Drosophila melanogaster. In Drosophila Cytogenetics Protocols, leading drosophilists describe, in step-by-step detail, all the essential techniques for studying Drosophila chromosomes and suggest new avenues for scientific exploration. General areas covered include analyses of nuclei and chromosomes in spermatogenesis and oogenesis, fluorescence-activated cell sorting of ovarian follicle cells, analyses of nuclear division cycles in embryos, and analyses of programmed cell death. In addition, there are conventional light-, electron-, and immunofluorescence-microscopic analyses of polytene chromosomes; cytological analyses of larval brains, imaginal discs, and histoblasts; and nonfluorescent and fluorescent in situ hybridization (FISH) to both polytene and mitotic chromosomes. A cutting-edge combined FISH and immunolocalization protocol will be important for discovering how DNA sequence influences higher-order chromosome architecture and ultimately gene expression. Each readily reproducible protocol offers a background introduction, equipment and reagent lists, and tips on troubleshooting and avoiding pitfalls. Readily accessible and highly practical, Drosophila Cytogenetics Protocols provides a comprehensive cytogenetics laboratory manual for Drosophila investigators, one that is suitable not only for novices, but also highly informative for seasoned investigators.

Title from ebook title screen (viewed on Mar. 30, 2005).

In this updated and expanded second edition of an established classic, the editors have added critical reviews to a fresh collection of cutting-edge protocols for gene expression in bacteria, fungi, plants, plant cells, animals, and animal cells. The review articles survey new directions in recombinant gene expression research, technique, and application, and point the way to using recombinant gene expression for metabolic engineering and the production of nonprotein molecules. The readily reproducible protocols offer new host-vector systems, such as Antarctic bacteria and moderately halophytic bacteria, vectors for chromosomal editing and metabolic engineering, and information to aid in problem solving, strategy planning, and the promotion of protein folding. There are also novel heterologous gene expression schemes for cell-free systems, plant chloroplasts, microplates, hairy roots, and animal milk, and new developments in consolidated heterologous systems. The protocols follow the successful Methods in Molecular Biology'!series format, each one offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. State-of-the-art and forward looking, Recombinant Gene Expression: Reviews and Protocols, Second Edition offers investigators seeking an overview of this critically important field not only the understanding, but also the tools needed to begin producing nonprotein products and altering the central metabolic pathways of cells to enhance heterologous gene expression

Transposable elements (TEs)-DNA sequences that are capable of moving from one chromosome location to another-are found in all living organisms. They have been increasingly investigated in a wide spectrum of species, including bacteria, plants, fungi, and animals. In Mobile Genetic Elements: Protocols and Genomic Applications, leading experts describe in step-by-step detail their most productive transposon-based methods and strategies for studying genome structure, function, and evolution. These readily reproducible techniques cover a broad range, including mutagenesis, transgenesis, gene silencing, and molecular systematics. Among the highlights are a series of DNA hybridization methods for analyzing the distribution and dynamics of mobile DNA at the hosts'genomic level, techniques for studying LTR retrotransposons in heterologous host systems, and mutagenesis protocols for investigating gene functions in a broad range of organisms. Additional methods deal with highly informative sets of polymorphic markers, RNAi technology in gene silencing, and applications during transgenesis. The protocols presented follow the successful Methods in Molecular Biology™ series format, each one offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. State-of-the-art and highly practical, Mobile Genetic Elements: Protocols and Genomic Applications offers investigators powerful genetic tools for dissecting the function of a specific gene, elaborating on the mechanisms leading to genetic change and diversity, and studying the evolutionary impact of mobile DNA on the biology and evolution of organisms.

The transcription of messenger RNA from a DNA template is a key process in a wide variety of biological systems. In Gene Expression Profiling: Methods and Protocols, leaders in gene expression methodology and bioinformatics data analysis share their best methods for measuring RNA levels in cells and tissues. Each proven protocol is described in step-by-step detail and contains an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Special care has been taken to distill the basic underlying principles of each method to a few straightforward concepts so that investigators can easily choose the method most appropriate to their application. The readily reproducible techniques presented include new methods for applying the GeneChip®, SAR-SAGE, StaRT-PCR, SSH, the Invader Assay®, and ADGEM. The authors also provide critical bioinformatics insight and resources for data analysis and management. Cutting-edge and highly practical, Gene Expression Profiling: Methods and Protocols allows novice and experienced investigators alike not only to choose the best method to determine how genes are turned on and off in many different organisms, but also to use informatics resources to analyze and understand the results.

Recent advances in epigenetic research as well as the development of exciting new technologies have helped greatly in unraveling the many mysteries of nontraditional genetic processes. In Epigenetics Protocols, hands-on researchers describe state-of-the-art methods for epigenetic analysis, including recent breakthrough techniques that have great potential in the rapidly expanding field of non-Mendelian genetics. The authors provide techniques for the analysis of chromatin remodeling, such as histone acetylation and methylation. In addition, methods in newly developed and especially promising areas of epigenetics, such as telomere position effects, quantitative epigenetics, and ADP ribosylation are covered. There is also an updated analysis of techniques involving DNA methylation and its role in the modification, as well as the maintenance, of chromatin structure. Of special interest are potentially revolutionary techniques. These include methods for determining changes in native chromatin, methods of microarray analysis applied to epigenetics, and methylation-sensitive single-strand conformation techniques. The methods are suitable not only for studying fundamental biological processes, but also for investigating possible therapeutic interventions and such diseases as cancer. The protocols follow the successful Methods in Molecular Biology™ series format, each one 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. Comprehensive and easy to use, Epigenetics Protocols offers investigators readily reproducible techniques that will further promote progress in this critically important field.

As high-throughput methods become more available and routine, experimentalists and theoreticians must be prepared to take advantage of the analytical power these methods offer. In Functional Genomics: Methods and Protocols, leading experimentalists-many pioneers in the field-describe in step-by-step detail robust methods for microarray-based studies and provide expert guidance in optimal data analysis and informatics. Drawing on insights derived from their long experience in biology, neurobiology, cell biology, molecular biology, and statistics, the authors review each of the key methods and analytical techniques used in functional genomics. On the methods side, this includes proven techniques for monitoring subcellular RNA localization en masse, for mapping chromosomes at the resolution of a single gene, and for surveying the steady-state genome-wide distribution of DNA binding proteins in vivo. For those workers dealing with massive data sets, the book discusses the methodological aspects of data analysis and informatics in the design of microarray experiments, the choice of test statistic, and the assessment of observational significance, data reduction, and clustering. Additional chapters outline the many available approaches to data visualization and storage so crucial to the successful mining and examination of experimental results. Timely and cutting-edge, Functional Genomics: Methods and Protocols offers novice and advanced investigators in all biological fields a collection of highly practical, readily reproducible methods, data analysis techniques, and experimental design criteria that will ensure safe passage through one of the most demanding and fastest growing fields today.

Single nucleotide polymorphisms (SNPs) have become the markers of choice in elucidating the relationship between DNA sequence variation and susceptibility to disease and have clearly become the focus of the next phase of the human genome project. In Single Nucleotide Polymorphisms: Methods and Protocols, Pui-Yan Kwok, MD, PhD, has assembled a collection of robust techniques for the difficult process of SNP discovery and genotyping. These cutting-edge protocols for mutation/SNP detection utilize denaturing high-performance liquid chromatography (dHPLC), single-strand conformation polymorphism (SSCP), conformation-sensitive gel electrophoresis (CSGE), chemical cleavage, and direct sequencing. Equally powerful and up-to-date methods are given for genotyping SNPs, including molecular beacons, the Taqman assay, single-base extension approaches, pyrosequencing, ligation, the Invader assay, and primer extension with mass spectrometry detection. Described in step-by-step detail by their inventors, each method provides extensive notes on the technical steps critical for experimental success, time-saving techniques, and tips on avoiding pitfalls. Comprehensive and authoritative, Single Nucleotide Polymorphisms: Methods and Protocols provides in a readily reproducible format all the major SNP discovery and genotyping techniques in use today, whether for using DNA diagnostics to identify a pathogen, for studying the genetic basis of human disease, or for molecular breeding programs in agriculture.

Gene expression using E. coli as a host is carried out over a wide range of disciplines in academic and industrial laboratories. In E. coli Gene Expression Protocols, Peter E. Vaillancourt presents a collection of popular and emerging methodologies that take advantage of E. coli's ability to quickly and inexpensively express recombinant proteins. The authors focus on two areas of interest: the use of E. coli vectors and strains for production of pure, functional protein, and the use of E. coli as host for the functional screening of large collections of proteins and peptides. Among the cutting-edge techniques demonstrated are those for rapid high-level expression and purification of soluble and functional recombinant protein, and those essential to functional genomics, proteomics, and protein engineering. Described in step-by-step detail to ensure robust, trouble-free results, each proven method has been written by a hands-on expert and includes extensive notes and practical tips for avoiding pitfalls. Even highly skilled researchers will find many time-saving techniques. Authoritative and highly practical, E. coli Gene Expression Protocols provides a state-of-the-art collection of tested methods for this powerful gene expression technology, offering today's investigators proven tools for success in the emerging fields of functional genomics and proteomics.

Since the discovery of p53 as a tumor suppressor, numerous methods have evolved to reveal its unique structural features and biochemical functions. In p53 Protocols, Sumitra and Swati Palit Deb have assembled an indispensable collection of novel techniques that have proven most useful for studying the physiological properties of p53 both in vitro and in vivo. Described by leading basic and clinical researchers who have successfully used the methods, the techniques provide proven solutions to problems in studying the purification, target identification, gene expression, quantitation, interaction, signaling, transactivation, and transrepression of p53. The methods are also useful for delineating the functions of other proteins that may act as tumor or growth suppressors. Each technique includes step-by-step instructions, troubleshooting notes, a theoretical review, and discussion of associated problems that might arise during the course of investigation. Special effort has been made to discuss problems and their solutions to help ease the learning curve when standardizing a new method. Diverse and highly practical, p53 Protocols offers both beginning and experienced researchers in cancer biology a gold-standard compendium of readily reproducible techniques for studying p53 and related proteins.

Directed evolution, the application of evolutionary design to enzyme engineering, requires effective screening strategies to isolate those proteins that perform a desired function from the libraries generated by the techniques. In Directed Enzyme Evolution: Screening and Selection Methods, seasoned practitioners from many leading laboratories describe their leading and readily reproducible screening strategies for isolating useful clones. These techniques have been optimized for sensitivity, high throughput, and robustness, and are of proven utility for directed evolution purposes. The assays presented use a variety of techniques, including genetic complementation, microtiter plates, solid-phase screens with colorimetric substrates, and flow cytometric screens. There are also representative examples of how phage libraries may be interrogated for enzymatic activity. Each protocol contains detailed step-by-step instructions and many notes on how best to deal with the problems that may occur. An accompanying volume, Directed Evolution Library Creation: Methods and Protocols (ISBN 1-58829-285-1), describes readily reproducible methods for the creation of mutated DNA molecules and DNA libraries. Taken together, Directed Enzyme Evolution: Screening and Selection Methods and Directed Evolution Library Creation: Methods and Protocols capture for newcomers and more experienced investigators alike all the key methods for using directed protein evolution to better understand protein structure-function relationships, to discover new enzymes and therapeutic proteins, and to design new assays suitable for specific applications.

This book offers a wide ranging and review of cutting edge developments along with tried and tested methods for isolation, resolution and quantification of inositol phospholipids and inositol polyphosphates in both cells and tissues. It includes detailed and rigorous methodology for identification of molecular species of inositol phospholipids, including their phosphates and glycans, with numerous examples of specific applications. Wherever available, both chromatographic and mass spectrometricevidence is presented for specific phospholipid involvement in the biochemical transformations accompanying metabolic signalling events and, where possible, controversies have been resolved on basis of analytical ambiguity. The review of the lipidanalyses extends to products of phosphatidylinositol kinases, phosphatases and lipases. A general review chapter is included on metabolic signalling with special emphasis on the lipid products of 3-kinases. In view of the general lack of commercially available standards for inositol phospholipids and inositol phosphates, the book concludes with a chapter on the preparation and collection of chromatographic and mass spectrometric proof of their purity and identity.This book complements recent books and reviews on the mechanisms metabolic signalling, receptor binding and the polypeptide structure of the proteins involved in the various signalling pathways. It also complements those texts that deal exclusively with the chemical synthesis of the phosphatidylinositols and their polyphosphates and glycans.

With the decoding of the human genome and the potential application of its information to gene chips, the ability to generate cDNA libraries for functional assays of specific gene sequences has become critical in gene manipulation and genetic research. In Generation of cDNA Libraries: Methods and Protocols, expert researchers and inventors in the field describe their own proven techniques for generating cDNA/mRNA libraries to identify the functions of specific decoded gene sequences. A wide variety of techniques is presented for enhancing the generation of complete and full-length libraries, and for confirming the quality of the cDNAs generated. Among the applications detailed are electrophoresis, Northern blotting, single-cell microarray analysis, subtractive hybridization, subtractive cloning, gene cloning, and peptide library generation. Each method includes background information, step-by-step instructions, a list of reagents, operational tips, and notes on instrumentation. A background tutorial provides essential information: the definition of a cDNA library, the various types of cDNA libraries, the differences between methods for cDNA library generation using either conventional approaches or novel strategies, and the quality of cDNA libraries. State-of-the-art and highly practical, Generation of cDNA Libraries: Methods and Protocols provides a variety of powerful and readily reproducible techniques for the generation of the entire range of complete, full-length cDNA/mRNA libraries needed in today's forefront genetic research.

Molecular Microbiology Laboratory is designed to teach molecular biology techniques to upper level undergraduates majoring in the life sciences. An extremely detailed lab preparation manual for teaching assistants accompanies the lab book and contains a general discussion of scientific writing and critical reading, as well as detailed instructions for preparation and peer review of lab reports. Each experimental unit is accompanied by a number of additional writing exercises based upon primary journal articles. The studies in these articles employ the techniques that the students are learning in the lab exercises, which reinforces their understanding of the material. These are techniques that students in any biological science will need to know, making this manual applicable to any life science curriculum.Key Features• Not a typical cookbook lab exercise, offers students the excitement and intellectual challenge of characterizing true unknowns. They could discover a new species! • Success rate greater than 85% for the entire experiment, even with very inexperienced students. • The ONLY manual that incorporates writing exercises into the curriculum. • Co-authored by Dr. Janine Trempy, one of four senior editors of the Journal of Microbiology Education, published by the American Society for Microbiology.

Application of DNA technology to the identification of disease-causing mutations has become widespread in recent years. PCR Mutation Detection Protocols, provides biological and clinical investigators with a comprehensive collection of new, recent, and updated PCR-based screening methods suitable for detecting the presence of both known and novel mutations. The methods cover point mutations (e.g., ASO-PCR, SSCP, DGGE, chemical cleavage), deletions (multiplex PCR, FISH, blotting), non-sense mutations (PTT), and more. The new and exciting techniques of DNA array analysis, along with such recently developed experimental methods as conformation-sensitive gel electrophoresis, are also included. Additional coverage is given to the direct use of DNA sequencing as a detection method in its own right and to the characterization of mutations previously located by other screening techniques. Each chapter explains the basic theory behind the technique and provides valuable notes essential for its successful execution. Comprehensive and highly practical, PCR Mutation Detection Protocols assures both seasoned and novice investigators access to the highly productive and readily reproducible PCR-based mutation detection methods, techniques that are laying the groundwork for many of today's major scientific and medical advances.