In the post-genome era, in vitro mutagenesis has emerged as the critically important tool used by molecular biologists in establishing the functions of components of the proteome. In this second edition of In Vitro Mutagenesis Protocols, active researchers with proven track records describe in stepwise fashion their advanced mutagenesis techniques. Each contributor focuses on improvements to conventional site-directed mutagenesis, with chapters being devoted to chemical site-directed mutagenesis; PCR-based mutagenesis and the modifications that allow high-throughput experiments; and mutagenesis based on gene disruption that is both in vitro- and in situ-based. Additional methods are provided for in vitro gene evolution; for gene disruption based on transposon, recombination, and cassette mutagenesis; and for facilitating the introduction of multiple mutations. Each readily reproducible technique includes detailed step-by-step instructions, tips on pitfalls to avoid, and notes on reagents and suppliers. Time-tested and highly practical, the techniques in In Vitro Mutagenesis Protocols, Second Edition offer today's molecular biologists a rich compendium of reliable and powerful techniques with which to illuminate the proteome.
In In Vitro Mutagenesis Protocols leading experts from industrial and academic laboratories describe easily reproducible procedures for site-directed and random mutagenesis. Site-directed protocols include those based on strand-selection, PCR (including "splicing by overlap extension" and the "megaprimer" procedure), the ligase chain reaction, positive antibiotic selection, unique restriction site elimination, gapped heteroduplex formation, and solid-phase capture with the biotin/ strepavidin system. Many techniques can be used with virtually any double-stranded DNA plasmid. The random mutagenesis protocols include methods based on PCR, degenerate oligonucleotides, cassette mutagenesis, nested deletion mutagenesis, and a specialized E. coli mutator strain. These invaluable protocols facilitate the study of gene regulation and structure/function relationships in proteins and permit modification of DNA sequences for purposes such as vector construction.
Hands-on researchers with proven track records describe in stepwise fashion their advanced mutagenesis techniques. The contributors focus on improvements to conventional site-directed mutagenesis, including a chapter on chemical site-directed mutagenesis, PCR-based mutagenesis and the modifications that allow high throughput mutagenesis experiments, and mutagenesis based on gene disruption (both in vitro- and in situ-based). Additional methods are provided for in vitro gene evolution; for gene disruption based on recombination, transposon, and casette mutagenesis; and for facilitating the introduction of multiple mutations. Time-tested and highly practical, the protocols in In Vitro Mutagenesis Protocols, 2nd Edition offer today's molecular biologists reliable and powerful techniques with which to illuminate the proteome.
In vitro mutagenesis remains a critical experimental approach for investigating gene and protein function at the cellular level. This volume provides a wide variety of updated and novel approaches for performing in vitro mutagenesis using such methods as genome editing, transposon (Tn) mutagenesis, site-directed, and random mutagenesis. In Vitro Mutagenesis: Methods and Protocols guides readers through methods for gene and genome editing, practical bioinformatics approaches for identifying mutagenesis targets, and novel site-directed and random mutagenesis approaches aimed at gaining a better understanding of protein-protein and protein-cofactor interactions. Written in the highly successful Methods in Molecular Biology series format, 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 cutting-edge, In Vitro Mutagenesis: Methods and Protocols aims to provide a highly accessible and practical manual for current and future molecular biology researchers, from the beginner practitioner to the advanced investigator in fields such as molecular genetics, biochemistry, and biochemical and metabolic engineering.
The purpose of this volume is to highlight some current areas of poxvirus research which are likely to be particularly fruitful in the upcoming few years. The first chapter, by Drs. Condit and Niles, discusses poxvirus genetics. Work in this area has provided mutants, produced practical procedures to simplify the manipulation of viral genes, and generated information about the molecular architecture and organization of genes characteristic of pox viruses. One of the most intensively studied regions of the viral genome is the HindIII D region of vaccinia, in which a combination of classical and molecular genetic analysis of the region has been particularly revealing. Within this region are open reading frames, some of which are expressed early and others late, organized in a fashion which is now known to be typical of these viruses. Other studies, related to temperature sensitive, drug resistant, and drug dependent mutants, are also discussed. Each of the other reviews included in this volume summarizes areas of research which have depended heavily on the genetics of the system. The intracellular site of a poxvirus infection is mostly, if not exclusively, limited to the cytoplasm which dictates several interesting biological ramifications. For example, poxvirus transcription must occur in the cytoplasm, rather than in the nucleus. The virus copes with this situation by incorporating into the virion the enzymatic machinery necessary to initiate transcription from input virus.
Proteins: Analysis and Design focuses solely on individual experimental approaches, rather than on specific classes of proteins. The book provides insight into the important issues in protein science and how one can cope with them. These include all issues which explore the detailed relationship of protein structure to function. Provides problems and technical solutions Includes posttranslational modifications Uses synthetic peptides as biological models Details mutagenesis and protein engineering Covers design of protein structure and function
Comprehensive Biomaterials II, Second Edition brings together the myriad facets of biomaterials into one expertly-written series of edited volumes. Articles address the current status of nearly all biomaterials in the field, their strengths and weaknesses, their future prospects, appropriate analytical methods and testing, device applications and performance, emerging candidate materials as competitors and disruptive technologies, research and development, regulatory management, commercial aspects, and applications, including medical applications. Detailed coverage is given to both new and emerging areas and the latest research in more traditional areas of the field. Particular attention is given to those areas in which major recent developments have taken place. This new edition, with 75% new or updated articles, will provide biomedical scientists in industry, government, academia, and research organizations with an accurate perspective on the field in a manner that is both accessible and thorough. Reviews the current status of nearly all biomaterials in the field by analyzing their strengths and weaknesses, performance, and future prospects Covers all significant emerging technologies in areas such as 3D printing of tissues, organs and scaffolds, cell encapsulation; multimodal delivery, cancer/vaccine - biomaterial applications, neural interface understanding, materials used for in situ imaging, and infection prevention and treatment Effectively describes the many modern aspects of biomaterials from basic science, to clinical applications
In the second edition of this bestselling textbook, new materials have been added, including a new chapter on real time polymerase chain reaction (RTPCR) and a chapter on fungal solid state cultivation. There already exist a number of excellent general textbooks on microbiology and biotechnology that deal with the basic principles of microbial biotechnology. To complement them, this book focuses on the various applications of microbial-biotechnological principles. A teaching-based format is adopted, whereby working problems, as well as answers to frequently asked questions, supplement the main text. The book also includes real life examples of how the application of microbial-biotechnological principles has achieved breakthroughs in both research and industrial production. Although written for polytechnic students and undergraduates, the book contains sufficient information to be used as a reference for postgraduate students and lecturers. It may also serve as a resource book for corporate planners, managers and applied research personnel.
A collection of basic cutting-edge techniques for studying the mechanisms underlying cell cycle regulation and checkpoint control. Using mammalian, yeast, and frog systems, these readily reproducible methods can be used to induce cell cycle checkpoints, detect changes in cell cycle progression, identify and analyze genes and proteins that regulate the process, and characterize chromosomal status as a function of cell cycle phase and progression. Each fully tested technique includes step-by-step instructions written by an investigator who routinely performs it, an introduction explaining the principle behind the method, equipment and reagent lists, and tips on troubleshooting and avoiding known pitfalls.
Comprehensive Biomaterials brings together the myriad facets of biomaterials into one, major series of six edited volumes that would cover the field of biomaterials in a major, extensive fashion: Volume 1: Metallic, Ceramic and Polymeric Biomaterials Volume 2: Biologically Inspired and Biomolecular Materials Volume 3: Methods of Analysis Volume 4: Biocompatibility, Surface Engineering, and Delivery Of Drugs, Genes and Other Molecules Volume 5: Tissue and Organ Engineering Volume 6: Biomaterials and Clinical Use Experts from around the world in hundreds of related biomaterials areas have contributed to this publication, resulting in a continuum of rich information appropriate for many audiences. The work addresses the current status of nearly all biomaterials in the field, their strengths and weaknesses, their future prospects, appropriate analytical methods and testing, device applications and performance, emerging candidate materials as competitors and disruptive technologies, and strategic insights for those entering and operational in diverse biomaterials applications, research and development, regulatory management, and commercial aspects. From the outset, the goal was to review materials in the context of medical devices and tissue properties, biocompatibility and surface analysis, tissue engineering and controlled release. It was also the intent both, to focus on material properties from the perspectives of therapeutic and diagnostic use, and to address questions relevant to state-of-the-art research endeavors. Reviews the current status of nearly all biomaterials in the field by analyzing their strengths and weaknesses, performance as well as future prospects Presents appropriate analytical methods and testing procedures in addition to potential device applications Provides strategic insights for those working on diverse application areas such as R&D, regulatory management, and commercial development