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.
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.
Micropropagation has become a reliable and routine approach for large-scale rapid plant multiplication, which is based on plant cell, tissue and organ culture on well defined tissue culture media under aseptic conditions. A lot of research efforts are being made to develop and refine micropropagation methods and culture media for large-scale plant multiplication of several number of plant species. However, many forest and fruit tree species still remain recalcitrant to in vitro culture and require highly specific culture conditions for plant growth and development. The recent challenges on plant cell cycle regulation and the presented potential molecular mechanisms of recalcitrance are providing excellent background for understanding on totipotency and what is more development of micropropagation protocols. For large-scale in vitro plant production the important attributes are the quality, cost effectiveness, maintenance of genetic fidelity, and long-term storage. The need for appropriate in vitro plant regeneration methods for woody plants, including both forest and fruit trees, is still overwhelming in order to overcome problems facing micropropagation such as somaclonal variation, recalcitrant rooting, hyperhydricity, polyphenols, loss of material during hardening and quality of plant material. Moreover, micropropagation may be utilized, in basic research, in production of virus-free planting material, cryopreservation of endangered and elite woody species, applications in tree breeding and reforestation.
Elizabeth M.J. Gillam,Janine N. Copp,David F. Ackerley
Author: Elizabeth M.J. Gillam,Janine N. Copp,David F. Ackerley
Directed Evolution Library Creation: Methods and Protocols, Second Edition presents user-friendly protocols for both proven strategies and cutting-edge approaches for the creation of mutant gene libraries for directed evolution. As well as experimental methods, information on current computational approaches is provided in a user-friendly format that will allow researchers to make informed choices without needing to comprehend the full technical details of each algorithm. Directed evolution has become a fundamental approach for engineering proteins to enhance activity and explore structure-function relationships, and has supported the rapid development of the field of synthetic biology over the last decade. Divided into three convenient sections, topics include point mutagenesis strategies, recombinatorial methods wherein genetic diversity is sourced from multiple parental genes that are combined via either homology-dependent or -independent techniques and a variety of computational methods to guide the design and analysis of mutant libraries. Written in the 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 protocols and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Directed Evolution Library Creation: Methods and Protocols, Second Edition will serve as a reliable manual for both novice and experienced protein engineers and synthetic biologists and will enable further technical innovation and the exploitation of directed evolution for a deeper understanding of protein design and function.
Recombinant DNA methods are powerful, revolutionary techniques that allow the isolation of single genes in large amounts from a pool of thousands or millions of genes and the modification of these isolated genes or their regulatory regions for reintroduction into cells for expression at the RNA or protein levels. These attributes lead to the solution of complex biological problems and the production of new and better products in the areas of medicine, agriculture, and industry. Recombinant DNA Methodology, a volume in the Selected Methods in Enzymology series produced in benchtop format, contains a selection of key articles from Volumes 68, 100, 101, 153, 154, and 155 of Methods in Enzymology. The essential and widely used procedures provided at an affordable price will be an invaluable aid to the graduate student and the researcher. Enzymes in DNA research DNA isolation, hybridization, and cloning DNA sequence analysis cDNA cloning Gene products Identification of cloned genes and mapping of genes Monitoring cloned gene expression Cloning and transferring of genes into yeast cells Cloning and transferring of genes into plant cells Cloning and transferring of genes into animal cells Site-directed mutagenesis Protein engineering Expression vectors
Ashutosh Kumar,Vasily N. Dobrovolsky,Alok Dhawan,Rishi Shanker
Author: Ashutosh Kumar,Vasily N. Dobrovolsky,Alok Dhawan,Rishi Shanker
Publisher: Academic Press
Mutagenicity: Assays and Applications presents an extensive examination of the detection, assessment and future of mutagenicity, particularly as it concerns human health and the environment. Chapters focused on specific types of mutagens or testing methods for their detection collectively explore the current state of human and environmental mutagenesis, future perspectives and regulatory needs. The test procedures for measuring mutagenicity, their advantages and limitations are described with practical and procedural detail, along with their presentation and data processing aspects. It is an essential reference covering the breadth and depth of the field of mutagenicity studies and regulation. By providing both important introductory material and practical assays and applications, this book is useful to graduate students, academic and industry researchers and regulators at various stages of their careers, leading to improved risk assessment and regulation. Presents an up-to-date and in-depth review of the current state of mutagenesis research Draws upon the combined experience and expertise of an international group of highly respected editors and chapter authors Provides an introduction to the concept of mutagenesis with particular consideration given to novel chemicals and materials
The authors present a comprehensive collection of readily reproducible techniques for the manipulation of recombinant plasmids using the bacterial host E. coli. The authors describe proven methods for cloning DNA into plasmid vectors, transforming plasmids into E. coli, and analyzing recombinant clones. They also include protocols for the construction and screening of libraries, as well as specific techniques for specialized cloning vehicles, such as cosmids, bacterial artificial chromosomes, 1 vectors, and phagemids. Common downstream applications such as mutagenesis of plasmids and the use of reporter genes, are also described.
The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity. In this new edition, authors Joseph Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology. Handsomely redesigned and presented in new bindings of proven durability, this three–volume work is essential for everyone using today’s biomolecular techniques. The opening chapters describe essential techniques, some well–established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small. These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing. The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein–protein interactions. The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information. As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved.
The Condensed Protocols From Molecular Cloning: A Laboratory Manualis a singleâ€“volume adaptation of the threeâ€“volume third edition of Molecular Cloning: A Laboratory Manual.This condensed book contains only the stepâ€“byâ€“step portions of the protocols, accompanied by selected appendices from the world's bestâ€“selling manual of molecular biology techniques. Each protocol is crossâ€“referenced to the appropriate pages in the original manual. This affordable companion volume, designed for bench use, offers individual investigators the opportunity to have their own personal collection of short protocols from the essential Molecular Cloning.
In this second edition of a much praised laboratory manual devoted to eukaryotic systems, Daryl S. Henderson has refocused the book on mammalian cells, adding fourteen entirely new chapters and extensively revising many of the remaining chapters. The authors address a broad range of questions about practical mammalian DNA repair, including such arcana as "what is radioresistant DNA synthesis and how is it measured?" The techniques presented are readily reproducible and offer cutting-edge methods for cytogenetic analysis, measuring the cellular response to ionizing radiation, detecting single-strand (nicks) and double-strand DNA breaks, detecting the presence of "adducted" bases in DNA, and preparing mismatch repair (MMR) plasmid substrates. Among the highlights are excellent coverage of both base excision repair (BER) and nucleotide excision repair (NER), useful assays for identifying and quantifying UV-induced DNA lesions and DNA breakage, gene therapy, environmental mutagenesis and cancer, and gene targeting. 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. Authoritative and highly practical, DNA Repair Protocols: Mammalian Systems, Second Edition, offers investigators a wide variety of productive methods to explore and make new discoveries in the world of mammalian DNA repair.
Seasoned practitioners from many leading laboratories describe their best 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. An accompanying volume, Directed Evolution Library Creation: Methods and Protocols, describes readily reproducible methods for the creation of mutated DNA molecules and DNA libraries.
In this book, a panel of expert researchers and established group leaders describe in step-by-step detail the key methodologies of contemporary peptide systhesis and illustrate numerous applications that employ peptides as unique and essential materials. Techniques presented include protocols for chemical ligation, the systhesis of cyclic and phosphotyrosine-containing peptides, lipoamino acid- and sugar-conjugated peptides, and peptide purification and analysis.
As applied life science progresses, becoming fully integrated into the biological, chemical, and engineering sciences, there is a growing need for expanding life sciences research techniques. Anticipating the demands of various life science disciplines, Laboratory Protocols in Applied Life Sciences explores this development. This book covers a wide spectrum of areas in the interdisciplinary fields of life sciences, pharmacy, medical and paramedical sciences, and biotechnology. It examines the principles, concepts, and every aspect of applicable techniques in these areas. Covering elementary concepts to advanced research techniques, the text analyzes data through experimentation and explains the theory behind each exercise. It presents each experiment with an introduction to the topic, concise objectives, and a list of necessary materials and reagents, and introduces step-by-step, readily feasible laboratory protocols. Focusing on the chemical characteristics of enzymes, metabolic processes, product and raw materials, and on the basic mechanisms and analytical techniques involved in life science technological transformations, this text provides information on the biological characteristics of living cells of different origin and the development of new life forms by genetic engineering techniques. It also examines product development using biological systems, including pharmaceutical, food, and beverage industries. Laboratory Protocols in Applied Life Sciences presents a nonmathematical account of the underlying principles of a variety of experimental techniques in disciplines, including: Biotechnology Analytical biochemistry Clinical biochemistry Biophysics Molecular biology Genetic engineering Bioprocess technology Industrial processes Animal Plant Microbial biology Computational biology Biosensors Each chapter is self-contained and written in a style that helps students progress from basic to advanced techniques, and eventually design and execute their own experiments in a given field of biology.
The critically acclaimed laboratory standard for forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerlyawaited, frequently consulted, and praised by researchers and reviewers alike. More than 250 volumes have been published (all of them still in print) and much of the material is relevant even today--truly an essential publication for researchers in all fields of life sciences. * Methods for: * DNA isolation and cloning * Synthesizing complementary DNA (cDNA) * Cleaving and manipulating DNA * Selecting useful reporter genes * Constructing vectors for cloning genes * Constructing expression vectors * Site-directed mutagenesis and gene disruption * Identifying and mapping genes * Transforming animal and plant cells * Sequencing DNA * Amplifying and manipulating DNA and PCR * Detecting DNA - protein interaction
Annotation 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.
In Vitro Toxicology details the protocols and methods of in vitro testing, highlighting the usefulness of models, methods and the cost-effectiveness and reproducibility of such methodologies. The current approaches and strategies required to develop an easy, reliable, validated and high throughput system for use in alternative animal models to circumvent in vivo testing are discussed in detail. The book also includes chapters on the principles involved in the general selection and use of models that address safety concerns, regulatory acceptance and the current understandings and strategies for the identification of biomarkers in toxicity testing. Furthermore, principles involved in the general selection and use of models that address the issues of safety concerns and regulatory acceptance of these models are discussed, making the book beneficial to students, scientists, and regulators working in toxicology, as well as those in the field of chemicals and the safety assessment of novel materials. Discusses new techniques and protocols in a clear and concise manner includes examinations of nanotoxicity, genotoxicity and carcinogenicity Explains practical laboratory methods and the theories behind in vitro testing
Author: Radhakrishnan Padmanabhan,Subhash G. Vasudevan
Publisher: Humana Press
Infection by flaviviruses such as dengue virus serotypes (DENV 1-4), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBE), yellow fever virus (YFV) and West Nile virus (WNV) impact millions of lives and cause tens of thousands of mortalities each year. Dengue is a global public health emergency especially since there is no preventative vaccine or antiviral treatment for dengue disease. Dengue: Methods and Protocols offers the increasing number of dengue researchers a one-stop protocol book with techniques compiled from the leading laboratories working on dengue. Chapters cover topics such as dengue virus isolation from clinical samples, quantification of human antibodies against the virus, assays to quantify the virus particles, the widely used mouse model to study dengue pathogenesis, vaccine and antiviral efficacies. Written in the 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 protocols and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Dengue: Methods and Protocols seeks to serve both professionals and novices with its well-honed methodologies on dengue research.
A comprehensive source of technical guidance for experienced investigators and essential resource for newcomers to mammalian genetics and embryology. This edition (1st ed., 1986) is revised and expanded to incorporate improvements to existing methods as well as new technologies. It contains new sect