This is not just a cookbook for real-time quantitative PCR (qPCR). Admittedly, there are lots of recipes from distinguished contributors and Bustin has attempted to collect, sift through and rationalize the vast amount of information that is available on this subject. And yes, this book was conceived as a comprehensive hands-on manual to allow both the novice researcher and the expert to set up and carry out qPCR assays from scratch.
However, this book also sets out to explain as many features of qPCR as possible, provide alternative viewpoints and methods and, perhaps most importantly, aims to stimulate the researcher into generating, interpreting and publishing data that are reproducible, reliable and biologically meaningful.
In this book, leading scientists from academia as well as biotech and pharma companies introduce the revolutionary concept of designing RNA and DNA oligonucleotides with novel functions by in vitro selection. These functions comprise high affinity binding (aptamers), catalytic activity (ribozymes and deoxyribozymes) or combinations of binding and catalytic properties (aptazymes).
Basic concepts and technologies describing in detail how these functional oligonucleotides can be identified are presented. Numerous examples demonstrate the versatility of in vitro selected oligonucleotides. Special emphasis has been put on a section that shows the broad applicability of aptamers, e. g. in target validation, for analytics, or as new therapeutics. This first overview in the field is of prime interest for a broad audience of scientists both in academia and in industry who wish to expand their knowledge on the potential of new oligonucleotide functions and their applications.
This book is the first comprehensive guide devoted exclusively to calculations encountered in the genetic engineering laboratory. This guide, written for students, technicians, and scientists, provides example calculations for the most frequently confronted problems encountered in gene discovery and analysis.
The text and sample calculations are written in an easy-to-follow format. It is the perfect laboratory companion for anyone working in DNA manipulation and analysis.
This resource presents protocols for isolating a gene, cloning and characterizing it, expressing its encoded protein, and purifying and characterizing the protein′s physical properties. It includes background and procedures and is structured around 20 experiments that demonstrate how to prepare, manipulate, and analyze plasmids, produce fusion proteins in bacteria, and purify these proteins based on chemical properties or substrate affinities.
It describes topics such as the use of antibodies and techniques developed to transform their structures, and approaches designed to manipulate structure and functions of proteins and nucleic acids.
This book offers a set of readily reproducible protocols of the analysis of DNA methylation and methylases. These powerful methods provide the tools necessary for studying methylation at both the global level and the level of sequence, and include many techniques for identifying genes that might be aberrantly methylated in cancer and aging. Additional methods cover genome-wide analysis of abnormal DNA methylation and the isolation and measurement of demethylases and related proteins.
This is a novel compilation of methods for studying the interactions of proteins with DNA. Multidisciplinary approaches are valuable for solving problems and obtaining a detailed understanding of the molecular regulatory interactions involved. It covers all of the major tools that are required for the study of the large macromolecular enzymatic machines that manipulate DNA, with particular emphasis on biophysical techniques applied to the analysis of transcription and its relation to chromatin structure.
In Volume 2 of Essential Molecular Biology, procedures for preparing gene libraries and identifying genes are described, together with methods for studying the structure of a cloned gene and the way it is expressed in the cell.
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. This book covers expression across a broad range of systems, including the following. *Baculovirus expression vectors, *CHO cells, * E. coli, *HEK293-EBNA1 cells, * Lactococcus lactis, * 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. In addition, the book describes the optimisation of expression strategies, expression engineering using ribosome display, and how to select protein variants with improved expression.
Highly skilled investigators in gene targeting and mouse genetics describe their best techniques for the design of targeting constructs and for the analysis of the mouse phenotype. These include embryo transplantation, in vitro embryonic stem cell differentiation, creation of aggregation chimeras, mouse pathology, embryo cryopreservation, and transplantation. State-of-the-art and highly practical, it constitutes an invaluable source of readily reproducible techniques for genetic researchers today.
Gene transfer to animal cells was first achieved more than 30 years ago. Since then, transformation technology has developed rapidly, resulting in a multitude of techniques for cell transformation and the creation of transgenic animals. As with any expanding technology, it becomes difficult to keep track of all the developments and to find a concise and comprehensive source of information that explains all the underlying principles. This book describes the principles behind gene transfer technologies, how gene expression is controlled in animal cells and how advanced strategies can be used to add, exchange or delete sequences from animal genomes in a conditional manner. The final chapter provides an overview of all the applications of animal cell transformation.
This new laboratory protocols manual provides investigators with up-to-date RT-PCR based methods for gene cloning and analysis. Since RT-PCR was first described for the detection of low-abundance mRNAs, the method has been extended to quantitative measurements of relative and absolute levels of single and multiple mRNAs. Methods for obtaining RNA for RT-PCR have also been extended to include analysis of mRNA levels in clinical samples and even in single cells.
This book contains a comprehensive generic set of protocols for gene hunting and genome analysis. Drawing on emerging technologies in the fields of bioinformatics and proteomics, these protocols cover traditional genomics, and early therapeutic approaches exploiting the potential of gene therapy. Highlights include methods for the analysis of differential gene expression, SNP detection, comparative genomic hybridization, and the functional analysis of genes, as well as the use of bioinformatics for gene identification and the prediction of protein function.
This revised and updated edition of a recognized classic emphasizes tissue and cell in situ hybridization methods. Among the new techniques detailed are PNA probes for viral diagnostics, plant in situ hybridization, cell proliferation detection, and quantitation of in situ hybridization. There are also cutting-edge techniques for tissue microarrays, expanded embryology–developmental gene detection, and expanded cell culture. Derivative techniques presented include identification of transplanted cells, histones, nick-end labeling for apoptosis, the use of peptide nucleic acid probes, and in situ hybridization of plant specimens. The protocols include step-by-step laboratory instructions, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
The major histocompatibility complex is the subject of much research in the immunology field. There is a great deal of interest in MHC proteins and their function as antigen presenting molecules, and many immunology laboratories are investigating biochemical and genetic techniques to study these molecules. The identification of peptide transporter genes and the elution of peptides from MHC molecules illustrate how rapidly our understanding of the MHC locus has advanced. These volumes bring together the technologies, which make these advances possible. It explains in detail the process such as peptide translocation into the ER, the application of mass spectrometry to the analysis of peptides bound to MHC, and signal detection via MHC class II molecules, which lead to either activation or cell death.
Regarded as the "Bible" of mammalian embryo manipulation techniques, the third edition has been completely reorganized and rewritten. The result is a compilation of new, cutting-edge protocols that include embryonic stem cell production and genetic manipulation, mouse chimeras, mouse cloning, assisted reproduction strategies (including intracytoplasmic sperm injection and in vitro fertilization), whole embryo culture systems, electroporation, embryo and gamete cryopreservation and rederivation, and gene expression, as well as more extensive background information on the use of these techniques.
Molecular biology and genetic engineering have made a great impact on the methods and strategies of modern pharmaceutical and medicinal chemistry, transforming every step in the drug development process, from target finding to drug design and ADMET analysis. Even the synthesis and manufacture of pharmaceutically active compounds has benefited from novel approaches based on molecular biology tools. Topics include: Cellular assays in drug discovery; Transgenic models; Reporter gene assay systems; Stereoselective synthesis with recombinant enzymes; Nucleic acid drugs; Enantioseparation of chiral drugs; NMR-based drug discovery; Pharmacogenomics and toxicogenomics.
For nearly a quarter century Molecular Biology of the Cell has been the leading cell biology textbook. This tradition continues with the new Fifth Edition, which has been completely revised and updated to describe our current, rapidly advancing understanding of cell biology. To list but a few examples, a large amount of new material is presented on epigenetics; stem cells; RNAi; comparative genomics; the latest cancer therapies; apoptosis (now its own separate chapter); and cell cycle control and the mechanics of M phase (now integrated into one chapter). The hallmark features of Molecular Biology of the Cell have been retained, such as its consistent and comprehensive art program, clear concept headings, and succinct section summaries. Additionally, in response to extensive feedback from readers, the Fifth Edition now includes several new features. It is now more portable. Chapters 1-20 are printed and Chapters 21-25, covering multicellular systems, are provided as pdf files on the free Media DVD-ROM which accompanies the book.* And for the first time, Molecular Biology of the Cell now contains end-of-chapter questions. These problems, written by John Wilson and Tim Hunt, emphasize a quantitative approach and the art of reasoning from experiments, and -they will help students review and extend their knowledge derived from reading the textbook. The Media DVD-ROM, which is packaged with every copy of the book, contains PowerPoint® presentations with all of the figures, tables and micrographs from the text (available as JPEGs too). Also included is the Media Player, which plays over 125 movies—animations, videos, and molecular models—all with voiceover narration. A new reader-friendly feature is the integration of media codes throughout the text that link directly to relevant videos and animations. The Media DVD-ROM holds the multicellular systems chapters (21-25) of the text as well. By skillfully extracting the fundamental concepts from this enormous and ever-growing field, the authors tell the story of cell biology, and thereby create a coherent framework through which readers may approach and enjoy this subject that is so central to all of biology.
This edition is a massive revision of the previous edition. It is the classic in-depth text reference in cell biology, yet puts the latest hot topics sensibly in context - including genomics, protein structure, array technology, stem cells and genetic diseases. It incorporates and emphasized new genomic data. It not only sets forth the current understanding of cell biology, but also explores the intriguing implications and possibilities of that which remains unknown. It contains over 1,600 illustrations, electron micrographs, and photographs, of which over 1,000 are originally conceived by the authors. "Cell Biology Interactive", a CD-ROM, is packaged with every copy.
This sixth edition of James D. Watson′s classic textbook Molecular Biology of the Gene has been thoroughly revised and updated. Accessible to anyone interested in molecular biology and genetics, the book provides a historical basis for the field, concise descriptions of fundamental chemical concepts, a comprehensive survey of genome maintenance and expression, and a discussion of standard techniques and model organisms commonly used in molecular biology studies. It includes all new chapters on the regulatory RNAs and genomics and systems biology. The book has an accompanying Web site (www.aw-bc.com/watson/), which contains interactive tutorials, animations, and critical-thinking exercises designed to help students explore and visualize complex concepts.
In this new edition, authors Joe 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. 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. It includes 240 laboratory protocols in DNA science in which over 35% were created especially for this edition, along with coverage of bioinformatics and DNA microarrays.
A methods manual for studying all aspects of NO and its metabolism. Readily reproducible techniques form a core of effective experimental methods for researchers new to the field and serve as a ready reference for experienced investigators. Cloning and expression of NO synthase isoforms, measurement of NO synthase activity, direct and indirect quantitation of NO, and the role of NO in DNA damage and apoptosis are all covered. In addition to stepwise instructions, each protocol also summarizes pros and cons and describes pitfalls and troubleshooting.
This book provides a view of the molecular structures of DNA and RNA and how they are recognized by small molecules and proteins. Source material is provided, including information on relevant web sites and computer programs. The major methods of structural investigation for nucleic acids, x-ray crystallography, NMR, and molecular modelling, are reviewed and their scope and limitations discussed. Also covered are the conformational features of nucleic acid building blocks. Both covalent and non-covalent nucleic acid interactions with small molecules are described, with the emphasis on recognition principles and sequence specific gene recognition.
This edition update and expands Bruce White′s best-selling "PCR 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 PCR and GC-rich template amplification. Powerful applications of PCR in library construction and sublibrary generation and screening are presented.
This second edition of a much praised and widely used manual has been entirely revised and updated. Each technique is presented with extensive background information, advice, and troubleshooting. All current applications of PCR are covered in protocols that have the hallmark reliability of the previous edition.
The spectres of completely new infectious diseases and multiple drug-resistant pathogens are frightening reminders of our frailty. Modern PCR techniques provide the best methods for identifying the causes. This book presents 12 detailed protocols that can serve as models for development of new protocols when they are needed.
This book covers gene analysis at the level of restriction fragment polymorphism, point mutation, and sequence analysis. Methods are given for using different source materials: cloned DNA, genomic DNA, RNA, nucleic acid from archive material, and PCR products themselves.
A detailed practical guide for laboratory use aimed at those who wish to use efficient and reliable protocols in their work and at researchers checking the validity and interpretation of published data. These molecular cytogenetic methods can be used to characterize and locate nucleic acid sequences within the cell. It is presented in a user-friendly, open format for easy access to information with an extensive troubleshooting guide.
This hand-on guide to RNA interference makes the power of targeted gene silencing available to any laboratory with a basic equipment for the handling of nucleic acids. In easy to follow step-by-step protocols you will learn: * How RNAi works in plants, flies and mammals * How to design the most efficient RNAi constructs * How to achieve transient, stable and conditional RNAi in cell cultures * How to determine the efficiency of an RNAi experiment * How to use RNAi for gene therapy. All protocols have been thoroughly tested in the author′s laboratory. Examples of successful experiments and troubleshooting hints are provided to help in establishing your own successful RNAi experiments.
With a variety of detection chemistries, an increasing number of platforms, multiple choices for analytical methods and the jargon emerging along with these developments, real-time PCR is facing the risk of becoming an intimidating method, especially for beginners. Real-time PCR provides the basics, explains how they are exploited to run a real-time PCR assay, how the assays are run and where these assays are informative in real life. It addresses the most practical aspects of the techniques with the emphasis on ′how to do it in the laboratory′.
The new edition expanded to 2 volumes provides condensed descriptions of more than 700 methods compiled from Current Protocols in Molecular Biology. The books are specifically designed to provide quick access to step-by-step instructions for the essential methods used in every major area of molecular biological research. Includes new chapters on chromatin assembly and analysis, nucleic acid arrays, generation and use of combinatorial libraries, discovery and analysis of differentially expressed genes in single cells and cell populations.
As molecular and cellular biologists move toward nano–techniques for performing experiments on single molecules rather than on populations of molecules, a comprehensive manual on how (and why) to carry out such experiments is needed. Single-Molecule Techniques: A Laboratory Manual fills this requirement—it is the first to take researchers who know nothing about single–molecule analyses to the point where they can successfully design and execute appropriate experiments. Geared toward research scientists in structural and molecular biology, biochemistry, and biophysics, the manual will be useful to all who are interested in observing, manipulating, and elucidating the molecular mechanisms and discrete properties of macromolecules. Techniques range from in vivo and in vitro fluorescent–based methods to the use of atomic force microscopy, optical and magnetic tweezers, and nanopores. The book is edited by Paul R. Selvin and Taekjip Ha, two pioneers in the field of experimental biophysics who have made significant contributions to the development and application of single–molecule technologies.
This update presents techniques for generation and analysis of transgenic animals, with emphasis on manipulation of the mammalian genome. It concentrates on creation and maintenance of genetically modified murine strains, providing access to the germline by conventional pronuclear injection, and by retroviral and adenoviral infection. Emphasis is given to the generation, maintenance, and manipulation of embryonic stem cell lineages, with protocols for constitutive and conditional gene targeting. Chapters include techniques for cryopreservation of male and female germlines and for generation of transgenic sheep by nuclear transfer.
Using Whole Genome Amplification (WGA) methods, it is possible to create microgram quantities of DNA starting with as little as one nanogram of genomic DNA and in some cases even a single eukaryotic or bacterial cell. The implementation of such WGA methods provides an ample supply of DNA for large-scale genetic studies. This title provides a comprehensive overview of the field and will be welcomed by all researchers looking to take advantage of the latest developments.
This volume, part of the Advances in Molecular Biology series, presents detailed protocols and trouble-shooting advice on the yeast two-hybrid system, one of the most powerful and versatile methods for characterizing a protein′s functions. Topics include: characterizing hormone/receptor complexes, identifying peptide ligands, how to identify mutations that disrupt an interaction, how to construct an activation domain hybrid library, and how to dissect the cell cycle and other complex genetic networks.
