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DNA sequencing has become increasingly efficient over the years, resulting in an enormous increase in the amount of data gener ated. In recent years, the focus of sequencing has shifted, from being the endpoint of a project, to being a starting point. This is especially true for such major initiatives as the human genome project, where vast tracts of DNA of unknown function are sequenced. This sheer volume of available data makes advanced computer methods essen tial to analysis, and a familiarity with computers and sequence analy sis software a vital requirement for the researcher involved with DNA sequencing. Even for nonsequencers, a familiarity with sequence analysis software can be important. For instance, gene sequences already present in the databases can be extremely useful in the design of cloning and genetic manipulation experiments. This two-part work on Computer Analysis of Sequence Data is designed to be a practical aid to the researcher who uses computers for the acquisition, storage, or analysis of nucleic acid (and/or pro tein) sequences. Each chapter is written such that a competent scien tist with basic computer literacy can carry out the procedure successfully at the first attempt by simply following the detailed prac tical instructions that have been described by the author. A Notes section, which is included at the end of each chapter, provides advice on overcoming the common problems and pitfalls sometimes encoun tered by users of the sequence analysis software.
DNA sequencing has become increasingly efficient over the years, resulting in an enormous increase in the amount of data gen- ated. In recent years, the focus of sequencing has shifted, from being the endpoint of a project, to being a starting point. This is especially true for such major initiatives as the human genome project, where vast tracts of DNA of unknown function are sequenced. This sheer volume of available data makes advanced computer methods ess- tial to analysis, and a familiarity with computers and sequence ana- sis software a vital requirement for the researcher involved with DNA sequencing. Even for nonsequencers, a familiarity with sequence analysis software can be important. For instance, gene sequences already present in the databases can be extremely useful in the design of cloning and genetic manipulation experiments. This two-part work on Analysis of Data is designed to be a practical aid to the researcher who uses computers for the acquisition, storage, or analysis of nucleic acid (and/or p- tein) sequences. Each chapter is written such that a competent sci- tist with basic computer literacy can carry out the procedure successfully at the first attempt by simply following the detailed pr- tical instructions that have been described by the author. A Notes section, which is included at the end of each chapter, provides advice on overcoming the common problems and pitfalls sometimes enco- tered by users of the sequence analysis software. Software packages for both the mainframe and personal computers are described.
Probabilistic models are becoming increasingly important in analysing the huge amount of data being produced by large-scale DNA-sequencing efforts such as the Human Genome Project. For example, hidden Markov models are used for analysing biological sequences, linguistic-grammar-based probabilistic models for identifying RNA secondary structure, and probabilistic evolutionary models for inferring phylogenies of sequences from different organisms. This book gives a unified, up-to-date and self-contained account, with a Bayesian slant, of such methods, and more generally to probabilistic methods of sequence analysis. Written by an interdisciplinary team of authors, it aims to be accessible to molecular biologists, computer scientists, and mathematicians with no formal knowledge of the other fields, and at the same time present the state-of-the-art in this new and highly important field.
Chromosomes, as the genetic vehicles, provide the basic material for a large proportion of genetic investigations, from the construction of gene maps and models of chromosome organization, to the inves tigation of gene function and dysfunction. The study of chromosomes has developed in parallel with other aspects of molecular genetics, beginning with the first preparations of chromosomes from animal cells, through the development of banding techniques, which permitted the unequivocal identification of each chromosome in a karyotype, to the present analytical methods of molecular cytogenetics. Although some of these techniques have been in use for many years, and can be learned relatively easily, most published scientific reports—as a result of pressure on space from editors, and the response to that pressure by authors—contain little in the way of technical detail, and thus are rarely adequate for a researcher hoping to find all the necessary information to embark on a method from scratch. A new user needs not only a detailed description of the methods, but also some help with problem solving and sorting out the difficulties en countered in handling any biological system. This was the require ment to which the series Methods in Molecular Biology is addressed, and Chromosome Analysis Protocols forms a part of this series.
The study of protein-nucleic acid interactions is currently one of the most rapidly growing areas of molecular biology. DNA binding proteins are at the very heart of the regulation and control of gene expression, replication, and recombination: Enzymes that recognize and either modify or cleave specific DNA sequences are equally important to the cell. Some of the techniques reported in this volume can be used to identify previously unknown DNA binding proteins from crude cell extracts. Virtually all are capable of giving direct information on the molecular basis of the interaction—the location of the DNA binding site; the strength and specificity of binding; the identities of individual groups on specific bases involved in binding; the specific amino acid residues of the protein that interact with the DNA; or the effects of protein binding on gross conformation and local structure of DNA. The recognition of DNA sequences by proteins is a complex phenomenon, involving specific hydrogen bonding contacts to the DNA bases ("direct readout") and/or interactions with the sugar-phos phate backbone ("indirect readout"). The latter interactions can also be highly specific because of sequence-dependent conformational changes in the DNA. In addition, intercalation of planar aromatic amino acid side-chains between the DNA bases can occur, most notably with single-stranded DNA binding proteins. Furthermore, when bound, many DNA binding proteins induce drastic structural changes in the DNA as an integral part of their function.
The storage of biological material for regular or future use is a fundamental requirement in many biological and medical sciences. Cryopreservation and freeze-drying are the preferred techniques for achieving long-term storage, and have been applied to a diverse range of biological materials. Though the basis for many methodologies is common, laboratories frequently lack expertise with the correct storage procedures, so that many apply outdated or inappropriate protocols for storing their samples or cultures. Cryopreservation and Freeze-Drying Protocols is a compilation of the many and varied methodologies that have been developed in expert laboratories. The protocols are reproducible, robust, and in most instances have been transferred quite successfully to other laboratories. Our intended readers are those proposing to establish or improve biostorage systems in their own laboratories or units, whether concerned with culture collections, animal husbandry, aquaculture, or human fertilization programs. Because the emphasis of Cryopreservation and Freeze-Drying Protocols is on methodology, it is our intention to provide readers with the tools to make practical progress without reference to other sources. Each chapter deals with an organelle, cell, or tissue type: a short int- duction on the status of its biostorage development is followed by a detailed description of the materials required and a methodological p- tocol to be followed, with explanatory notes. This is very much a first edition; we hope and trust that future editions will contain cryopreservation and freeze-drying protocols for ceils, tissues, and organs that are at present still recalcitrant to succe- ful preservation.
Most laboratories conducting studies that use molecular biology techniques employ in vitro transcription and translation systems as a routine part of their day-to-day research. The commercial availability of purified bacterial RNA polymerase and the availability of robust tra- lation systems has made in vitro systems attractive not only as an alt- native to the in vivo expression of genes, but also as good model systems for studying specific aspects of transcription and translation. Although fairly efficient eukaryotic translation systems have been established for a number of years, reconstitution of transcription in vitro has proved to be more difficult. Recent improvements in fractionation techniques and the cloning of proteins involved in transcription have made this a fast moving area of research. Considerable progress has also been made in recent years in developing in vitro systems to study transcription and translation in chloroplasts and mitochondria, together with systems for the study of protein import. In Vitro Transcription and Translation Protocols provides many detailed experimental procedures for prokaryotic transcription and translation systems, together with protocols for many key techniques used in the analysis of eukaryotic transcription. In keeping with the successful format of preceding volumes of the Methods in Molecular Biology series, step-by-step instructions are provided, together with extensive notes that cover troubleshooting and special tips considered important.
The principle that antibodies can be used as cytochemical agents provided they are tagged with suitable markers has been evident for over 50 years. During this time the use of immunocytochemical meth ods has spread to a wide array of biological disciplines. Early applica tions focused on the detection of microbial antigens in tissues, while more recent applications have used monoclonal antibodies to study cell differentiation during embryonic development. For a select few disci plines, volumes have been published focusing on the specific applica tion of immunocytochemical techniques to that discipline. What distinguishes the present book, Immunocytochemical Meth ods and Protocols, from earlier books is its broad appeal to researchers in all disciplines, including those in both research and clinical settings. The methods and protocols presented here are designed to be general in their application and the accompanying "Notes" provide invaluable assistance in adapting or troubleshooting the protocols. Interspersed throughout the book are chapters providing overviews of select topics related to immunocytochemistry.
As our understanding of the biological sciences expands, the bou- aries between traditional disciplines tend to blur at the edges. Physio- gists and pharmacologists, for instance, now need to embrace techniques that until recently were the strict preserves of biochemists and mole- lar biologists. However, the acquisition of new technologies can be a time-consuming and frustrating business, and unless an expert is on hand to give instruction, precious hours can be spent poring over half-described Methods sections with no guarantee of eventual success. The aim of Signal Transduction Protocols has been to get experts with "hands-on" experience in particular techniques to give detailed accounts of experimental protocols in a recipe-type format, which we hope will circumvent the problems of ambiguity often encountered when reading the literature. The techniques described in Signal Transduction Protocols are those that we think will be most useful in addressing questions in the area of receptor-mediated cell signaling, with particular regard to those receptors that are part of the G-protein-linked superfamily. To keep it to a manageable size, we have omitted any reference to electrophysi- ogy and have instead concentrated on more biochemical approaches.
ELISA: Theory and Practice introduces to scientists at all levels of expertise the principles of the most commonly used assay technique known as the Enzyme Linked Immunosorbent Assay. The book provides readers with full descriptions of the basic systems that make ELISA one of the most powerful techniques in science today, and also examines in detail the data obtained by ELISA and their analysis and actual manipulation. ELISA: Theory and Practice is designed not only to train novices in the science of ELISA, but also to aid investigators experienced in any of the biological sciences in performing independently assays of antibodies and antigens. Mastery of the book's contents will allow readers to fully appreciate exactly how and why assays function, as well as permit the efficient development of individual assays that are both rapid and accurate.