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Die Kapillarelektrophorese (CE) ist heute in biochemischen Laboratorien noch nicht als Routinemethode zur DNA-Trennung etabliert, obwohl sie diverse Vorzüge gegenüber den derzeitigen Methoden besitzt. In diesem Band stellen verschiedene Wissenschaftler die theoretischen Aspekte der Trennung von Oligonucleinsäuren mit der CE, die instrumentellen Grundlagen und deren Grenzen und schließlich die praktische Anwendung in der Biochemie und Molekularbiologie vor. This book on capillary electrophoresis is unique in its focus on the separation of nucleic acids. The importance of electrophoretic separation in every molecular biology laboratory justifies this specialization, which is also reflected in the development of instrumentation. This book is aimed to help to implement this rather new and promising technology in the biological laboratories and to help to overcome typical problems that can occur when starting with a new technique. It should also trigger further development in the field. It covers the theoretical background as well as practical examples of usual applications. The authors are all experts in their field, having many years of experience with capillary electrophoresis and their application to nucleic acids.
The development of PCR, which enables extremely small amounts of DNA to be amplified, led to the rapid development of a multiplicity of a- lytical procedures that permit use of this new resource for the analysis of genetic variation and for the detection of disease-causing mutations. The advent of capillary electrophoresis (CE), with its power to separate and a- lyze very small amounts of DNA, has also stimulated researchers to develop analytical procedures for the CE format. The advantages of CE in terms of speed and reproducibility of analyses are manifold. Furthermore, the high s- sitivity of detection, and the ability to increase sample throughput with par- lel analysis, has led to the creation of a full range of analysis of DNA molecules, from modified DNA adducts and single-strand oligonucleotides through PCR-amplified DNA fragments and whole chromosomes. Capillary Elect- phoresis of Nucleic Acids focuses on analytical protocols that can be used for detection and analysis of mutations and modification, from precise DNA loci through entire genomes of organisms. Important practical considerations for CE, such as the choice of separation media, electrophoresis conditions, and the influence of buffer additives and dyes on DNA mobility, are discussed in several key chapters and within particular applications.
Over the last decade there has been a rapid development of molecular techniques, with an increasing range of instrumentation now available. The development of accompanying reference literature has not kept pace with technological advances and this poses significant challenges to the analyst. Essentials of Nucleic Acid Analysis sets out to guide the analyst through the steps needed to obtain good quality results in DNA analysis. The underlying principles for achieving this goal were formulated by LGC (formerly the Laboratory of the Government Chemist) as the six principles for ensuring valid analytical measurement, which are detailed in the introduction. The reader is also provided with guidelines for method validation and quality control of established and emerging DNA measurement techniques. The authors of each chapter are practitioners of the art of DNA analysis in areas where the quality of the result is critical. Technical details and examples of application of key techniques in nucleic acid analysis are provided while highlighting best practice, available standards and practical advice on improving measurement quality. This book provides an indispensable handbook and premier reference for those working in the widely varying areas and specifically in the fields of food analysis and forensic applications.
Various sophisticated techniques such as capillary electrophoresis, pulsed-field electrophoresis, fingerprinting using RFLP and RAPD, DNA sequencing, and mobility shift assay are described here in detail. Leading experts present the required apparatus, appropriate use, preparation of probes, gel staining, interpretation of results, tricks for troubleshooting, manufacturers' addresses, helpful Internet resources, as well as specific applications, e.g. in legal medicine, microbiology and agriculture.
In Clinical Applications of Capillary Electrophoresis, Stephen Palfrey brings together for first time a collection of detailed capillary electrophoresis protocols designed exclusively for clinical applications. Written by the leading scientists who have often perfected these methods in their own laboratories, the protocols furnish new and more powerful assays for many routine serum and blood tests now regularly performed in clinical laboratories, including urine protein analysis, hemoglobin separation, and the detection of CSF proteins, lipoproteins, myoglobin, cryoglobulins, HbA1c, and cathepsin. The protocols offered for DNA studies include double-stranded DNA analysis, the prenatal diagnosis of Down's syndrome, Rh D/d genotyping, the identification of mutated p53 oncogene, and the detection of microsatellite instability in cancers. Many of the methods can be automated to replace the more costly and labor-intensive tests that are currently used in most clinical laboratories. Clinical Applications of Capillary Electrophoresis demonstrates clearly the simplicity, versatility, and power of CE over conventional methods. It offers to beginning clinical investigators, as well as established laboratories new to the technique, a representative range of highly practical CE methods-assays that are not only certain to become ever more productive, but are already eminently useful today.
Over the last decade, high performance Capillary electrophoresis (HPCE) has emerged as a powerful and versatile separation technique that promises to rival high performance liquid chromatography when applied to the separation of both charged and neutral species. The high speed and high separation efficiency which can be attained using any of the various modes of HPCE has resulted in the increased use of the technique in a range of analytical environments. The procedures are, however, still in the early stages of development and several barriers remain to their adoption as the technique of choice for a range of analytical problems. One such barrier is the selection and optimization of the conditions required to achieve reproducible separations of analytes and it is in this area that this new book seeks to give assistance. The book is written by an international team of authors, drawn from both academic and industrial users, and the manufacturers of instruments. At its heart are a number of tables, divided into specific application areas. These give details of published separations of a wide range of archetypal analytes, the successful separation conditions and the matrix in which they were presented. These tables are based on separations reported since 1992 and are fully referenced to the original literature. The tables are supported by discussions of the problems that a particular area presents and the strategies and solutions adopted to overcome them. The general areas covered are biochemistry, pharmaceutical science, bioscience, ion analysis, food analysis and environmental science.
The development of PCR, which enables extremely small amounts of DNA to be amplified, led to the rapid development of a multiplicity of a- lytical procedures that permit use of this new resource for the analysis of genetic variation and for the detection of disease-causing mutations. The advent of capillary electrophoresis (CE), with its power to separate and a- lyze very small amounts of DNA, has also stimulated researchers to develop analytical procedures for the CE format. The advantages of CE in terms of speed and reproducibility of analyses are manifold. Furthermore, the high s- sitivity of detection, and the ability to increase sample throughput with par- lel analysis, has led to the creation of a full range of analysis of DNA molecules, from modified DNA adducts and single-strand oligonucleotides through PCR-amplified DNA fragments and whole chromosomes. Capillary Elect- phoresis of Nucleic Acids focuses on analytical protocols that can be used for detection and analysis of mutations and modification, from precise DNA loci through entire genomes of organisms. Important practical considerations for CE, such as the choice of separation media, electrophoresis conditions, and the influence of buffer additives and dyes on DNA mobility, are discussed in several key chapters and within particular applications.
Through its clear presentation of the basic concepts, Gel Electrophoresis: Nucleic Acids breaks new ground by describing the principles of the technique without resorting to complicated protocols and recipes.
Providing current information and guidance on the uses of various nucleic acid amplification technologies for clinical laboratory diagnosis, this book goes beyond the Polymerase Chain Reaction to explore a broader range of important alternative DNA/RNA amplification methods including the Ligase Chain Reaction, Q[beta] Replicase Assays and TMA. There are many examples of specific applications of these technologies, discussions of yet unresolved issues and demonstrations of the relevance of these technologies to medical research and disease diagnostics. Individual chapters cover uses of these methods in clinical situations such as detection of food pathogens, viral infections, STDs, Mycobacteria drug resistance mutations, and heritable diseases. Automation, diagnostic test evaluation, and the synthesis of artificial DNA are also discussed. This book is designed for all biomedical scientists interested in the application of molecular biology to clinical diagnosis.