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The in situ hybridization and PCR technologies are now well-established molecular techniques for studying chromosomal aneuploidy and rearran- ments, gene localization and expression, and genomic organization. Over the last decade, we have seen increasing applications in these fields. By combining the high sensitivity of the PCR reaction and the cytological localization of target sequences, both PRINS and in situ PCR techniques have provided highly powerful complements to FISH for in situ cellular and molecular investigations. Both these approaches have several advantages in terms of sensitivity and specificity, owing to the use of primers and to the fast kinetics of annealing and elongation reactions in situ. In the first edition of PRINS and In Situ PCR Protocols edited by John R. Gosden, experts in the field presented in detail a variety of applications of PRINS and in situ PCR techniques, in a wide range of clinical conditions. Since the publication of this successful reference book, there have been s- nificant improvements in in situ detection techniques. This completely revised and updated second edition presents a compreh- sive selection of new procedures developed in the field of PRINS and in situ PCR technologies. The book has two sections. Part I, Basic Methodology, contains chapters that provide useful protocols for many variations of PRINS and in situ PCR, including a new fast multicolor PRINS method, and protocols for PRINS detection of unique sequences in situ.
The in situ hybridization and PCR technologies are now well-established molecular techniques for studying chromosomal aneuploidy and rearran- ments, gene localization and expression, and genomic organization. Over the last decade, we have seen increasing applications in these fields. By combining the high sensitivity of the PCR reaction and the cytological localization of target sequences, both PRINS and in situ PCR techniques have provided highly powerful complements to FISH for in situ cellular and molecular investigations. Both these approaches have several advantages in terms of sensitivity and specificity, owing to the use of primers and to the fast kinetics of annealing and elongation reactions in situ. In the first edition of PRINS and In Situ PCR Protocols edited by John R. Gosden, experts in the field presented in detail a variety of applications of PRINS and in situ PCR techniques, in a wide range of clinical conditions. Since the publication of this successful reference book, there have been s- nificant improvements in in situ detection techniques. This completely revised and updated second edition presents a compreh- sive selection of new procedures developed in the field of PRINS and in situ PCR technologies. The book has two sections. Part I, Basic Methodology, contains chapters that provide useful protocols for many variations of PRINS and in situ PCR, including a new fast multicolor PRINS method, and protocols for PRINS detection of unique sequences in situ.
In this new edition, the editors have thoroughly updated and dramatically expanded the number of protocols to take advantage of the newest technologies used in all branches of research and clinical medicine today. These proven methods include real time PCR, SNP analysis, nested PCR, direct PCR, and long range PCR. Among the highlights are chapters on genome profiling by SAGE, differential display and chip technologies, the amplification of whole genome DNA by random degenerate oligonucleotide PCR, and the refinement of PCR methods for the analysis of fragmented DNA from fixed tissues. Each fully tested protocol is described in step-by-step detail by an established expert in the field and includes a background introduction outlining the principle behind the technique, equipment and reagent lists, tips on trouble shooting and avoiding known pitfalls, and, where needed, a discussion of the interpretation and use of results.
The technique of in situ hybridization, in its various forms, has been used routinely in many laboratories for a number of years. In the post-genome era, gene arrays and proteomics have allowed us to identify hitherto unknown unrecognized pathways and mechanisms. However, rather than diminish the importance of in situ hybridization, the now widespread use of screening te- nologies has increased the need to temporally and spatially localize the dist- bution of mRNA expression. Our intention, in In Situ Hybridization Protocols is to provide ample inf- mation for novices planning to set up the in situ hybridization technique and use it in their laboratory for the first time, as well as giving updates of recent developments for those laboratories where in situ hybridization techniques are already in use. Despite its widespread significance, in situ hybridization has retained a re- tation as one of the more difficult and capricious molecular biological te- niques. This may in part be because of the hybrid nature of the technique, which often requires a mixture of molecular biological and histological skills. The two techniques are usually taught and acquired in different streams of biolo- cal science. The step-by-step and detailed protocols provided in In Situ Hybridization Protocols by researchers active in the field should make it p- sible for both the molecular biologist with little experience of histology and the histologist with little experience of molecular biology to use the technique s- cessfully in their laboratories.
Book & CD. Advances in molecular biotechnology have greatly improved the sensitivity and the efficiency of methods utilised for genetic investigations and diagnosis. In the domain of chromosome analysis, the introduction of molecular techniques has led to the development of a new approach, called Molecular Cytogenetics, which has surpassed previously available techniques to become a foremost biological method. The fluorescence in situ hybridisation (FISH) is quickly became the standard technique for in situ chromosomal investigations, as illustrated by its large variety of applications in research and diagnosis. However, during the last decade, alternative methods to FISH have been introduced and have shown to be valuable in detecting chromosomes and quantifying chromosomal abnormalities. These alternative procedures are the Primed IN Situ (PRINS) labelling and the Peptide Nucleic Acid (PNA) probes. The two procedures present several advantages for the in situ detection of nucleic acid sequences, such as the small size of PNA probes and PRINS primers, or the fast kinetics of PRINS and PNA labelling reactions, that make them very attractive for a number of cytogenetic purposes. This book provides a valuable introduction and overview of the principles and the applications of alternative approaches in the field of molecular cytogenetics.
A technique used to amplify the number of copies of a specific region of DNA, the polymerase chain reaction (PCR) is at the forefront of the dramatic development of biochemistry. This text provides the tools for developing innovative approaches to using this leading technology. It includes theoretical considerations, discussions, and a selection of
PCR is the most powerful technique currently used in molecular biology. It enables the scientist to quickly replicate DNA and RNA on the benchtop. From its discovery in the early 80's, PCR has blossomed into a method that enables everything from ready mutation of DNA/RNA to speedy analysis of tens of thousands of nucleotide sequences daily.PCR Applications examines the latest developments in this field. It is the third book in the series, building on the previous publications PCR Protocols and PCR Strategies. The manual discusses techniques that focus on gene discovery, genomics, and DNA array technology, which are contributing factors to the now-occurring bioinformatics boom.Key Features* Focuses on gene discovery, genomics, and DNA array technology* Covers quantitative PCR techniques, including the use of standards and kinetic analysisincludes statistical refinement of primer design parameters* Ilustrates techniques used in microscopic tissue samples, such as single cell PCR, whole cell PCR, laser capture microdissection, and in situ PCREntries provide information on:* Nomenclature* Expression* Sequence analysis* Structure and function* Electrophysiology* Parmacology* Information retrieval
Major advancements in the field of in situ molecular pathology have occurred since publication of the first edition. In Situ Molecular Pathology and Co-expression Analyses, Second Edition, continues to teach both the molecular basis for the improvements and the actual protocols. This is the unique feature that separates it from the pack of other "cook-book" type approaches. The fields of in situ hybridization and immunohistochemistry have expanded rapidly where computer-based analyses systems have greatly expanded the power of these methods. Further, knowledge of the marked improvements in the reagents themselves since the first edition can make the difference of excellent versus misleading data. The automated platforms require that researchers and diagnostic biomedical investigators have a good understanding of the basics of in situ based tests, protocols, and biochemistry for troubleshooting in order to maximize the use of these platforms. This second edition focuses attention on straightforward protocols used to simultaneously detect two or more proteins/nucleic acids within intact tissue by doing co-expression analyses. Practicing molecular pathologists, diagnostic pathologists, laboratory directors, and toxicologists, as well as clinicians and researchers in training, will benefit from this clear presentation of protocols and theoretical framework. Data derived from in situ hybridization and immunohistochemistry. - Explains the theory and foundation of immunohistochemistry and in situ hybridization and presents easy-to-follow experimental protocols with tricks of the trade - Includes two new chapters: Recent improvements in immunohistochemistry and in situ hybridization, Quality control for immunohistochemistry and in situ hybridization: How to know if the color change is signal or background - The second edition also includes a detailed test to help one learn the basics of histologic interpretation of tissues and a separate detailed test in how to differentiate signal from background - Includes chapter-ending summaries of Key Points to Remember, bringing beginners up to speed with any seasoned veteran in the field - Thoughtfully tackles the molecular basis if IHC and ISH, along with application of that knowledge to improving the techniques is significant
Endocrine Pathology: Differential Diagnosis and Molecular Advances, Second Edition provides detailed coverage of endocrine pathology with extensive discussion of the differential diagnosis as well as presentation of molecular pathobiology of the major endocrine organs. Revised and expanded from the first edition, each chapter, written by leaders in their respective field, has been updated with the latest advances that are transforming the field of endocrine pathology. Richly illustrated with color photomicrographs, useful diagrams and line drawings, each chapter includes differential diagnosis of common and uncommon lesions as well as material on molecular developments, with emphasis on the molecular findings that are most helpful in the diagnosis of specific disorders. Endocrine Pathology: Differential Diagnosis and Molecular Advances, Second Edition, provides a useful and well-organized resource designed not only for the endocrine pathologist and the general surgical pathologist, but also for the clinical endocrinologist and the endocrine surgeon.
This book is a unique source of information on the present state of the exciting field of molecular cytogenetics and how it can be applied in research and diagnostics. The basic techniques of fluorescence in situ hybridization and primed in situ hybridization (PRINS) are outlined, the multiple approaches and probe sets that are now available for these techniques are described, and applications of them are presented in 36 chapters by authors from ten different countries around the world. The book not only provides the reader with basic and background knowledge on the topic, but also gives detailed protocols that show how molecular cytogenetics is currently performed by specialists in this field. The FISH Application Guide initially provides an overview of the (historical) development of molecular cytogenetics, its basic procedures, the equipment required, and probe generation. The book then describes tips and tricks for making different tissues available for molecular cytogenetic studies. These are followed by chapters on various multicolor FISH probe sets, their availability, and their pot- tial for use in combination with other approaches. The possible applications that are shown encompass the characterization of marker chromosomes, cryptic cytogenetic aberrations and epigenetic changes in humans by interphase and metaphase cyto- netics, studies of nuclear architecture, as well as the application of molecular cytogenetics to zoology, botany and microbiology.