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The polymerase chain reaction (PCR) is a powerful research tool used in many scientific disciplines. It is also used for detection and testing in areas such as food microbiology, environmental microbiology, biotechnology, industrial microbiology, veterinary and medical diagnostics. This indispensable manual is a compilation of review articles written by experts in the field of PCR technology. Topics covered include: principles of PCR, fluorescent chemistries, instrumentation, quantification strategies, extraction and purification of nucleic acids, sample preparation, controls for validation, primers and probes, standardization of methods, MIQE guidelines, mRNA expression and PCR arrays. This book provides a comprehensive overview of PCR theory, instrumentation and methods. The book represents an excellent, detailed guide for anyone interested in the development and use of PCR technology. It is a recommended purchase for all microbiology and molecular biology laboratories and university libraries.

Understanding PCR: A Practical Bench-Top Guide gives you all of the information you need to plan your first PCR, from reagents to conditions to analysis and beyond. It is a user friendly book that has step-by-step basic protocols, which can be adapted to your needs. Includes helpful information such as where to order your reagents and basic troubleshooting hints and tips. Includes resources for reagents Explains basic laboratory preparation Provides straightforward experimental protocols Incorporates fundamental analytical techniques Contains a troubleshooting guide

Molecular Analysis and Genome Discovery, Second Edition is a completely revised and updated new edition of this successful book. The text provides a comprehensive overview of recent developments in the fast moving field of molecular based diagnostics of disease markers. Key concepts and applications are provided alongside practical information on current techniques currently being researched and developed. Each chapter offers an up-to-date analysis of the subject encompassing the very latest technology platforms and is an essential reference for researchers in the field looking for an up-to-date overview of the subject. The book will also be an indispensable resource for those working in the biotechnology and pharmaceutical industries. New for this edition: chapters on Genotyping through Mutation Detection; Differential Gene Expression; Haplotyping and Molecular Profiling.

Real-time PCR (RT-PCR) technology is highly flexible and many alternative instruments and fluorescent probe systems have been developed recently. The decreased hands-on time, increased reliability and improved quantitative accuracy of RT-PCR methods have contributed to the adoption of RT-PCR for a wide range of new applications. This essential manual presents a comprehensive guide to the most up-to-date technologies and applications as well as providing an overview of the theory of this increasingly important technique. Renowned experts in the field describe and discuss the latest PCR platforms, fluorescent chemistries, validation software, data analysis, and internal and external controls. This timely and authoritative volume also discusses a wide range of RT-PCR applications including: clinical diagnostics, biodefense, RNA expression studies, validation of array data, mutation detection, food authenticity and legislation, NASBA, molecular halotyping, and much more. An essential book for all laboratories using PCR.

The aim of Single Cell Diagnostics: Methods and Protocols is for all readers to extend their knowledge and expertise in analysis of single cells. The book starts with laser assisted cell collection, non-invasive assessment of single cells and moves through the techniques of standard fluorescence in situ hybridization and polymerase chain reaction (PCR) As the reader moves through the book, the scope and complexity of each technique gradually increases as real-time quantitative PCR, isothermal whole genome amplification, comparative genomic hybridization, real-time gene expression analysis and the production of RNA and cDNA libraries are covered. The book closes with the application of customized microarrays to the study of single cells.

PCR is the most widely used technique in molecular biology. New PCR variants offering substantial benefits to existing protocols appear on a frequent basis. PCR: Methods Express describes the very latest PCR-based methodologies and approaches to provide the most up-to-date practical advice on how to tackle a broad range of biological problems including: •real time qRT-PCR •rapid generation of gene targeting constructs •PCR multiplexes •PCR-based mutagenesis •identification of microdeletions and microduplications •DNA methylation analysis •forensic genetic DNA typing •genotyping •identification of mutations in single cells •whole genome amplification •diagnosis of infectious diseases •inverse PCR-based RFLP This book is a comprehensive research guide; every chapter discusses the merits and limitations of the available approaches and then provides fully-proven protocols with hints and tips for success. PCR: Methods Express is an essential laboratory manual for researchers in all life science fields and at all levels, from postgraduate student to principal investigator.

Carrying on the high standards of the much-praised first edition, leading researchers and clinical investigators have completely revised and updated PRINS and In Situ PCR Protocols to take advantage of the many new developments and applications that have occurred for cellular and molecular investigations. The authors contribute proven protocols for many variations of PRINS and in situ PCR, including a new, fast multicolor PRINS method for identifying human chromosomes, PRINS protocols for in situ detection of unique sequences and point mutations, a new dideoxy PRINS procedure, and in situ PCR and PRINS protocols for plants. They describe, in detail, various applications of PRINS in human cells (detection of gene deletions in cancer, detection of fetal cells in maternal blood, and assessment of aneuploidy in brain tissues and embryos) as well as in plant cells. Readily reproducible in situ PCR techniques are also presented for the detection of cytomegalovirus and for use in combination with microdissection. In situ RT-PCR techniques are also given for use in plant or cancer investigation. 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. Comprehensive and highly practical, PRINS and In Situ PCR Protocols offers molecular biologists, cytogeneticists, and cytologists a cutting-edge guide to today's new in situ labeling technology.

A thoroughly updated version of the successful first edition with a new chapter on Real-Time PCR, more prokaryotic applications, and more detail in the complex mutagenesis sections. Information on PCR applications in genomics and proteomics have been expanded and integrated throughout the text. There is also advice on available products a

Carrying on the high standards of the first edition of Differential Display Methods, Peng Liang et al. have based their second edition on a new mathematical model of differential display (DD) that takes advantage of automation, as well as digital data acquisition and analysis. These well-versed authors explain and highlight all the recent methodological refinements, including automated liquid handling of hundreds of DD PCR reaction setups combined with capillary electrophoresis, a prototype computer program to automatically allow positive band identification from a fluorescence differential display image, and restriction fragment-based DD screenings that can link any cDNA fragment directly to a given gene once the sequence information of all transcripts becomes available. Other improvements discussed are combining DD and DNA microarrays by reducing the complexity of cDNA probes while increasing the sensitivity of detection, and a DD approach to detect prokaryotic mRNA expression. The authors also demonstrate the power of DD technology with a collection of outstanding examples of DD applications and detailed experimental procedures. The elegant studies described here have led to the discovery of many important genes involved in viral infection, Prion disease, cancer, ovulation, circadian clock, floral color, transcription repression gene silencing, mRNA polymorphism, and protein-RNA interaction. State-of-the-art and highly practical, Differential Display Methods, Second Edition offers gene hunters the possibility of genome-wide comprehensive DD screening, as well as a proven road map for any successful'gene fishing'expedition

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'. Keeping with the spirit of the Advanced Methods Series, most chapters provide an experimental protocol as an example of a specific assay.

This advanced level textbook provides a comprehensive overview of recent developments in the area of molecular based diagnostics (including nucleic acids, biosensors and immunoessays) of disease markers. It also covers the impact of techniques such as in vitro nucleic acid amplifications (e.g. PCR) and other amplification methods, as well as gene and biochip production and automated techniques such as fluorescent sequencing. The book discusses key concepts where new and merging areas, including pharmacogenomics, proteomics and functional genomics, are being researched and developed. In addition, examples are given where this new area of bioscience has or may be successfully applied.

Although the polymerase chain reaction has revolutionized genetic analysis by amplifying rare nucleic acid sequences, the in situ application is the only method that allows the localization of amplified signal within tissue structure. The applications of in situ polymerase chain reaction have greatly enhanced the field of investigation in many disc

Drawing on the proven qualities of the much praised and widely used first edition, John M.S. Bartlett and David Stirling 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 successful 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. In situ PCR methods and their application in parallel with other methods, such as immunohistochemistry, are also included. 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 troubleshooting and avoiding known pitfalls, and, where needed, a discussion of the interpretation and use of results. Cutting-edge and highly practical, PCR Protocols, Second Edition provides both novice and experienced investigators with an up-to-date compendium of powerful PCR methods for easy reference and consultation in the day-to-day performance of PCR-based experimentation, one that will enhance understanding of PCR, satisfy current needs, and point to powerful future applications.

The past several decades have witnessed an impressive array of conceptual and techno­ logical advances in the biomedical sciences. Much of the progress in this area has developed directly as a result of new morphology-based methods that have permitted the assessment of chemical, enzymatic, immunological, and molecular parameters at the cellular and tissue levels. Additional novel approaches including laser capture microdissection have also emerged for the acquisition of homogeneous cell popula­ tions for molecular analyses. These methodologies have literally reshaped the approaches to fundamental biological questions and have also had a major impact in the area of diagnostic pathology. Much of the groundwork for the development of morphological methods was estab­ th lished in the early part of the 19 century by Francois-Vincent Raspail, generally acknowledged as the founder of the science of histochemistry. The earliest work in the field was primarily in the hands of botanists and many of the approaches to the under­ standing of the chemical composition of cells and tissues involved techniques such as microincineration, which destroyed structural integrity. The development of aniline th dyes in the early 20 century served as a major impetus to studies of the structural rather than chemical composition of tissue. Later in the century, however, the focus returned to the identification of chemical constituents in the context of intact cell and tissue structure.

In Quantitative PCR Protocols, Bernd Kochanowski and Udo Reischl assemble a significant collection of established and novel methods for the successful quantitation of nucleic acids. Each method has been refined and tested by its developer and proven to work in such problems as the analysis of eukaryotic gene expression, the quantitation of viral loads in clinical specimens, reporter gene expression, and quantitative oncogene analysis. Particular emphasis is placed on the underlying principles of the design of competitive or noncompetitive standards, as well as on the optimization of the amplification process. In important cases several methods are given for the same problem so that readers may set up test systems tailored to their specific practical needs. Quantitative PCR Protocols provides a collection of detailed methods that offers comprehensive and up-to-date information on how to control PCR to obtain reliable quantitation. With its step-by-step instructions researchers can really master the complexities of PCR and address otherwise inaccessible biological and diagnostic questions.

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

The polymerase chain reaction (PCR) is a technique used to replicate specific pieces of DNA millions of times, which permits the detection and analysis of minute amounts of nucleic acids. Since its introduction in the late 1980s, this technique has been applied not only in molecular biology research but also in fields as diverse as anthropology, phylogeny, and forensics. However, despite the large impact of PCR, many of its applications remain within the confines of research and the academic environment. Now, in A Low-Cost Approach to PCR: Appropriate Transfer of Biomolecular Techniques, Dr. Eva Harris makes this elegantly simple technique more accessible to researchers, physicians, and laboratory workers throughout the world. She provides a description of the theoretical basis of the technique, the practical details of the method, and the philosophy behind the technology transfer program that she developed over the last ten years. The book serves as a guide for potential users in developing countries and for scientists in developed countries who may wish to work abroad. In addition, the low-cost approach outlined in this book can be useful for high school, undergraduate, or continuing education programs in the United States. While the specific applications of PCR outlined in the book are immediately useful to the study of infectious diseases, the approach presented can be generalized to a number of other technologies and situations. The book will help laboratories in many areas of the world generate information on site for use by physicians, epidemiologists, public health workers, and health policy professionals to develop new strategies for disease control.

Molecular toxicology is an emerging discipline that utilizes molecular and cell biology to understand how drugs and chemicals result in their unwanted effects. PCR Protocols in Molecular Toxicology is a practical guide to the use of polymerase chain reaction (PCR) to help examine, on a molecular and cellular level, how toxic responses are manifested. It offers a basic understanding of PCR and its optimization, as well as describing specific, high-impact areas of molecular toxicology and recent advances. The following techniques are described in detail: Quantitative reverse transcriptase PCR and methods to examine gene expression Differential display cloning Cloning and library screening by PCR Genotype and polymorphism analysis of drug and toxicant metabolizing enzymes Basic, non-PCR based molecular biology methods PCR Protocols in Molecular Toxicology will aid both novices and experienced PCR practitioners in using PCR to its fullest potential.

The volumes in this series include contemporary techniques significant to a particular branch of neuroscience. They are an invaluable aid to the student as well as the experienced researcher not only in developing protocols in neuroscience but in disciplines where research is becoming closely related to neuroscience. Each volume of Methods in Neurosciences contains an index, and each chapter includes references. Dr. Conn became Editor-in-Chief of the series beginning with Volume 15, so each subsequent volume could be guest-edited by an expert in that specific field. This further strengthens the depth of coverage in Methods in Neurosciences for students and researchers alike.Key Features• Direct application of PCR to fresh or frozen clinical specimens (e.g., blood and solid tissue)• Complete retrieval of novel expressed genes by PCR without screening a library• Quantitation by PCR• Mutagenesis by PCR• PCR in AIDS research• Simple and effective protocols for PCR on archival specimens

PCR Strategies expands and updates the landmark volume PCR Protocols. It is a companion laboratory manual that provides a completely new set of up-to-date strategies and protocols for getting the most from PCR. The editors have organized the book into four sections, focusing on principles, analyses, research applications, and alternative strategies for a wide variety of basic and clinical needs. If you own PCR Protocols, you will want PCR Strategies. If you don't own PCR Protocols, you will want to buy both! Concepts explained Methods detailed Trouble-shooting emphasized Novel applications highlighted Key concepts for PCR Analysis of PCR products Research applications Alternative amplification strategies

Molecular diagnostic procedures have been described in a number of recent books and articles. However, these publications have not focused on virus detection, nor have they provided practical protocols for the newer molecular methods. Written by the inventors or principal developers of these technologies, Molecular Methods for Virus Detection provides both reviews of individual methods and instructions for detecting virus nucleic acid sequences in clinical specimens. Each procedure includes quality assurance protocols that are often ignored by other methodology books. Molecular Methods for Virus Detection provides clinically relevant procedures for many of the newer diagnostic methodologies. Provides state-of-the-art PCR methods for amplification, quantitation, in situ hybridization, and multiplex reactions Goes beyond PCR with protocols for 3SR, NASBA, LCR, SDA, and LAT Covers important virus detection methods such as in situ hybridization; Southern, dot, and slot blots; branched chain signal amplification; and chemiluminescence Includes quality control information crucial in research and clinical laboratories Most chapters are written by the inventors and principal developers of the methodologies Includes color plates, 77 figures, and 18 tables