A comprehensive treasury of all the key molecular biology methods-ranging from DNA extraction to gene localization in situ-needed to function effectively in the modern laboratory. Each of the 120 highly successful techniques follows the format of the much acclaimed Methods in Molecular BiologyOao series, providing an introduction to the scientific basis of each technique, a complete listing of all the necessary materials and reagents, and clear step-by-step instruction to permit error-free execution. Included for each technique are notes about pitfalls to avoid, troubleshooting tips, alternate methods, and explanations of the reasons for certain steps-all key elements contributing significantly to success or failure in the lab. The Nucleic Acid Protocols Handbook constitutes today's most comprehensive collection of all the key classic and cutting-edge techniques for the successful isolation, analysis, and manipulation of nucleic acids by both experienced researchers and those new to the field."

Good methods must be reliable, well-tested, and honed to minimize the time and expense required to achieve the desired results. CPNC provides a continuously growing and evolving set of protocols that allows researchers to benefit from the experience of other researchers around the world. The core manual provides a comprehensive set of protocols that have been compiled, revised, and streamlined over the last 6 years. Quarterly updates provide new protocols in emerging areas of research as well as continued advances and new applications for fundamental methods. The book is designed to grow and change with the field of nucleic acid chemistry. Fundamental nucleoside chemistry methods include sugar-base condensation, phosphorylation, and nucleoside protection. Methods for oligonucleotide synthesis include H-phosphonate and phosphoramidite approaches, solid-phase and solution-phase synthesis, large-scale synthesis, synthesis for modified and unmodified oligonucleotides, conjugation of oligonucleotides, synthesis without base protection, and synthesis on microarrays. More specialized synthetic methods include synthesis of biologically active nucleosides and prodrugs. Purification and characterization methods are detailed. Advanced methods include biophysical analysis, combinatorial methods, and nanotechnology. Each protocol includes rationale for choosing appropriate methods, step-by-step procedures, complete recipes, anticipated results, characterization data, and troubleshooting, as well as background and recommended reading. The level of procedural detail is far beyond that found in the research literature, and tips and comments from authors are geared towards ensuring reliable duplication in the laboratory.

Peptide nucleic acids (PNAs) have now existed for slightly more than ten years, with the interest in and applications of this pseudopeptide DNA mimic steadily increasing during the entire period. PNAs have rapidly attracted the attention of scientists from a diversity of fields ranging from (bio)organic and biophysical chemistry to prebiotic evolution, and from molecular biology to genetic diagnostics and drug development. Many of the applications take advantage of the unique properties of PNA—an uncharged pseudopeptide—that distinguish this DNA mimic from more traditional DNA analogs. Rather than trying to create a comprehensive collection of all published methods and protocols involving PNA—many of which have not yet been validated— I have decided to concentrate on select protocols that are either very well established by several groups around the world, such as PCR-clamping and in situ hybridization, or on new methods that may have broader future impact. Basic methods for PNA oligomer synthesis and analyses have also been included. I am very grateful to those friends and colleagues who have enthusiastically contributed their work, discussions, and writing, and thereby made this book possible. Peter E. Nielsen v Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix IINTRODUCTION 1 PNA Technology Peter E. Nielsen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 II CHEMISTRY 2 Solid Phase Synthesis of PNA Oligomers Frederik Beck. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3 Synthesis of PNA-Peptide Conjugates Satish Kumar Awasthi and Peter E. Nielsen. . . . . . . . . . . . . . . . . . 43 4 Parallel Synthesis of PNA-Peptide Conjugate Libraries Satish Kumar Awasthi and Peter E. Nielsen. . . . . . . . . . . . . . . . . .

An Indispensable Roadmap for Nucleic Acid Preparation Although Friedrich Miescher described the first isolation of nucleic acid in 1869, it was not until 1953 that James Watson and Francis Crick successfully deciphered the structural basis of DNA duplex. Needless to say, in the years since, enormous advances have been made in the study of nucleic a

The authors are commonly the techniques" originators, and each has demonstrated a hands-on mastery of the methods described, always fine-tuning them here for optimal productivity.

Peptide Nucleic Acids, Second Edition has been extensively revised, updated, and enlarged to contain many new chapters covering the most recent topics and applications in this fast-moving field. The book contains state-of-the-art protocols and applications on all aspects of peptide nucleic acids. Concepts are clearly explained with each chapter containing concise background information. Written by leading experts in the field, the book is an invaluable and complete reference work on this novel and exciting area.

Ribonucleic acids are central to cellular and molecular processes and perform vital functions in both structural and functional roles. RNA molecules form the bridge between the stable genetic information contained within DNA and enzymes and proteins that carry out much of the metabolism within the cell. Many of the sites of protein synthesis, the ribosomes within the cell, are composed of these ribonucleic acids as are the tRNA molecules that deliver the amino acid building blocks to the ribosomes. Of all the RNA species, the nucleic acid intermediate, messenger RNA, is a desirable source of material to biologists, since this reflects much of, what ultimately, is translated into enzymes and proteins. In order to determine the qualitative and quantitative changes in mRNA expression, a vast number of molecular biological techniques have been developed. Key molecular methods that provide the means to initially isolate and analyze RNA molecules are the focus of this volume. In putting together this collection of protocols, we have tried to provide techniques that are most applicable and widely used. In particular, there are a number of iso- tion techniques included that have been developed, modified, or adapted to enable extraction from a variety of cell types, organisms, or subcellular organelles. Successful isolation of intact RNA is an essential starting point for any sub- quent analysis. This is why we have aimed to make this section comprehensive. The analysis of RNA is the focus of the following chapters.

Recent advances in the biosciences have led to a range of powerful new technologies, particularly nucleic acid, protein and cell-based methodologies. The most recent insights have come to affect how scientists investigate and define cellular processes at the molecular level. This book expands upon the techniques included in the first edition, providing theory, outlines of practical procedures, and applications for a range of techniques. Written by a well-established panel of research scientists, the book provides an up-to-date collection of methods used regularly in the authors’ own research programs.

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.

An indispensable handbook of the highest standard for those working in the fields of food analysis and forensic applications.