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When first conceived, not only was the aim of Protocols for Oligo nucleotides and Analogs to provide wide coverage of the ohgonuc- otide chemistry field for readers who are well versed within the field, but also to give investigators just entering into the field a new perspective. The very first book on this topic was edited and published by Michael Gait in 1984, in whose laboratory I encountered the newer aspects of oligonucleotide chemistry. Since then, oligonucleotide research has developed to such an extent that its uses extend far beyond basic studies, and now find wide application throughout clinical science as well. Until recently, the major application of oligonucleotides has been in the area of DNA-based diagnostic and "antisense oligonucleotid- based therapeutic approaches. However, oligonucleotides are now also being used as therapeutic agents and are thus frequently found in clinical trials in humans. Synthesis of unmodified oligonucleotides using automated synthe sizers has become a common practice in numerous laboratories. How ever, improvements on the existing techniques and the introduction of ever newer methods for oligonucleotide synthesis is constantly driving ahead in the leading research laboratories. And several new oligonucle otide analogs have been synthesized and studied for their individual prop erties in recent years. The present volume strives to bring the readers the most up-to-date information on the newest aspects of synthesis of oligo nucleotides and their analogs. A separate volume covers synthesis of oligonucleotide conjugates, along with most of the analytical techniques presently used for analysis of oligonucleotides.
This comprehensive collection of recently developed methods for producing new antibody reagents by immunization and recombinant DNA techniques contains ready-to-use protocols that illuminate current areas of research on antibody structure, functions, and applications. The methods can be applied in basic immunological studies involving antibody specificity, catalysis, and evolution, and in the isolation of rare antibodies by phage display technology and the engineering of new antibodies by mutagenesis. They offer insight into new ways of developing clinically useful antibody reagents. Antibody Engineering Protocols constitutes a single-source volume for laboratory investigators who want to minimize extensive literature and methodology searches and to work productively in their fields with reproducible step-by-step protocols.
Hans Neurath has written that this is the second golden era of enzymology {Protein Science [1994], vol. 3, pp. 1734—1739); he could with justice have been more general and referred to the second golden age of protein chemistry. The last two decades have seen enormous advances in our understanding of the structures and functions of pro teins arising on the one hand from improvements and developments in analytical techniques {see the companion volume, Basic Protein and Peptide Protocols, in this series) and on the other hand from the tech nologies of molecular genetics. Far from turning the focus away from protein science, the ability to isolate, analyze, and express genes has increased interest in proteins as gene products. Hence, many laborato ries are now getting involved in protein isolation for the first time, either as an essential adjunct to their work in molecular genetics or because of a curiosity to know more about the products of the genes that they have been studying. Protein Purification Protocols is aimed mainly at these newcom ers to protein purification, but it is hoped that it will also be of value to established practitioners who may find here techniques that they have not tried, but which might well be most applicable in their work. With the exception mainly of the first and last chapters, the format of the contributions to the present book conform to the established format of the Methods in Molecular Biology series.
An unprecedented collection of all the most up-to-date techniques for gene isolation and mapping, including the latest methods for gene characterization using database analyses. This collection of thoroughly tested recipes also includes chapters for the computational analysis of novel cDNA sequences with up-to-the-minute information on basic sequence analysis, sequence similarity searches, exon detection and similarity searches, and the prediction of gene function. Its state-of-the-art methods constitute indispensable tools for all scientists engaged in the search for specific disease genes, or in the general advancement of the human genome project.
The ability to introduce macromolecules into animal cells, includ ing DNA, RNA, proteins, and other bioactive compounds has facili tated a broad range of biological studies, from biochemistry and biophysics to molecular biology, cell biology, and whole animal stud ies. Gene transfer technology in particular will continue to play an essential role in studies aimed at improving our understanding of the relationships between the gene structure and function, and it has impor tant practical applications in both biotechnology and biomedicine, as evidenced by the current intense interest in gene therapy. Although DNA and other macromolecules may be introduced into cells by a variety of methods, including chemical treatments and microinjection, el- troporation has proven to be simpler to perform, more efficient, and effective with a wider variety of cell types than other techniques. The early and broad success of electric field-mediated DNA transfer soon prompted researchers to investigate electroporation for transferring other types of molecules into cells, including RNA, enzymes, antibodies, and analytic dyes. Animal Cell Electroporation and Electrofusion Protocols begins with three chapters that describe the theoretical and practical aspects of electroporation, including a review of the commercially available instrumentation. These introductory chapters will be of particular inter est to those new to electric field technologies and to those developing protocols for as yet untested species or cell types. Nineteen chapters follow that present well-tested protocols for electroporation of proteins and DNA into insect, fish, and mammalian cells.
The first libraries of complementary DNA (cDNA) clones were con structed in the mid-to-late 1970s using RNA-dependent DNA polymerase (reverse transcriptase) to convert poly A* mRNA into double-stranded cDNA suitable for insertion into prokaryotic vectors. Since then cDNA technology has become a fundamental tool for the molecular biologist and at the same time some very significant advances have occurred in the methods for con structing and screening cDNA libraries. It is not the aim of cDNA Library Protocols to give a comprehensive review of all cDNA library-based methodologies; instead we present a series of up-to-date protocols that together should give a good grounding of proce dures associated with the construction and use of cDNA libraries. In deciding what to include, we endeavored to combine up-to-date versions of some of the most widely used protocols with some very usefiil newer techniques. cDNA Library Protocols should therefore be especially useful to the investigator who is new to the use of cDNA libraries, but should also be of value to the more experienced worker. Chapters 1—5 concentrate on cDNA library construction and manipula tion, Chapters 6 and 7 describe means of cloning difficult-to-obtain ends of cDNAs, Chapters 8-18 give various approaches to the screening of cDNA libraries, and the remaining chapters present methods of analysis of cDNA clones including details of how to analyze cDNA sequence data and how to make use of the wealth of cDNA data emerging from the human genome project.
Now completely revised and updated from the original, much-acclaimed and bestselling first edition, Basic Cell Culture Protocols, 2nd ed. offers today's most comprehensive collection of easy-to-follow, cutting-edge protocols for the culture of a wide range of animal cells. Its authoritative contributors provide explicit, step-by-step instructions, along with extensive notes and tips that allow both experts and beginners to successfully achieve their desired results. Topics range from basic culture methodology to strategies for culturing previously uncultured cell types and hard-to-culture differentiated cells. Methods are also provided for the analysis of living cells by FACS, video microscopy, and confocal microscopy. Like the first edition, this book should be in every cell culture laboratory and be of use to all who use cell cultures in research.
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.
This book is intended to be a working guide to the operation of capillary electrophoresis (CE) instrumentation. Since CE is still a rap idly maturing technique, detailed validated protocols are not widely established. Therefore, extensive experimental procedures are not pro vided for individual analyses. The intention is to provide general guide lines on the principles and practice of CE and to give an overview of the specific technologies and important application areas. Part I provides operating instructions for standard commercially available instruments. Guidelines are included for activities such as changing capillaries, method development, quantitative procedures, optimization of precision and sensitivity, and the validation of meth ods, fraction collection, and troubleshooting, as well as a quick guide to running a separation. The application range of CE is possibly the most diverse of all analytical techniques and ranges from large, complex macromolecules, such as proteins and nucleic acids, to small solutes, such as organic drugs and inorganic anions and cations.
Gene transfer is an essential technology for improving our under standing of gene structure and function. Although there are many meth ods by which DNA may be introduced into cells—including heat and chemical treatments, and microinjection—electroporation has been found to be the most versatile gene transfer technique. Electroporation is effective with a wide variety of cell types, including those that are difficult to transform by other means. For many cell types, electroporation is either the most efficient or the only means known to effect gene transfer. The early and broad success of electric field-medi ated DNA transfer soon prompted researchers to investigate electroporation for transferring other types of molecules into cells, in cluding RNA, enzymes, antibodies, and analytic dyes. The first section of Plant Cell Electroporation and Electrofusion Protocols includes two chapters that serve as a guide to theoretical and practical aspects of electroporation, and will be of particular interest to those developing protocols for as yet untested species or cell types, and a third chapter that describes commercially available electroporation instruments. The remaining chapters describe well-tested protocols for DNA electrotransfection, electroporation of other biomolecules, or cell electrofusion. These chapters also include brief discussions of alterna tives to electric field-based methods, citing the advantages and limita tions of the various methods for achieving specific goals.