cDNA Library Protocols

by Ian G. Cowell (Editor), Caroline A. Austin (Editor)

Overview

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 Prools to give a comprehensive review of all cDNA library-based methodologies; instead we present a series of up-to-date prools that together should give a good grounding of procedures 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 prools with some very usefiil newer techniques. cDNA Library Prools 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 manipulation, 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.

Product Details

ISBN-13:	9780896033832
Publisher:	Springer-Verlag New York, LLC
Publication date:	10/01/1996
Series:	Methods in Molecular Biology , #69
Edition description:	1997
Pages:	321
Product dimensions:	6.10(w) x 9.25(h) x 0.03(d)

Table of Contents

cDNA Library Construction from Small Amounts of RNA Using Paramagnetic Beads and PCR.- Increasing the Average Abundance of Low-Abundance cDNAs by Ordered Subdivision of cDNA Populations.- Isolation of Messenger RNA from Plant Tissues.- cDNA Library Construction for the Lambda ZAP®-Based Vectors.- Clone Excision Methods for the Lambda ZAP®-Based Vectors.- Using Rapid Amplification of cDNA Ends (RACE) to Obtain Full-Length cDNAs.- Inverse PCR Approach to Cloning cDNA Ends.- Cloning Gene Family Members Using the Polymerase Chain Reaction with Degenerate Oligonucleotide Primers.- Subtractive Hybridization for the Isolation of Differentially Expressed Genes Using Magnetic Beads.- Preparation of Competent Cells for High-Efficiency Plasmid Transformation of Escherichia coli.- The Tetramethylammonium Chloride (TMAC) Method for Screening cDNA Libraries with Highly Degenerate Oligonucleotide Probes Obtained by Reverse Translation of Amino Acid Sequences.- Screening cDNA Libraries by Hybridization with Double-Stranded DNA Probes and Oligonucleotides.- Immunological Screening of—-Phage cDNA Expression Libraries.- Cloning Sequence-Specific DNA-Binding Factors from cDNA Expression Libraries Using Oligonucleotide Binding Site Probes.- Protein Interaction Cloning by Far-Western Screening of—-Phage cDNA Expression Libraries.- Yeast Two-Hybrid Library Screening.- Signal Sequence Trap.- Isolation of Genetic Suppressor Elements (GSEs) from Random Fragment cDNA Libraries in Retroviral Vectors.- Expression and Preparation of Fusion Proteins from Recombinant—gt1.1 Phages.- Computer Analysis of Cloned Sequences.- The TIGR Human cDNA Database.- Searching the dbEST Database.- Reference cDNA Library Facilities Available from European Sources.

Editorial Reviews

This is a good overview of the essentials for preparing a cDNA library, for both the novice and the expert...the book is a true laboratory manual for the experimental scientist. It Should represent a useful and up-to-date addition for any lab that, for whatever reason, needs to generate its own libraries.-Hoffman-LaRoche, Inc.

"...their fine layout and ease of use suggest that the lab copy won't stay on the bookshelf for long."-SGM Quarterly

From the Publisher

This book presents a series of protocols for those interested in constructing and analyzing cDNA libraries. This book is not a comprehensive review of all cDNA library based technology; such a daunting task is beyond the scope of this or any other book. However, the authors attempted, and succeeded, in creating a book that covers the "basics" of cDNA cloning and many advanced techniques. It is written for both the advanced molecular biology researcher as well as those with little experience. The book begins with several chapters on constructing high quality, full-length cDNA libraries. Numerous strategies are discussed, including PCR-based approaches that use paramagnetic beads to purify cDNA before cloning. Next, there are several chapters that concentrate on problems that arise during cDNA cloning experiments, such as failing to obtain full length cDNA. Finally, there is an extensive discussion of cDNA library screening techniques, including hybridization with labeled probes, antibody screening of expression libraries and several specialty methods (e.g., yeast two-hybrid screening and isolation of genetic suppressor elements from random fragment cDNA libraries). The book contains a sufficient number of illustrations to complement the text. This book will benefit those researchers interested in constructing and screening cDNA libraries. I would highly recommend this book for libraries and to individuals that want to apply cDNA cloning technology to their research.

Philip M. Sass

This collection of 23 protocols covers all areas of cDNA work, from library construction and manipulation to screening and analysis of resulting clones. Care has been taken to combine versions of the most widely used protocols with useful newer techniques. The protocols describe methods for cloning difficult-to-obtain ends of cDNAs, methods for analyzing cDNA sequence data, and methods for using the wealth of cDNA data emerging from the human genome project. Annotation c. by Book News, Inc., Portland, Or.

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