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Full Description
Since its invention and subsequent development nearly 20 years ago, po- merase chain reaction (PCR) has been extensively utilized to identify numerous gene probes in vitro and in vivo. However, attempts to generate complete and full-length complementary cDNA libraries were, for the most part, fruitless and remained elusive until the last decade, when simple and rapid methods were developed. With current decoding and potential application of human genome information to genechips, there are urgent needs for identification of functional significance of these decoded gene sequences. Inherent in bringing these app- cations to fruition is the need to generate a complete and full-length cDNA library for potential functional assays of specific gene sequences. Generation of cDNA Libraries: Methods and Protocols serves as a laboratory manual on the evolution of generation of cDNA libraries, covering both ba- ground information and step-by-step practical laboratory recipes for which p- tocols, reagents, operational tips, instrumentation, and other requirements are detailed. The first chapter of the book is an overview of the basics of generating cDNA libraries, which include the following: (a) the definition of a cDNA library, (b) different kinds of cDNA libraries, (c) differences between methods for cDNA library generation using conventional approaches and novel stra- gies, including reverse generation of RNA repertoires from cDNA libraries, and (d) the quality of cDNA libraries.
Contents
Complementary DNA Libraries.- Rapid Amplification of cDNA Ends.- cDNA Generation on Paramagnetic Beads.- Construction of a Normalized cDNA Library by mRN-cDNA Hybridization and Subtraction.- Amplification of cDNA Ends Using PCR Suppression Effect and Step-Out PCR.- Use of Inverse PCR to Clone cDNA Ends.- Construction of Size-Fractionated cDNA Library Assisted by an In Vitro Recombination Reaction.- Construction of a Full-Length Enriched and a 5?-End Enriched cDNA Library Using the Oligo-Capping Method.- cDNA Library Construction Using In Vitro Transcriptional Amplification.- Amplification of Representative cDNA Pools from Microscopic Amounts of Animal Tissue.- Single-Cell cDNA Library Construction Using Cycling aRNA Amplification.- mRNA/cDNA Library Construction Using RNA—Polymerase Cycling Reaction.- Quality Assessment of cDNA Libraries.- Assessment of the Quality of mRNA Libraries by Agarose Gel Electrophoresis.- PACS RT-PCR.- Single-Cell mRNA Library Analysis by Northern Blot Hybridization.- Generation of cDNA Libraries for Profiling Gene Expression of Given Tissues or Cells.- Screening Poly [dA/dT(?)] cDNA for Gene Identification.- Generation of Longer cDNA Fragments from SAGE Tags for Gene Identification.- Generation of Full-Length cDNA Libraries Enriched for Differentially Expressed Genes.- Subtractive Hybridization for the Identification of Differentially Expressed Genes Using Uraci-DNA Glycosylase and Mung-Bean Nuclease.- Subtractive Cloning of Differential Genes Using RNA-PCR.- Strategy for Construction of a cDNA Encoding a Repetitive Amino Acid Sequence.- Preparing Lambda Libraries for Expression of Proteins in Prokaryotes or Eukaryotes.- Peptide Library Construction from RNA-PCR-Derived RNAs.- Identifying Interacting Proteins in an Escherichiacoli-Based Two-Hybrid System.- Future Perspectives.
