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Generation of cDNA Libraries : Methods and Protocols - Shao-Yao Ying

Generation of cDNA Libraries

Methods and Protocols

By: Shao-Yao Ying (Editor)

Hardcover Published: 19th February 2003
ISBN: 9781588290663
Number Of Pages: 338

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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.

...the ready-for-use protocols are excellent...A good buy. - Cell Biochem Funct

Prefacep. v
Contributorsp. xi
Complementary DNA Libraries: An Overviewp. 1
Rapid Amplification of cDNA Endsp. 13
cDNA Generation on Paramagnetic Beadsp. 25
Construction of a Normalized cDNA Library by mRNA-cDNA Hybridization and Subtractionp. 33
Amplification of cDNA Ends Using PCR Suppression Effect and Step-Out PCRp. 41
Use of Inverse PCR to Clone cDNA Endsp. 51
Construction of Size-Fractionated cDNA Library Assisted by an In Vitro Recombination Reactionp. 59
Construction of a Full-Length Enriched and 5'-End Enriched cDNA Library Using the Oligo-Capping Methodp. 73
cDNA Library Construction Using In Vitro Transcriptional Amplificationp. 93
Amplification of Representative cDNA Pools from Microscopic Amounts of Animal Tissuep. 103
Single-Cell cDNA Library Construction Using Cycling aRNA Amplificationp. 117
mRNA/cDNA Library Construction Using RNA-Polymerase Cycling Reactionp. 129
Quality Assessment of cDNA Librariesp. 145
Assessment of the Quality of mRNA Libraries by Agarose Gel Electrophoresisp. 155
PACS RT-PCR: A Method for the Generation and Measurement of any Poly(A)-Containing mRNA Not Affected by Contaminating Genomic DNAp. 161
Single-Cell mRNA Library Analysis by Northern Blot Hybridizationp. 169
Generation of cDNA Libraries for Profiling Gene Expression of Given Tissues or Cellsp. 179
Screening Poly [dA/dT(-)] cDNA for Gene Identificationp. 197
Generation of Longer cDNA Fragments from SAGE Tags for Gene Identificationp. 207
Generation of Full-Length cDNA Libraries Enriched for Differentially Expressed Genesp. 223
Subtractive Hybridization for the Identification of Differentially Expressed Genes Using Uracil-DNA Glycosylase and Mung-Bean Nucleasep. 239
Subtractive Cloning of Differential Genes Using RNA-PCRp. 253
Strategy for Construction of a cDNA Encoding a Repetitive Amino Acid Sequencep. 261
Preparing Lambda Libraries for Expression of Proteins in Prokaryotes or Eukaryotesp. 271
Peptide Library Construction from RNA-PCR-Derived RNAsp. 289
Identifying Interacting Proteins in an Escherichia coli-Based Two-Hybrid Systemp. 295
Future Perspectivesp. 311
Indexp. 331
Table of Contents provided by Syndetics. All Rights Reserved.

ISBN: 9781588290663
ISBN-10: 1588290662
Series: Methods in Molecular Biology
Audience: Professional
Format: Hardcover
Language: English
Number Of Pages: 338
Published: 19th February 2003
Publisher: Humana Press Inc.
Country of Publication: US
Dimensions (cm): 23.5 x 15.5  x 2.41
Weight (kg): 0.75