Generation of cDNA Libraries

Generation of cDNA Libraries : Methods and Protocols

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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.
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Product details

  • Hardback | 338 pages
  • 154.9 x 231.1 x 25.4mm | 557.93g
  • Totowa, NJ, United States
  • English
  • 2003 ed.
  • XIV, 338 p.
  • 1588290662
  • 9781588290663

Back cover copy

With the decoding of the human genome and the potential application of its information to gene chips, the ability to generate cDNA libraries for functional assays of specific gene sequences has become critical in gene manipulation and genetic research. In Generation of cDNA Libraries: Methods and Protocols, expert researchers and inventors in the field describe their own proven techniques for generating cDNA/mRNA libraries to identify the functions of specific decoded gene sequences. A wide variety of techniques is presented for enhancing the generation of complete and full-length libraries, and for confirming the quality of the cDNAs generated. Among the applications detailed are electrophoresis, Northern blotting, single-cell microarray analysis, subtractive hybridization, subtractive cloning, gene cloning, and peptide library generation. Each method includes background information, step-by-step instructions, a list of reagents, operational tips, and notes on instrumentation. A background tutorial provides essential information: the definition of a cDNA library, the various types of cDNA libraries, the differences between methods for cDNA library generation using either conventional approaches or novel strategies, and the quality of cDNA libraries.
State-of-the-art and highly practical, Generation of cDNA Libraries: Methods and Protocols provides a variety of powerful and readily reproducible techniques for the generation of the entire range of complete, full-length cDNA/mRNA libraries needed in today's forefront genetic research.
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Table of contents

Complementary DNA Libraries: An Overview
Shao-Yao Ying

Rapid Amplification of cDNA Ends
Yue Zhang

cDNA Generation on Paramagnetic Beads
Zhaohui Wang and Michael G. K. Jones

Construction of a Normalized cDNA Library by mRNA-cDNA Hybridization and Subtraction
Ye-Guang Chen

Amplification of cDNA Ends Using PCR Suppression Effect and Step-Out PCR
Mikhail V. Matz, Naila O. Alieva, Alex Chenchik, and Sergey Lukyanov

Use of Inverse PCR to Clone cDNA Ends
Sheng-He Huang, Steven H. M. Chen, and Ambrose Y. Jong

Construction of Size-Fractionated cDNA Library Assisted by an In Vitro Recombination Reaction
Osamu Ohara

Construction of a Full-Length Enriched and 5'-End Enriched cDNA Library Using the Oligo-Capping Method
Yutaka Suzuki and Sumio Sugano

cDNA Library Construction Using In Vitro Transcriptional Amplification
Shi-Lung Lin and Henry Ji

Amplification of Representative cDNA Pools from Microscopic Amounts of Animal Tissue
Mikhail V. Matz

Single-Cell cDNA Library Construction Using Cycling aRNA Amplification
Shi-Lung Lin

mRNA/cDNA Library Construction Using RNA-Polymerase Cycling Reaction
Shi-Lung Lin and Shao-Yao Ying

Quality Assessment of cDNA Libraries
Hans-Jurgen Fulle

Assessment of the Quality of mRNA Libraries by Agarose Gel Electrophoresis
Tsen-Yin Lin and Shao-Yao Ying

PACS RT-PCR: A Method for the Generation and Measurement of any Poly(A)-Containing mRNA Not Affected by Contaminating Genomic DNA
Igor Nepluev and Rodney J. Folz

Single-Cell mRNA Library Analysis by Northern Blot Hybridization
Shi-Lung Lin

Generation of cDNA Libraries for Profiling Gene Expression of Given Tissues or Cells
Xin Zhang, Qiu-Hua Huang, and Ze-Guang Han

Screening Poly [dA/dT(-)] cDNA for Gene Identification
San MingWang, Scott C. Fears, Lin Zhang, Jian-Jun Chen, and Janet D. Rowley

Generation of Longer cDNA Fragments from SAGE Tags for Gene Identification
Jian-Jun Chen, Sanggyu Lee, Guolin Zhou, Janet D. Rowley, and San Ming Wang

Generation of Full-Length cDNA Libraries Enriched for Differentially Expressed Genes
Bakhyt Zhumabayeva, Cynthia Chang, Joseph McKinley, Luda Diatchenko, and Paul D. Siebert

Subtractive Hybridization for the Identification of Differentially Expressed Genes Using Uracil-DNA Glycosylase and Mung-Bean Nuclease
Tsen-Yin Lin and Shao-Yao Ying

Subtractive Cloning of Differential Genes Using RNA-PCR
Shao-Yao Ying and Shi-Lung Lin

Strategy for Construction of a cDNA Encoding a Repetitive Amino Acid Sequence
Masahiro Asada and Toru Imamura

Preparing Lambda Libraries for Expression of Proteins in Prokaryotes or Eukaryotes
Rebecca L. Mullinax and Joseph A. Sorge

Peptide Library Construction from RNA-PCR-Derived RNAs
Shi-Lung Lin

Identifying Interacting Proteins in an Escherichia coli-Based Two-Hybrid System
Bonnie Wu, Rebecca L. Mullinax, and Joseph A. Sorge

Future Perspectives, Shao-Yao Ying

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Review quote

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