Cdna
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Generation of cDNA Libraries
Author: Shao-Yao Ying
language: en
Publisher: Springer Science & Business Media
Release Date: 2008-02-03
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.
cDNA Library Protocols
Author: Ian G. Cowell
language: en
Publisher: Springer Science & Business Media
Release Date: 2008-02-02
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.
Positional Cloning by Exon Trapping and cDNA Selection
Tremendous strides in decoding the human genome have been made over the last ten years. Large, chromosome-scale, genome-scale EST-sequencing and mapping techniques have dramatically expanded the base of identified genes and sequenced DNA. Gene isolation and cloning techniques that emerged earlier on, however, still remain effective mainstays for constructing dense transcript maps to isolate coding sequences and disease-associated genes contained within a specific chromosomal region. Positional Cloning by Exon Trapping and cDNA Selection examines two powerful methods for locating the coding region of a given gene by isolating gene fragments from individual clones or pools of genomic clones. This comprehensive guide details the exon-trapping and cDNA selection processes step by step-from isolating genomic templates and nuclear splicing, to verifying generated clones and sequenced data, to analyzing exon libraries and cDNA sublibraries. Procedures covered include: * Exon-trapping systems and descriptions of pSPL1 and pSPL3 vectors. * Exon amplification protocols for preparing vectors for cloning; subcloning genomic DNA into vectors; transformation, analysis, and transfection of sublibraries; RNA transcription; and PCR amplification and PCR product cloning. * Evaluation of exon libraries by PCR colony testing, identifying artifactual clones using Southern blot, and sequencing and mapping back candidate exons. * Isolation of genomic templates, such as COSMID-, P1-, PAC, and YAC DNA. * cDNA selection experiment protocols including cDNA screening, hybridization, and biotinylation; preparation of genomic and cDNA sources; and PCR cloning. * Clone analysis and analysis by hybridization. Positional Cloning by Exon Trapping and cDNA Selection offers researchers, scientists, and graduate students an invaluable tool for probing gene distribution and molecular organization. Most importantly, it provides a critical approach to isolating specific disease genes within a targeted genomic area.