Cdna Libraries
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Generation Of Cdna Libraries
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Author : Shao-Yao Ying
language : en
Publisher: Springer Science & Business Media
Release Date : 2008-02-03
Generation Of Cdna Libraries written by Shao-Yao Ying and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2008-02-03 with Science categories.
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
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Author : Ian G. Cowell
language : en
Publisher: Springer Science & Business Media
Release Date : 2008-02-02
Cdna Library Protocols written by Ian G. Cowell and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2008-02-02 with Science categories.
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.
Cdna Libraries
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Author : Chaofu Lu
language : en
Publisher: Humana Press
Release Date : 2016-08-23
Cdna Libraries written by Chaofu Lu and has been published by Humana Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2016-08-23 with Medical categories.
The numerous vital applications of complementary DNA (cDNA) technology have changed dramatically as the technology has advanced over recent years. In cDNA Libraries: Methods and Protocols, expert researchers provide current techniques that reflect the latest advances in the construction and application of cDNA libraries. The first half of the volume covers improved approaches to some of the most basic elements of creating cDNA libraries, while the second half casts a much wider net and includes visionary applications of cDNA technology which were either unforeseen or technically impractical until recently. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, cDNA Libraries: Methods and Protocols serves as an ideal guide to all scientists seeking to advance this important technology and provide answers to the enduring fundamental questions of biology.
Efficient And Simpler Method To Construct Normalized Cdna Libraries With Improved Representations Of Full Length Cdnas
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Author :
language : en
Publisher:
Release Date : 1998
Efficient And Simpler Method To Construct Normalized Cdna Libraries With Improved Representations Of Full Length Cdnas written by and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 1998 with categories.
This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.
Normalized Cdna Libraries
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Author :
language : en
Publisher:
Release Date : 1997
Normalized Cdna Libraries written by and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 1997 with categories.
This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.
Genetic Library Construction And Screening
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Author : R.C. Bird
language : en
Publisher: Springer Science & Business Media
Release Date : 2012-12-06
Genetic Library Construction And Screening written by R.C. Bird and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2012-12-06 with Science categories.
Designed as an introductory text the authors cover all core strategies in the application of modern recombinant DNA technology. The first chapters directly address the applications of polymerase chain reaction to a variety of problems in DNA cloning that are, or have been, extremely challenging using more traditional approaches and technologies. These include cDNA cloning and transcript mapping, mutagenesis as well as the cloning of very long transcripts and protocols using limiting amounts of total RNA. Further chapters describe approaches to subtractive cloning technologies as well as novel specialized expression cloning and library screening strategies. The handbook contains detailed step-by-step protocols and extensive hands-on advice.
High Throughput Plasmid Cdna Library Screening
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Author :
language : en
Publisher:
Release Date : 2006
High Throughput Plasmid Cdna Library Screening written by and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2006 with categories.
Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins. Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions. Expressed sequence tag (EST) sequencing samples cDNA libraries at random, which is most useful at the beginning of large-scale screening projects. However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.
Requirements In Screening Cdna Libraries For New Genes And Solutions Offered By Sbh Technology
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Author :
language : en
Publisher:
Release Date : 1993
Requirements In Screening Cdna Libraries For New Genes And Solutions Offered By Sbh Technology written by and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 1993 with categories.
Under different assumptions about the total number of genes, the number of housekeeping and tissue-specific genes, and the difference in the number of mRNAs per cell for functional and nonfunctional genes, significantly different results can be expected from screening random cDNA clones. We have developed gene expression models as a guide for interpretation of experimental results. For statistical, biological, and technical reasons, the search for 100,000 plus genes and discrimination between nonfunctional, housekeeping, and tissue-specific genes requires the analysis of up to 10 million clones from 20 to 50 tissues. Oligonucleotide hybridization of dense clone blots is an inexpensive and fast way to screen such large clone sets. Our preliminary results on control clones and thousands of cDNA clones from an infant brain library demonstrate the feasibility of the method. We present several models of gene expression and analyze the main factors which can influence the hunt for new genes via the screening of random cDNA libraries. The basic steps in the preparation and use of dense DNA dot arrays are described, and some results that demonstrate the feasibility and efficiency of gene inventorying by oligonucleotide hybridization are presented. Furthermore, partial SBH and single-pass gel sequencing are compared and a gene analysis scheme that combines the two approaches is discussed.
Method For Construction Of Normalized Cdna Libraries
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Author :
language : en
Publisher:
Release Date : 1998
Method For Construction Of Normalized Cdna Libraries written by and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 1998 with categories.
This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.
Positional Cloning By Exon Trapping And Cdna Selection
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Author : Bernhard Korn
language : en
Publisher: John Wiley & Sons
Release Date : 1999-06-02
Positional Cloning By Exon Trapping And Cdna Selection written by Bernhard Korn and has been published by John Wiley & Sons this book supported file pdf, txt, epub, kindle and other format this book has been release on 1999-06-02 with Science categories.
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.