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9781588292858

Directed Evolution Library Creation

by ;
  • ISBN13:

    9781588292858

  • ISBN10:

    1588292851

  • Format: Hardcover
  • Copyright: 2003-05-01
  • Publisher: Humana Pr Inc

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Summary

A comprehensive compendium of cutting-edge protocols for the generation of molecular diversity. Described in step-by-step detail to ensure experimental success, these protocols include readily reproducible methods for random mutagenesis of entire genes or segments of genes, for homologous and nonhomologus recombination, and for constructing in vivo libraries in bacteria and yeast. In addition to the various protocols for creating libraries, this volume also describes ways to analyze libraries, particularly those made by recombination. An accompanying volume, Directed Enzyme Evolution: Screening and Selection Methods (ISBN: 1-58829-286-X), is devoted entirely to selection and screening methods that can be applied to the directed evolution of enzymes. Copy for Both Volumes Directed Evolution Library Creation: Methods and Protocols and Directed Enzyme Evolution: Screening and Selection Methods constitute an extraordinary collection of all the key methods used today for directed evolution research. Described in step-by-step detail to ensure robust experimental results, these methods will enable both newcomers and more experienced investigators to design and implement directed evolution strategies for the engineering of novel proteins. The first volume describes methods for the creation of mutated DNA molecules, or DNA libraries, encoding variants of desired proteins. The second volume describes methods for screening DNA libraries to isolate mutant proteins that exhibit a specified function.

Table of Contents

Prefacep. v
Contributorsp. ix
Mutagenesis and Recombination Methods
Generating Mutant Libraries Using Error-Prone PCRp. 3
Preparing Libraries in Escherichia colip. 11
Preparing Libraries in Saccharomyces cerevisiaep. 17
Creating Random Mutagenesis Libraries by Megaprimer PCR of Whole Plasmid (MEGAWHOP)p. 23
Construction of Designed Protein Libraries Using Gene Assembly Mutagenesisp. 29
Production of Randomly Mutated Plasmid Libraries Using Mutator Strainsp. 39
Evolution of Microorganisms Using Mutator Plasmidsp. 45
Random Insertion and Deletion Mutagenesisp. 53
Random Oligonucleotide Mutagenesisp. 65
Saturation Mutagenesisp. 75
DNA Shufflingp. 85
Family Shuffling with Single-Stranded DNAp. 91
In Vitro DNA Recombination by Random Primingp. 99
Staggered Extension Process (StEP) In Vitro Recombinationp. 105
RACHITT: Gene Family Shuffling by Random Chimeragenesis on Transient Templatesp. 111
The Creation of ITCHY Hybrid Protein Librariesp. 129
Preparation of SCRATCHY Hybrid Protein Libraries: Size- and In-Frame Selection of Nucleic Acid Sequencesp. 143
Sequence Homology-Independent Protein Recombination (SHIPREC)p. 153
Producing Chimeric Genes by CLERY: In Vitro and In Vivo Recombinationp. 165
Analysis of Library Diversity
Analysis of Shuffled Libraries by Oligonucleotide Probe Hybridizationp. 177
Sequence Mapping of Combinatorial Libraries on Macro- or Microarrays: Experimental Design of DNA Arraysp. 189
Sequence Mapping of Combinatorial Libraries on Macro- and Microarrays: Bioinformatic Treatment of Datap. 199
Indexp. 213
Table of Contents provided by Rittenhouse. All Rights Reserved.

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