In Vitro Mutagenesis Protocols

In the post-genomic era, in vitro mutagenesis has emerged as a critically important tool for establishing the functions of components of the proteome. The third edition of In Vitro Mutagenesis Protocols represents a practical toolbox containing protocols vital to advancing our understanding of the connection between nucleotide sequence and sequence function. Fully updated from the previous editions, this volume contains a variety of specialty tools successfully employed to unravel the intricacies of protein-protein interaction, protein structure-function, protein regulation of biological processes, and protein activity, as well as a novel section on mutagenesis methods for unique microbes as a guide to the generalization of mutagenesis strategies for a host of microbial systems. Written in the highly successful Methods in Molecular Biology ™ series format, chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and expert tips on troubleshooting and avoiding known pitfalls.

Authoritative and up-to-date, In Vitro Mutagenesis Protocols, Third Edition offers today's researchers a valuable compendium of reliable and powerful techniques with which to illuminate the proteome and its rich web of biological implications.

Establishes protein structure, function, activity, and interrelationships by authors of peer reviewed articles published in respected journals Covers computational methods to evaluate protein stability and the resultant change upon introducing mutations Utilizes animal models to identify loci that genetically interact with human disease-causing point mutations Includes detailed methods on protein structure and function mutagenesis Includes supplementary material: sn.pub/extras

Inhalt
Mutagenesis in Various Microbial Backgrounds.- Mutagenesis Protocols in Saccharomyces cerevisiae by In Vivo Overlap Extension.- In Vitro Mutagenesis of Brucella Species.- Random Mutagenesis Strategies for Campylobacter and Helicobacter Species.- Mutagenesis of the Repeat Regions of Herpesviruses Cloned as Bacterial Artificial Chromosomes.- An Efficient Protocol for VZV BAC-Based Mutagenesis.- A Method for Rapid Genetic Integration into Plasmodium falciparum Utilizing Mycobacteriophage Bxb1 Integrase.- PCR Mutagenesis.- Random Mutagenesis by Error-Prone PCR.- A Rapid and Versatile PCR-Based Site-Directed Mutagenesis Protocol for Generation of Mutations Along the Entire Length of a Cloned cDNA.- Rapid Sequence Scanning Mutagenesis Using In Silico Oligo Design and the Megaprimer PCR of Whole Plasmid Method (MegaWHOP).- Insertion and Deletion Mutagenesis by Overlap Extension PCR.- Targeted Amplification of Mutant Strands for Efficient Site-Directed Mutagenesis and Mutant Screening.- A Modified Inverse PCR Procedure for Insertion, Deletion, or Replacement of a DNA Fragment in a Target Sequence and Its Application in the Ligand Interaction Scan Method for Generation of Ligand-Regulated Proteins.- Amplification of Orthologous Genes Using Degenerate Primers.- Reviews.- Computational Evaluation of Protein Stability Change upon Mutations.- Approaches for Using Animal Models to Identify Loci That Genetically Interact with Human Disease-Causing Point Mutations.- Protein Evolution Mutagenesis.- Using Peptide Loop Insertion Mutagenesis for the Evolution of Proteins.- Massive Mutagenesis®: High-Throughput Combinatorial Site-Directed Mutagenesis.- Directed In Vitro Evolution of Reporter Genes Based on Semi-Rational Design and High-Throughput Screening.- Ribosome Display for RapidProtein Evolution by Consecutive Rounds of Mutation and Selection.- Protein Structure and Function Mutagenesis.- Fine-Tuning Enzyme Activity Through Saturation Mutagenesis.- Characterization of Structural Determinants of Type 1 Corticotropin Releasing Hormone (CRH) Receptor Signalling Properties.- Site-Directed Mutagenesis for Improving Biophysical Properties of VH Domains.- Phenotype Based Functional Gene Screening Using Retrovirus-Mediated Gene Trapping in Quasi-Haploid RAW 264.7 Cells.- Site-Directed Disulfide Cross-Linking to Probe Conformational Changes of a Transporter During Its Functional Cycle: Escherichia coli AcrB Multidrug Exporter as an Example.- Site-Specific Incorporation of Extra Components into RNA by Transcription Using Unnatural Base Pair Systems.- Random Mutagenesis.- Mutagen™: A Random Mutagenesis Method Providing a Complementary Diversity Generated by Human Error-Prone DNA Polymerases.- Random-Scanning Mutagenesis.- Easy Two-Step Method for Randomizing and Cloning Gene Fragments.- Mutator Bacterial Strain Mutagenesis.- Random Mutagenesis Using a Mutator Strain.- En Passant Mutagenesis: A Two Step Markerless Red Recombination System.