In Vitro Mutagenesis: Methods and Protocols

ISBN-13: 9781493964703 / Angielski / Twarda / 2016 / 511 str.

Andrew R. Reeves

Opis

In vitro mutagenesis remains a critical experimental approach for investigating gene and protein function at the cellular level. This volume provides a wide variety of updated and novel approaches for performing in vitro mutagenesis using such methods as genome editing, transposon (Tn) mutagenesis, site-directed, and random mutagenesis. In Vitro Mutagenesis: Methods and Protocols guides readers through methods for gene and genome editing, practical bioinformatics approaches for identifying mutagenesis targets, and novel site-directed and random mutagenesis approaches aimed at gaining a better understanding of protein-protein and protein-cofactor interactions. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, In Vitro Mutagenesis: Methods and Protocols aims to provide a highly accessible and practical manual for current and future molecular biology researchers, from the beginner practitioner to the advanced investigator in fields such as molecular genetics, biochemistry, and biochemical and metabolic engineering.

Spis treści

Design and Validation of CRISPR/Cas9 Systems for Targeted Gene Modification in Induced Pluripotent Stem Cells.- Mutagenesis and Genome Engineering of Epstein-Barr Virus in Cultured Human Cells by CRISPR/Cas9.- Use of CRISPR/Cas Genome Editing Technology for Targeted Mutagenesis in Rice.- All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells.- CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-free E8 Medium.- Development of CRISPR/Cas9 for Efficient Genome Editing in Toxoplasma gondii.- Generation of Stable Knockout Mammalian Cells By TALEN-Mediated Locus-Specific Gene Editing.- Efficient Generation of Gene-Modified Mice by Haploid Embryonic Stem Cell-

Mediated Semi-Cloned Technology. - Insertion of Group II Intron-Based Ribozyme Switches into Homing Endonuclease Genes.- Generating a Genome Editing Nuclease for Targeted Mutagenesis in Human Cells.- Use of Group II Intron Technology for Targeted Mutagenesis in Chlamydia trachomatis.- in silico Approaches to Identify Mutagenesis Targets to Probe and Alter Protein-Cofactor and Protein-Protein Functional Relationships.- in silico Prediction of Deleteriousness for Non-Synonymous and Splice-Altering Single Nucleotide Variants in the Human Genome.- in silico methods for Analyzing Mutagenesis Targets.- Methods for Detecting Critical Residues in Proteins.- A Method for Bioinformatic Analysis of Transposon Insertion Sequencing (INSeq) Results for Identification of Microbial Fitness Determinants.- Application of in vitro Transposon Mutagenesis to Erythromycin Strain Improvement in Saccharopolyspora erythraea.- Engineering Gram-Negative Microbial Cell Factories Using Transposon Vectors.- PERMutation Using Transposase Engineering (PERMUTE):  a Simple Approach for Constructing Circularly-Permuted Protein Libraries.- Transposon Insertion Mutagenesis for Archaeal Gene Discovery.- Genome-Wide Transposon Mutagenesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.- Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.- Seamless Ligation Cloning Extract (Slice) Method Using Cell Lysates from Laboratory Escherichia coli Strains and its Application to Slip Site-Directed Mutagenesis.- Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.- Revised Mechanism and Improved Efficiency of the Quikchange Site-Directed Mutagenesis Method.- An in vitro Single Primer Site-Directed Mutagenesis Method for Use in Biotechnology.- Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Site-Specifically Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function.- Step-by-Step in vitro Mutagenesis - Lessons from Fucose-Binding Lectin PA-IIL.- Analytical Methods for Assessing the Effects of Site-Directed Mutagenesis on Protein-Cofactor and Protein-Protein Functional Relationships.- Biochemical and Biophysical Methods to Examine the Effects of Site-Directed Mutagenesis on Enzymatic Activities and Interprotein Interactions.- Use of Random and Site-Directed Mutagenesis to Probe Protein Structure-Function Relationships: Applied Techniques in the Study of Helicobacter pylori.- Novel Random Mutagenesis Method for Directed Evolution.- Random Mutagenesis by Error-Prone Polymerase Chain Reaction Using a Heavy Water Solvent.- Development and Use of a Novel Random Mutagenesis Method: in Situ Error-Prone PCR (is-epPCR).