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We are entering a particularly fruitful period in evolutionary genetics, as rapid technological progress transforms the investigation of genetic variation within and between species. Molecular Methods for Evolutionary Genetics is a collection of advanced molecular biology protocols and general overviews intended to represent the essential methods currently bringing evolutionary genetics to fruition. Divided into six thematic sections, this volume covers methods for characterizing genomes, diverse approaches to enrich DNA for subsets of the genome prior to sequencing, and state-of-the-art protocols for sampling genetic variation for genetic mapping studies and population genetic studies (RAD sequencing, Sequenom, microarrays, etc.). The volume concludes by focusing on methods to study candidate genes, from obtaining their sequences and analyzing their transcripts to experimentally manipulating their activities in vivo. Written in the highly successful Methods in Molecular Biology(TM) series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.
Authoritative and accessible, Molecular Methods for Evolutionary Genetics serves as a rich resource to biologists interested in evolution, whether they be specialists or beginners in molecular biology.
Table des matières
Genome Size Determination using Flow Cytometry of Propidium Iodide-stained Nuclei.- Chromosome Analysis in Invertebrates and Vertebrates.- Genomic Libraries. I. Construction and Screening of Fosmid Genomic Libraries.- Genomic Libraries. II. Subcloning, Sequencing, and Assembling Large-insert Genomic DNA Clones.- Reduced Representation Methods for Sub-genomic Enrichment and Next-generation Sequencing.- Accessing the Transcriptome: How to Normalize mRNA Pools.- Transcriptome Sequencing Goals, Assembly, and Assessment.- Rapid Retrieval of DNA Target Sequences by Primer Extension Capture.- SNP Discovery and Genotyping for Evolutionary Genetics using RAD Sequencing.- DNA Microarray-based Mutation Discovery and Genotyping.- Genotyping with Sequenom.- Isolating Microsatellite Loci: Looking Back, Looking Ahead.- Design of Custom Oligonucleotide Microarrays for Single Species or Interspecies Hybrids using Array Oligo Selector.- Identification of Homologous Gene Sequences by PCR with Degenerate Primers.- Characterizing cDNA Ends by Circular RACE.- Identification of DNA Sequences that Flank a Known Region by Inverse PCR.- Quantification of Transcript Levels with Quantitative RT-PCR.- Using Pyrosequencing to Measure Allele-specific mRNA Abundance and Infer the Effects of Cis- and Trans-regulatory Differences.- Whole Mount In situ Hybridization of Sectioned Tissues of Species Hybrids to Detect Cis-Regulatory Changes in Gene Expression Pattern.- Identifying Fluorescently Labeled Single Molecules in Image Stacks using Machine Learning.- Experimental Approaches to Evaluate the Contributions of Candidate Cis-regulatory Mutations to Phenotypic Evolution.- Experimental Approaches to Evaluate the Contributions of Candidate Protein-coding Mutations to Phenotypic Evolution.- Making Reporter Gene Constructs to Analyze Cis-regulatory Elements.- PCR-directed In vivo Plasmid Construction using Homologous Recombination in Baker's Yeast.- Production of Fosmid Genomic Libraries Optimized for Liquid Culture Recombineering and Cross-species Transgensis.- Recombination-mediated Genetic Engineering of Large Genomic DNA Transgenes.- Overlap Extension PCR: An Efficient Method for Transgene Construction.- Gene Knockdown Analysis by Double-stranded RNA Injection
Résumé
Divided into six thematic sections, this volume covers methods for characterizing genomes, diverse approaches to enrich DNA for subsets of the genome prior to sequencing, and state-of-the-art protocols for sampling genetic variation for genetic mapping studies and population genetic studies (RAD sequencing, Sequenom, microarrays, etc.).
