Applied molecular genetics

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Title:

Author:

Miesfeld, Roger L.

ISBN:

9780471156765

Personal Author:

Miesfeld, Roger L.

Publication Information:

New York : Wiley-Liss , 1999.

Physical Description:

293 s. ; 25 sm.

Abstract:

Applied molecular genetics Roger L. Miesfield CONTENTS Preface xiii Acknowledgments xv SECTION 1 LABORATORY TECHNIQUES 1 CHAPTER 1 BIOCHEMICAL BASIS OF APPLIED MOLECULAR GENETICS 3 Flow of Genetic Information: DNA -" RNA -" Protein 3 DNA Synthesis 5 RNA Synthesis 5 Protein Synthesis 5 Nucleic Acid Biochemistry 6 Structure of Nucleic Acid Polymers 6 Denaturation and Renaturation of Nucleic Acid Duplexes 7 Chemical Synthesis of DNA and RNA 10 DNA Metabolizing Enzymes 12 Sequence-Specific DNA Restriction Enzymes 12 Ligases and Kinases 14 DNA and RNA Polymerases 16 Biochemical Methods to Study DNA and RNA 17 Purification of Nucleic Acids 17 Gel Electrophoresis 18 DNA sequencing 19 Membrane Blotting and Hybridization of Nucleic Acids 20 Laboratory Practicum 1: Identifying the transcriptional start site of a gene transcript 22 References 27 CHAPTER 2 LABORATORY TOOLS FOR MOLECULAR GENETIC APPLICATIONS 31 Escherichia coll K-12: The Bacterial Host for All Occasions 32 Biology of E.coliK-12 32 The lac Operon 33 Genetic Variants of E. coli K-12 Used for Gene Cloning 36 Bacterial Plasmids: The Biological Currency of Applied Molecular Genetics 38 Plasmid Biology 38 Antibiotic-Resistance Genes are Often Encoded on Plasmids 39 Plasmid DNA Cloning Vectors 39 Transferring DNA into E. coli by Transformation 42 Bacteriophage Cloning Vectors 44 Biology of Bacteriophage 44 X Phage Cloning Vectors 46 Ml3 Bacteriophage Cloning Vectors 47 Essential Laboratory Resources for Molecular Genetic Research 49 Laboratory P racticum 2: Regulated expression of a cloned gene product in E. coli 52 References 55 CHAPTER 3 OVERVIEW OF GENE ANALYSIS METHODS 59 DNA Libraries Contain a Collection of Gene Sequences 59 Representative DNA Libraries 61 Cloning Vectors Used for DNA Library Construction 63 Screening a X Phage Library by DNA Hybridization 63 Restriction Enzyme Mapping and Subcloning 64 In Vitro Mutagenesis of Cloned DNA Sequences 69 Deletion Mutagenesis 70 Oligonucleotide-Directed Mutagenesis 70 Random Point Mutagenesis 74 Laboratory Practicum 3: Alanine scanning mutagenesis of a gene coding sequence 75 References 78 SECTION 2 CORE METHODS 81 CHAPTER 4 CHARACTERIZATION OF GENOMIC DNA 83 Overview of Genome Organization 83 Organization of the E. coli K-12 Genome 83 Organization of the Yeast S. cerevisiae Genome 84 Organization of the Human Genome 85 Genomic Mapping 88 Linkage Mapping Using Meiotic Recombination Frequencies 89 Genomic Mapping Using Radiation-Induced Chromosome Rearrangements 90 Genomic Mapping Using DNA Sequence Polymorphisms as Genetic Markers 91 Using Genetic Data, DNA Markers, and Cloned Segments to Construct aContigMap 91 Manipulation of Large Genomic DNA Segments 95 FACS Sorting of Intact Chromosomes 95 Chromosome Microdissection 95 Pulsed-Field Gel Electrophoresis 95 Genomic DNA Cloning Vectors 96 Cosmid Vectors 97 Yeast Artificial Chromosome (YAC) Vectors 99 Bacterial Artificial Chromosome (BAG) Vectors 99 Screening Genomic Libraries Using DNA Pools 102 Mapping Gene Regulatory Sequences 102 Mapping Regulatory Sequences by in vivo Expression Assays 102 Mapping Protein Binding Sites by DNasel Protection 104 Electrophoretic Mobility Gel Shift Assays 106 Laboratory Practicum 4: Isolation of a human disease gene by positional cloning 109 References 111 CHAPTER 5 ISOLATION AND CHARACTERIZATION OF GENE TRANSCRIPTS 115 Converting mRNA Transcripts into cDNA 115 mRNA Purification 116 cDNA Synthesis 116 cDNA Cloning Vectors 121 Screening Representative cDNA Libraries 123 Degenerate Oligonucleotide Probes 123 Antibody Probes 124 Subtracted cDNA Probes 126 Functional Screening of cDNA Expression Libraries 128 Protein Activity Assays 128 Yeast Two-Hybrid System 128 cDNA Phage Display 130 Using Cloned cDNA as a Reagent 131 Northern Blotting 131 RNase Protection Assay 133 Nuclear Run-on Assay 133 Laboratory Practicum 5: In vitro expression cloning of genes encoding intracellular signaling proteins 134 References 138 CHAPTER 6 THE POLYMERASE CHAIN REACTION 143 Basic PCR Methodology 143 The PCR Amplification Cycle 144 PCR Requires a Thermostable DNA Polymerase 147 Design of PCR Primers 148 Optimizing a PCR Assay 150 Reverse Transcriptase-Mediated PCR 151 RACE: Rapid Amplification of cDNA Ends 152 Quantitative RT-PCR 152 Amplification of Differentially Expressed Genes 154 Diagnostic Applications of PCR 159 Detecting Pathogens in Tissue Samples 159 Identifying Genetic Mutations 160 PCR Genotyping Using Sequence Tagged Sites 160 Laboratory Applications of PCR 161 Subcloning DNA Targets Using PCR 162 PCR-Mediated in vitro Mutagenesis 163 Laboratory Practicum 6: Cloning cell-specific transcripts using RT-PCR 165 References 169 SECTIONS SPECIALIZED APPLICATIONS 173 CHAPTER 7 EXPRESSION OF CLONED GENES IN CULTURED CELLS 175 Gene Regulation Studies 175 Expression Vectors 176 Reporter Genes 176 Antisense Genetics 181 Methods to Express Genes in Cell Lines 183 Gene Transfer Methods 184 Transient Transfection Assays 186 Stable DNA Transfection 186 Using Yeast as a Model Eukaryotic Cell 191 Protein Expression in Cultured Cells 192 E. coli Expression Systems 193 Baculovirus Expression Systems 193 Laboratory Practicum 7: Characterization of a promoter-selective transcription factor 195 References 200 CHAPTER 8 CONSTRUCTION OFTRANSGENIC MULTICELLULAR ORGANISMS 205 Molecular Genetics of Drosophila Development 205 P Element-Mediated Transformation 206 P Element Enhancer Trap Vectors 208 Construction of Transgenic Crop Plants 208 Agmbacterium-Mediated Gene Transfer 210 Transgenesis of Rice and Corn with Biolistics 210 Agricultural Impact of Transgenic Crop Plants 212 Mouse Transgenesis 214 Generating Transgenic Mice Using Fertilized Egg Cells 214 Gene Knockouts by Homologous Recombination in ES Cells 215 Transgenic Mouse Models of Human Diseases 222 Development of Transgenic Livestock 223 Animal Pharming Using Transgenic Livestock 223 Cloning Animals by Nuclear Transfer 224 Laboratory Practicum 8: Creating cell-specific gene knockouts in transgenic mice 229 References 231 CHAPTER 9 CONTEMPORARY APPLIED MOLECULAR GENETICS 237 Automated Instrumentation for Genome Analysis 237 High-Throughput DNA Sequencing 237 Genome Probing Using DNA Microarrays 238 Robot-Driven Computerized Work Stations 241 Accessing Molecular Genetic Information through the Internet 244 DNA Sequence Analysis Programs and Databases 246 Integrated Biological Resource Databases 247 Bionet News Groups for Molecular Genetic Research 248 Applied Medical Molecular Genetics 250 DNA Forensics 250 Molecular Genetic Pathophysiology 251 Somatic Cell Gene Therapy 253 Laboratory Practicum 9: Identifying orthologous genes by searching Internet DNA databases. 258 References 262 SECTION 4 APPENDICES 267 Appendix A Standard Abbreviations 269 Appendix B Properties of Nucleic Acids 275 Appendix C Properties of Amino Acids 277 Appendix D Properties of Common Restriction Enzymes 279 Appendix E E. Coli Genetic Markers 281 Appendix F Gel Electrophoresis Information 285 Appendix G Useful Internet Resources 287 Index 289

Subject Term:

Genetik

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Library	Material Type	Item Barcode	Shelf Number	Status
Searching... Ferit Melen Library	Book	049547	575.1 MİEa 1999 k.1	Searching... Unknown

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