Mulhardt Molecular Biology and Genomics

1. Auflage 2010
ISBN: 978-0-08-046766-5
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 272 Seiten

Reihe: The Experimenter Series

Never before has it been so critical for lab workers to possess the proper tools and methodologies necessary to determine the structure, function, and expression of the corresponding proteins encoded in the genome. Mulhardt's Molecular Biology and Genomics helps aid in this daunting task by providing the reader with tips and tricks for more successful lab experiments. This strategic lab guide explores the current methodological variety of molecular biology and genomics in a simple manner, addressing the assets and drawbacks as well as critical points. It also provides short and precise summaries of routine procedures as well as listings of the advantages and disadvantages of alternative methods.
* Shows how to avoid experimental dead ends and develops an instinct for the right experiment at the right time
* Includes a handy Career Guide for researchers in the field
* Contains more than 100 extensive figures and tables

Autoren/Hrsg.

Mulhardt, Cornel

Inhaltsverzeichnis

1;Front Cover;1
2;Molecular Biology and Genomics;4
3;Copyright Page;5
4;Table of Contents;6
5;Foreword;12
6;Foreword to the First Edition;14
7;Acknowledgments;15
8;Chapter 1 What Is Molecular Biology?;16
8.1;1.1 The Substrate of Molecular Biology: The Molecular World for Beginners;17
8.2;1.2 What Is Required for This Work?;22
8.3;1.3 Safety in the Laboratory;23
9;Chapter 2 Fundamental Methods;26
9.1;2.1 Differences in Nucleic Acids;26
9.2;2.2 Precipitation and Concentration of Nucleic Acids;28
9.2.1;2.2.1 Alcohol Precipitation;28
9.2.2;2.2.2 Concentrators;31
9.2.3;2.2.3 Savant Speed Vac;32
9.2.4;2.2.4 Salting Out;32
9.3;2.3 Purification of Nucleic Acids;32
9.3.1;2.3.1 Phenol-Chloroform Extraction;32
9.3.2;2.3.2 Precipitation Using Polyethylene Glycol;34
9.3.3;2.3.3 Protein-Binding Filter Membranes;34
9.3.4;2.3.4 Anion-Exchange Columns;34
9.3.5;2.3.5 Glass Milk;35
9.3.6;2.3.6 Cesium Chloride Density Gradient;36
9.3.7;2.3.7 Dialysis;37
9.4;2.4 Determining the Concentration of Nucleic Acid Solutions;37
9.4.1;2.4.1 Optical Density Measurements with the Aid of Absorption Spectrometry;37
9.4.2;2.4.2 Determination of Concentration by Means of Agarose Gels;39
9.4.3;2.4.3 Dot Quantification;39
9.4.4;2.4.4 Fluorometric Determination;40
9.4.5;2.4.5 Nucleic Acid Dipsticks;40
9.4.6;2.4.6 Enzymatic Evidence;40
9.5;2.5 Methods of DNA Preparation;41
9.5.1;2.5.1 Preparation of Plasmid DNA on a Small Scale;41
9.5.2;2.5.2 Preparation of Plasmid DNA on a Large Scale;43
9.5.3;2.5.3 Bacterial Media;46
9.5.4;2.5.4 Preparation of Phage DNA;46
9.5.5;2.5.5 Preparation of Single-Stranded DNA with the Aid of Helper Phages;50
9.5.6;2.5.6 Preparation of Genomic DNA;50
10;Chapter 3 The Tools;52
10.1;3.1 Restriction Enzymes;52
10.1.1;3.1.1 Nomenclature;52
10.1.2;3.1.2 The Activity Test;54
10.1.3;3.1.3 Making a Restriction Digestion;55
10.1.4;3.1.4 Difficulties Associated with Restriction Digestion;56
10.1.5;3.1.5 Works of Reference for Restriction Digestion;59
10.2;3.2 Gels;60
10.2.1;3.2.1 Agarose Gels;60
10.2.2;3.2.2 Isolating DNA Fragments from Agarose Gels;65
10.2.3;3.2.3 Polyacrylamide Gels;68
10.2.4;3.2.4 Isolating DNA Fragments from Polyacrylamide Gels;70
10.2.5;3.2.5 Pulse-Field Gel Electrophoresis;71
10.2.6;3.2.6 Capillary Electrophoresis;71
10.3;3.3 Blotting;72
10.3.1;3.3.1 Southern Blots;73
10.3.2;3.3.2 Northern Blots;75
10.3.3;3.3.3 Dot Blots and Slot Blots;77
11;Chapter 4 The Polymerase Chain Reaction;80
11.1;4.1 Standard Polymerase Chain Reaction;80
11.2;4.2 Suggestions for Improving the Polymerase Chain Reaction;89
11.2.1;4.2.1 Nested Polymerase Chain Reaction;91
11.2.2;4.2.2 Multiplex Polymerase Chain Reaction;91
11.2.3;4.2.3 Amplification of Longer DNA Fragments;92
11.3;4.3 PCR Applications;93
11.3.1;4.3.1 Reverse Transcription-Polymerase Chain Reaction;93
11.3.2;4.3.2 Rapid Amplification of cDNA Ends;94
11.3.3;4.3.3 Amplification of Coincidental Products;95
11.3.4;4.3.4 Classic Quantitative Polymerase Chain Reaction;97
11.3.5;4.3.5 Real-Time Quantitative Polymerase Chain Reaction;99
11.3.6;4.3.6 Inverse Polymerase Chain Reaction;104
11.3.7;4.3.7 Biotin-RAGE Method and Supported Polymerase Chain Reaction;104
11.3.8;4.3.8 Mutagenesis with Modified Primers;105
11.3.9;4.3.9 Amplification Refractory Mutation System;106
11.3.10;4.3.10 In Situ Polymerase Chain Reaction;107
11.3.11;4.3.11 Cycle Sequencing;107
11.3.12;4.3.12 cDNA Synthesis;108
11.3.13;4.3.13 Single-Cell Polymerase Chain Reaction;109
11.4;Chapter 5 RNA;110
11.4.1;5.1 Inactivating RNases;110
11.4.2;5.2 Methods of RNA Isolation;111
11.4.2.1;5.2.1 Single-Step Method;111
11.4.2.2;5.2.2 Lysis with Nonidet P40;112
11.4.2.3;5.2.3 General Information;112
11.4.2.4;5.2.4 Determination of the RNA Concentration;113
11.4.3;5.3 Methods of mRNA Isolation;113
11.4.3.1;5.3.1 Purchasing RNA;114
11.4.4;5.4 Reverse Transcription: cDNA Synthesis;114
11.4.5;5.5 In Vitro Transcription: RNA Synthesis;115
11.4.6;5.6 RNA Interference;117
11.5;Chapter 6 Cloning DNA Fragments;120
11.5.1;6.1 The Basics of Cloning;120
11.5.1.1;6.1.1 The Vector;121
11.5.1.2;6.1.2 The DNA Fragment;122
11.5.1.3;6.1.3 Fill-in Reaction;123
11.5.1.4;6.1.4 DNA Quantities;124
11.5.1.5;6.1.5 The Ligation;125
11.5.1.6;6.1.6 Cloning Polymerase Chain Reaction Products;126
11.5.1.7;6.1.7 Cloning with Recombinase Systems;128
11.5.2;6.2 Choosing Vectors for Cloning;131
11.5.2.1;6.2.1 Plasmids;131
11.5.2.2;6.2.2 Phages;133
11.5.2.3;6.2.3 Cosmids;134
11.5.2.4;6.2.4 P1 Artificial Chromosomes and Bacterial Artificial Chromosomes;135
11.5.2.5;6.2.5 Yeast Artificial Chromosomes;136
11.5.3;6.3 Choosing the Bacteria;137
11.5.4;6.4 Construction of Competent Cells and Their Transformation;137
11.5.4.1;6.4.1 Calcium Chloride Method;138
11.5.4.2;6.4.2 Rubidium Chloride Method;139
11.5.4.3;6.4.3 TSS Method;139
11.5.4.4;6.4.4 Electroporation;139
11.5.4.5;6.4.5 Testing the Competency of Bacteria;141
11.5.5;6.5 Problems Associated with Cloning;142
11.5.6;6.6 Storage of Clones;143
11.6;Chapter 7 Hybridization: How to Track Down DNA;146
11.6.1;7.1 Production of Probes;146
11.6.1.1;7.1.1 Methods for the Production of Labeled Probes;147
11.6.2;7.2 Hybridization;151
11.6.2.1;7.2.1 The Hybridization Buffer;151
11.6.2.2;7.2.2 The Hybridization Vessels;152
11.6.2.3;7.2.3 The Hybridization Temperature;152
11.6.2.4;7.2.4 Washing;153
11.6.3;7.3 Verification of Labeled DNA;153
11.6.3.1;7.3.1 Autoradiography;153
11.6.3.2;7.3.2 Nonradioactive Methods of Detection;155
11.6.4;7.4 Screening of Recombinant DNA Banks;158
11.6.4.1;7.4.1 Plating Out the Bank;158
11.6.4.2;7.4.2 Filter Transfer;158
11.6.4.3;7.4.3 Filter Hybridization;159
11.6.4.4;7.4.4 Filter Exposure;159
11.6.4.5;7.4.5 Clone Detection;160
11.6.4.6;7.4.6 Bank Screening in the Future;161
11.6.5;7.5 Two-Hybrid System;161
11.7;Chapter 8 DNA Analysis;166
11.7.1;8.1 Sequencing;166
11.7.1.1;8.1.1 Radioactive Sequencing;168
11.7.1.2;8.1.2 Nonradioactive Sequencing and Automatic Sequencing Units;169
11.7.1.3;8.1.3 Minisequencing;171
11.7.1.4;8.1.4 Pyrosequencing;172
11.7.1.5;8.1.5 Idiosyncrasies in Sequencing: Octamers;174
11.7.2;8.2 Methods of Analyzing DNA for Mutations;175
11.7.2.1;8.2.1 Restriction Fragment Length Polymorphism;175
11.7.2.2;8.2.2 Single-Strand Conformation Polymorphism;176
11.7.2.3;8.2.3 Denaturing Gradient Gel Electrophoresis;177
11.7.2.4;8.2.4 Temporal Temperature Gradient Electrophoresis;179
11.7.2.5;8.2.5 Heteroduplex Analysis;180
11.7.2.6;8.2.6 Amplification Refractory Mutation System;180
11.7.2.7;8.2.7 Enzyme Mismatch Cleavage;180
11.7.2.8;8.2.8 Protein Truncation Test;182
11.8;Chapter 9 Investigating the Function of DNA Sequences;184
11.8.1;9.1 Investigating Transcription in Tissues;185
11.8.1.1;9.1.1 Ribonuclease Protection Assay;185
11.8.1.2;9.1.2 Real-Time Quantitative Polymerase Chain Reaction;185
11.8.1.3;9.1.3 In Situ Hybridization;186
11.8.1.4;9.1.4 Fluorescence In Situ Hybridization of Chromosomes;188
11.8.1.5;9.1.5 In Situ Polymerase Chain Reaction;190
11.8.1.6;9.1.6 Microarrays;190
11.8.2;9.2 Mutagenesis;195
11.8.3;9.3 In Vitro Translation;204
11.8.4;9.4 Expression Systems;205
11.8.4.1;9.4.1 Bacterial Expression Systems;206
11.8.4.2;9.4.2 Baculoviral Expression Systems;206
11.8.4.3;9.4.3 Additional Expression Systems;208
11.8.4.4;9.4.4 Heterologous Expression in Mammalian Cells;209
11.8.4.5;9.4.5 Transfection Methods;210
11.8.4.6;9.4.6 Cotransfection of Several Genes;217
11.8.4.7;9.4.7 Transient and Stabile Transfections;218
11.8.4.8;9.4.8 Reporter Genes;219
11.8.5;9.5 Transgenic Mice;224
11.8.5.1;9.5.1 Methods of Gene Transfer;225
11.8.6;9.6 Regulation of Transgenic Expression;230
11.8.6.1;9.6.1 The Tet System;230
11.8.6.2;9.6.2 The Ecdysone System;231
11.8.7;9.7 Gene Therapy;232
11.8.8;9.8 Genomics;234
11.9;Chapter 10 Using Computers;238
11.9.1;10.1 Something Quite Earnest;238
11.9.2;10.2 A Matter of Practice;240
11.10;Chapter 11 Suggestions for Career Planning: The Machiavelli Short Course for Young Researchers;242
11.11;Chapter 12 Concluding Thoughts;248
11.12;Appendix 1;250
11.12.1;Useful Figures and Tables;250
11.12.2;Standard Solutions and Bacterial Media;250
11.12.2.1;Solutions;250
11.12.2.2;Bacterial Media;251
11.12.3;Glossary;252
11.13;Appendix 2;256
11.13.1;Suppliers;256
11.13.2;Recommended Literature;259
11.13.3;Reading Material for Leisure Time;260
11.14;Index;262