Molecular Biology and Genomics (eBook)

Front Cover 1
Molecular Biology and Genomics 4
Copyright Page 5
Table of Contents 6
Foreword 12
Foreword to the First Edition 14
Acknowledgments 15
Chapter 1 What Is Molecular Biology? 16
1.1 The Substrate of Molecular Biology: The Molecular World for Beginners 17
1.2 What Is Required for This Work? 22
1.3 Safety in the Laboratory 23
Chapter 2 Fundamental Methods 26
2.1 Differences in Nucleic Acids 26
2.2 Precipitation and Concentration of Nucleic Acids 28
2.2.1 Alcohol Precipitation 28
2.2.2 Concentrators 31
2.2.3 Savant Speed Vac 32
2.2.4 Salting Out 32
2.3 Purification of Nucleic Acids 32
2.3.1 Phenol-Chloroform Extraction 32
2.3.2 Precipitation Using Polyethylene Glycol 34
2.3.3 Protein-Binding Filter Membranes 34
2.3.4 Anion-Exchange Columns 34
2.3.5 Glass Milk 35
2.3.6 Cesium Chloride Density Gradient 36
2.3.7 Dialysis 37
2.4 Determining the Concentration of Nucleic Acid Solutions 37
2.4.1 Optical Density Measurements with the Aid of Absorption Spectrometry 37
2.4.2 Determination of Concentration by Means of Agarose Gels 39
2.4.3 Dot Quantification 39
2.4.4 Fluorometric Determination 40
2.4.5 Nucleic Acid Dipsticks 40
2.4.6 Enzymatic Evidence 40
2.5 Methods of DNA Preparation 41
2.5.1 Preparation of Plasmid DNA on a Small Scale 41
2.5.2 Preparation of Plasmid DNA on a Large Scale 43
2.5.3 Bacterial Media 46
2.5.4 Preparation of Phage DNA 46
2.5.5 Preparation of Single-Stranded DNA with the Aid of Helper Phages 50
2.5.6 Preparation of Genomic DNA 50
Chapter 3 The Tools 52
3.1 Restriction Enzymes 52
3.1.1 Nomenclature 52
3.1.2 The Activity Test 54
3.1.3 Making a Restriction Digestion 55
3.1.4 Difficulties Associated with Restriction Digestion 56
3.1.5 Works of Reference for Restriction Digestion 59
3.2 Gels 60
3.2.1 Agarose Gels 60
3.2.2 Isolating DNA Fragments from Agarose Gels 65
3.2.3 Polyacrylamide Gels 68
3.2.4 Isolating DNA Fragments from Polyacrylamide Gels 70
3.2.5 Pulse-Field Gel Electrophoresis 71
3.2.6 Capillary Electrophoresis 71
3.3 Blotting 72
3.3.1 Southern Blots 73
3.3.2 Northern Blots 75
3.3.3 Dot Blots and Slot Blots 77
Chapter 4 The Polymerase Chain Reaction 80
4.1 Standard Polymerase Chain Reaction 80
4.2 Suggestions for Improving the Polymerase Chain Reaction 89
4.2.1 Nested Polymerase Chain Reaction 91
4.2.2 Multiplex Polymerase Chain Reaction 91
4.2.3 Amplification of Longer DNA Fragments 92
4.3 PCR Applications 93
4.3.1 Reverse Transcription-Polymerase Chain Reaction 93
4.3.2 Rapid Amplification of cDNA Ends 94
4.3.3 Amplification of Coincidental Products 95
4.3.4 Classic Quantitative Polymerase Chain Reaction 97
4.3.5 Real-Time Quantitative Polymerase Chain Reaction 99
4.3.6 Inverse Polymerase Chain Reaction 104
4.3.7 Biotin-RAGE Method and Supported Polymerase Chain Reaction 104
4.3.8 Mutagenesis with Modified Primers 105
4.3.9 Amplification Refractory Mutation System 106
4.3.10 In Situ Polymerase Chain Reaction 107
4.3.11 Cycle Sequencing 107
4.3.12 cDNA Synthesis 108
4.3.13 Single-Cell Polymerase Chain Reaction 109
Chapter 5 RNA 110
5.1 Inactivating RNases 110
5.2 Methods of RNA Isolation 111
5.2.1 Single-Step Method 111
5.2.2 Lysis with Nonidet P40 112
5.2.3 General Information 112
5.2.4 Determination of the RNA Concentration 113
5.3 Methods of mRNA Isolation 113
5.3.1 Purchasing RNA 114
5.4 Reverse Transcription: cDNA Synthesis 114
5.5 In Vitro Transcription: RNA Synthesis 115
5.6 RNA Interference 117
Chapter 6 Cloning DNA Fragments 120
6.1 The Basics of Cloning 120
6.1.1 The Vector 121
6.1.2 The DNA Fragment 122
6.1.3 Fill-in Reaction 123
6.1.4 DNA Quantities 124
6.1.5 The Ligation 125
6.1.6 Cloning Polymerase Chain Reaction Products 126
6.1.7 Cloning with Recombinase Systems 128
6.2 Choosing Vectors for Cloning 131
6.2.1 Plasmids 131
6.2.2 Phages 133
6.2.3 Cosmids 134
6.2.4 P1 Artificial Chromosomes and Bacterial Artificial Chromosomes 135
6.2.5 Yeast Artificial Chromosomes 136
6.3 Choosing the Bacteria 137
6.4 Construction of Competent Cells and Their Transformation 137
6.4.1 Calcium Chloride Method 138
6.4.2 Rubidium Chloride Method 139
6.4.3 TSS Method 139
6.4.4 Electroporation 139
6.4.5 Testing the Competency of Bacteria 141
6.5 Problems Associated with Cloning 142
6.6 Storage of Clones 143
Chapter 7 Hybridization: How to Track Down DNA 146
7.1 Production of Probes 146
7.1.1 Methods for the Production of Labeled Probes 147
7.2 Hybridization 151
7.2.1 The Hybridization Buffer 151
7.2.2 The Hybridization Vessels 152
7.2.3 The Hybridization Temperature 152
7.2.4 Washing 153
7.3 Verification of Labeled DNA 153
7.3.1 Autoradiography 153
7.3.2 Nonradioactive Methods of Detection 155
7.4 Screening of Recombinant DNA Banks 158
7.4.1 Plating Out the Bank 158
7.4.2 Filter Transfer 158
7.4.3 Filter Hybridization 159
7.4.4 Filter Exposure 159
7.4.5 Clone Detection 160
7.4.6 Bank Screening in the Future 161
7.5 Two-Hybrid System 161
Chapter 8 DNA Analysis 166
8.1 Sequencing 166
8.1.1 Radioactive Sequencing 168
8.1.2 Nonradioactive Sequencing and Automatic Sequencing Units 169
8.1.3 Minisequencing 171
8.1.4 Pyrosequencing 172
8.1.5 Idiosyncrasies in Sequencing: Octamers 174
8.2 Methods of Analyzing DNA for Mutations 175
8.2.1 Restriction Fragment Length Polymorphism 175
8.2.2 Single-Strand Conformation Polymorphism 176
8.2.3 Denaturing Gradient Gel Electrophoresis 177
8.2.4 Temporal Temperature Gradient Electrophoresis 179
8.2.5 Heteroduplex Analysis 180
8.2.6 Amplification Refractory Mutation System 180
8.2.7 Enzyme Mismatch Cleavage 180
8.2.8 Protein Truncation Test 182
Chapter 9 Investigating the Function of DNA Sequences 184
9.1 Investigating Transcription in Tissues 185
9.1.1 Ribonuclease Protection Assay 185
9.1.2 Real-Time Quantitative Polymerase Chain Reaction 185
9.1.3 In Situ Hybridization 186
9.1.4 Fluorescence In Situ Hybridization of Chromosomes 188
9.1.5 In Situ Polymerase Chain Reaction 190
9.1.6 Microarrays 190
9.2 Mutagenesis 195
9.3 In Vitro Translation 204
9.4 Expression Systems 205
9.4.1 Bacterial Expression Systems 206
9.4.2 Baculoviral Expression Systems 206
9.4.3 Additional Expression Systems 208
9.4.4 Heterologous Expression in Mammalian Cells 209
9.4.5 Transfection Methods 210
9.4.6 Cotransfection of Several Genes 217
9.4.7 Transient and Stabile Transfections 218
9.4.8 Reporter Genes 219
9.5 Transgenic Mice 224
9.5.1 Methods of Gene Transfer 225
9.6 Regulation of Transgenic Expression 230
9.6.1 The Tet System 230
9.6.2 The Ecdysone System 231
9.7 Gene Therapy 232
9.8 Genomics 234
Chapter 10 Using Computers 238
10.1 Something Quite Earnest 238
10.2 A Matter of Practice 240
Chapter 11 Suggestions for Career Planning: The Machiavelli Short Course for Young Researchers 242
Chapter 12 Concluding Thoughts 248
Appendix 1 250
Useful Figures and Tables 250
Standard Solutions and Bacterial Media 250
Solutions 250
Bacterial Media 251
Glossary 252
Appendix 2 256
Suppliers 256
Recommended Literature 259
Reading Material for Leisure Time 260
Index 262