Human molecular biology : laboratory manual

Preface xi Chapter 1 Preparation of Human Genomic DNA 1 Introduction 1 Background 1 Cell Breakage 3 Removal of Protein 3 Deproteinization using organic solvents 3 Deproteinization using enzymes 6 Removal of RNA 6 Concentrating the DNA 7 Determination of the Purity and Quantity of DNA 9 First Laboratory Period 11 Safety precautions 11 Technical tips 11 Protocol 13 Second Laboratory Period 16 Safety precautions 16 Technical tips 16 Protocol 16 Expected results 18 References 19 Chapter 2 DNA Fingerprinting: Multi-locus Analysis 20 Introduction 20 Background 20 First Laboratory Period 25 Experiment 1: Restriction Enzyme Digestion 25 Introduction 25 Background 25 Technical tips 26 Protocol 27 References 28 Experiment 2: agarose gel electrophoresis 29 Introduction 29 Background 29 Safety precautions 34 Protocol 34 References 36 Second Laboratory Period 37 Experiment 3: Southern blotting 37 Introduction 37 Background 37 Safety precautions 38 Technical tips 38 Protocol 38 References 41 Third Laboratory Period 43 Experiment 4: preparation of probe and hybridization 43 Introduction 43 Background 43 Technical tips 50 Protocol 51 References 54 Fourth Laboratory Period 56 Protocol 56 Fifth Laboratory Period 58 Protocol 58 Data analysis 59 Chapter 3 DNA Fingerprinting: Single-locus Analysis 62 Introduction 62 Background 63 First Laboratory Period 66 Protocol 66 Second Laboratory Period 69 Protocol 69 Data analysis 69 Expected results 72 References 75 General reading 75 Chapter 4 Out of Africa: Origin of Modern Humans 76 Introduction 76 Background 76 Origin of humans 76 PCR 80 First Laboratory Period 85 Technical tips 85 Protocol 85 Second Laboratory Period 89 Safety precautions 89 Protocol 89 Data analysis 92 Expected results 93 References 94 Chapter 5 DNA Sequencing 95 Introduction 95 Background 96 DNA sequencing methods 97 Sequencing strategies 98 References 99 First Laboratory Period 100 Experiment 1: nebulization shearing of DNA 100 Introduction 100 Background 100 Safety precautions 102 Technical tips 103 Protocol 104 Expected results 106 Experiment 2: repair of the ends of sheared DNA 106 Introduction 106 Safety precautions 106 Technical tips 107 Protocol 108 References 110 Second Laboratory Period 111 Experiment 3: ligation to sequencing vector 111 Introduction 111 Background 111 Technical tips 118 Protocol 119 References 121 Experiment 4: transformation of bacteria by electroporation 121 Introduction 121 Background 122 Technical tips 124 Protocol 126 Expected results 128 References 129 Third Laboratory Period 130 Experiment 5: preparation of plasmid for DNA sequencing 130 Introduction 130 Background 130 Technical tips 131 Protocol 132 Experiment 6: sequencing reactions for an ABI 3700 sequencer 134 Introduction 134 Technical tips 135 Protocol 136 Fourth Laboratory Period 137 Experiment 7: removing dideoxy terminators 137 Protocol 137 References 137 Chapter 6 Computer Analysis of Sequencing Data 139 Introduction 139 Background 139 Databases and sequence formats 140 Sequence alignments 143 BLAST 147 FASTA 150 BLAST versus FASTA 150 Single-sequence analysis 151 Dot matrix analysis 152 Technical tips 154 Protocol 155 References 157 Sequence Alignment with BLAST 158 Search of “nr” database 158 Search for an Alu SINE element 159 Search for expressed sequences 159 Search for the Chromosome Position of the Query Sequence 159 Single-sequence Analysis 161 Converting file formats 161 Base content analysis 162 Restriction enzyme site analysis 162 Dot matrix analysis 162 Chapter 7 Determination of Human Telomere Length 164 Introduction 164 Background 165 First Laboratory Period 170 Experiment 1: isolation of genomic DNA 170 Contents Introduction 170 Background 170 Safety precautions 171 Protocol 171 Second Laboratory Period 174 Experiment 2: determination of DNA concentration and purity 174 Protocol 174 Experiment 3: restriction enzyme digestion 174 Introduction 174 Background 175 Technical tips 175 Protocol 176 Experiment 4: agarose gel electrophoresis 177 Introduction 177 Background 177 Safety precautions 177 Technical tips 177 Protocol 178 Third Laboratory Period 180 Experiment 5: Southern transfer 180 Introduction 180 Background 180 Safety precautions 180 Technical tips 181 Protocol 181 Fourth Laboratory Period 185 Experiment 6: DNA hybridization 185 Introduction 185 Background 185 Technical tips 185 Protocol 186 Fifth Laboratory Period 187 Protocol 187 Sixth Laboratory Period 190 Experiment 7: analysis of TRF length 190 Introduction 190 Background 190 Technical tips 191 Protocol 192 References 194 Chapter 8 RT-PCR of Human Genes 196 Introduction 196 Background 196 First Laboratory Period 198 Experiment 1: purification of total RNA 198 Introduction 198 Background 198 Safety precautions 200 Technical tips 201 Protocol 202 References 203 Experiment 2: RNA agarose gel electrophoresis 204 Introduction 204 Background 204 Safety precautions 205 Technical tips 205 Protocol 205 References 207 Second Laboratory Period 209 Experiment 3: running an RT-PCR 209 Introduction 209 Background 209 Safety precautions 212 Technical tips 212 Protocol 213 Appendix 215 DNA Purification 215 Equipment and supplies 215 Solutions to prepare 216 DNA Fingerprinting: Multi-locus Analysis 217 Equipment and supplies 217 Solutions to prepare 218 DNA Fingerprinting: Single-locus Analysis 219 Equipment and supplies 219 Solutions to prepare 219 Out of Africa: Origin of Modern Humans 220 Equipment and supplies 220 Solutions to prepare 220 DNA Sequencing 220 Equipment and supplies 220 Solutions to prepare 221 Determination of Human Telomere Length 222 Equipment and supplies 222 RT-PCR of Human Genes 222 Equipment and supplies 222 Index 22