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分子生物学-PCR
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Abbreviations ix Preface xi Chapter 1 An Introduction to PCR 1 1.1 Introduction: PCR, a ‘DNA photocopier’ 1 1.2 PCR involves DNA synthesis 1 1.3 PCR is controlled by heating and cooling 3 1.4 PCR applications and gene cloning 5 1.5 History of PCR 6 Chapter 2 Understanding PCR 9 2.1 How does PCR work? 9 2.2 PCR: a molecular perspective 11 2.3 The kinetics of PCR 15 2.4 Getting started 18 2.5 Post-PCR analysis 18 Protocol 2.1: Basic PCR 20 Chapter 3 Reagents and Instrumentation 23 3.1 Technical advances in PCR 23 3.2 Reagents 23 3.3 PCR buffers 23 3.4 Nucleotides 25 3.5 Modified nucleotides 25 3.6 PCR premixes 26 3.7 Oligonucleotide primers 26 3.8 DNA polymerases for PCR 36 3.9 Early PCR experiments 37 3.10 Thermostable DNA polymerases 37 3.11 Properties of Taq DNA polymerase 37 3.12 Thermostable proofreading DNA polymerases 43 3.13 Tth DNA polymerase has reverse transcriptase activity 46 3.14 Red and green polymerases and reagents 47 3.15 Polymerase mixtures: high-fidelity, long-range and RT-PCRs 48 3.16 Nucleic acid templates 51 3.17 Mineral oil 54 3.18 Plasticware and disposables 54 3.19 Automation of PCR and thermal cyclers 55 Protocol 3.1: Phosphorylation of the 5-end of an oligonucleotide 63 Chapter 4 Optimization of PCR 65 4.1 Introduction 65 4.2 Improving specificity of PCR 65 4.3 Template DNA preparation and inhibitors of PCR 75 4.4 Nested PCR improves PCR sensitivity 76 4.5 Contamination problems 76 4.6 Preventing contamination 80 4.7 Troubleshooting guide 82 Chapter 5 Analysis, Sequencing and In Vitro Expression of PCR Products 87 5.1 Introduction 87 5.2 Analysis of PCR products 87 5.3 Verification of initial amplification product 89 5.4 Direct DNA sequencing of PCR products 93 5.5 Direct labeling of PCR products and homogenous assays 101 5.6 In vitro expression of PCR product 103 Protocol 5.1: Cycle sequencing – Applied Biosystems Big Dye terminators 108 Chapter 6 Purification and Cloning of PCR Products 111 6.1 Introduction 111 6.2 Purification of PCR products 111 6.3 Introduction to cloning of PCR products 115 6.4 Approaches to cloning PCR products 117 6.5 Confirmation of cloned PCR fragments 131 Protocol 6.1: Blunt-end polishing of PCR fragments 134 Protocol 6.2: PCR screening of bacterial colonies or cultures 135 Chapter 7 PCR Mutagenesis 137 7.1 Introduction 137 7.2 Inverse PCR mutagenesis 138 7.3 Unique sites elimination 144 7.4 Splicing by overlap extension (SOEing) 144 7.5 Point mutations 150 7.6 Deletions and insertions 151 7.7 Deletion mutagenesis 151 7.8 Insertion mutagenesis 151 7.9 Random mutagenesis 157 7.10 PCR misincorporation procedures 159 7.11 Recombination strategies 160 7.12 RACHITT 166 7.13 Gene synthesis 166 Protocol 7.1: Inverse PCR mutagenesis 171 Protocol 7.2: Quikchange mutagenesis of plasmid DNA 173 Protocol 7.3: Splicing by overlap extension (SOEing) 175 Protocol 7.4: ‘Sticky-feet’ mutagenesis 177 vi Contents Protocol 7.5: DNA shuffling 179 Protocol 7.6: Gene synthesis 182 Chapter 8 Analysis of Gene Expression 185 8.1 Introduction 185 8.2 Reverse transcriptase PCR (RT-PCR) 185 8.3 Semi-quantitative and quantitative RT-PCR 189 8.4 One-tube RT-PCR 194 8.5 Differential display 194 8.6 PCR in a cell: in situ RT-PCR 198 8.7 Microarrays 204 8.8 RNA interference (RNAi) 205 Protocol 8.1: Reverse transcriptase reaction 208 Chapter 9 Real-Time RT-PCR 209 9.1 Introduction 209 9.2 Basic principles of real-time RT-PCR 209 9.3 Detection methods 212 9.4 General guidelines for probe and primer design 221 9.5 Instruments and quantification of results 222 9.6 Normalization and control selection 225 9.7 A typical real-time RT-PCR experiment using SYBR® Green I 225 9.8 Common real-time RT-PCR pitfalls 228 9.9 Applications of real-time RT-PCR 229 Chapter 10 Cloning Genes by PCR 233 A Cloning genes of known DNA sequence 233 10.1 Using PCR to clone expressed genes 233 10.2 Express sequence tags (EST) as cloning tools 237 10.3 Rapid amplification of cDNA ends (RACE) 238 B Isolation of unknown DNA sequences 240 10.4 Inverse polymerase chain reaction (IPCR) 240 10.5 Multiplex restriction site PCR (mrPCR) 243 10.6 Vectorette and splinkerette PCR 244 10.7 Degenerate primers based on peptide sequence 248 Protocol 10.1: 5-RACE 253 Protocol 10.2: Inverse PCR from plant genomic DNA 255 Chapter 11 Genome Analysis 257 11.1 Introduction 257 11.2 Why map genomes? 258 11.3 Single-strand conformation polymorphism analysis (SSCP) 259 11.4 Denaturing-high-performance liquid chromatography (DHPLC) 263 11.5 Ligase chain reaction (LCR) 264 11.6 Amplification refractory mutation system (ARMS) 264 11.7 Cleaved amplified polymorphic sequence analysis (CAPS) 267 11.8 SNP genotyping using DOP-PCR 268 11.9 Random amplified polymorphic DNA (RAPD) PCR 269 11.10 Amplified fragment length polymorphisms (AFLPs) 270 11.11 Multiplex PCR analysis of Alu polymorphisms 270 11.12 Variable number tandem repeats in identity testing 271 11.13 Minisatellite repeat analysis 274 11.14 Microsatellites 276 11.15 Sensitive PCR for environmental and diagnostic applications 277 11.16 Screening transgenics 278 viii Contents |
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