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1949stone

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[资源] 分子生物学-PCR

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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