Principles and technical aspects of PCR amplification:
Saved in:
| Main Authors: | , , |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
[Dordrecht]
Springer
2008
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XII, 323 S. Ill., graph. Darst. |
| ISBN: | 9781402062407 9781402062414 |
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MARC
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| 100 | 1 | |a Pelt-Verkuil, Elizabeth van |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Principles and technical aspects of PCR amplification |c Elizabeth van Pelt-Verkuil ; Alex van Belkum ; John P. Hays |
| 264 | 1 | |a [Dordrecht] |b Springer |c 2008 | |
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Record in the Search Index
| _version_ | 1804138604158517248 |
|---|---|
| adam_text | Contents
Foreword v
Chapter 1 The Polymerase Chain Reaction 1
1.1 An Overview of the PCR Process 1
1.2 Before PCR and Beyond 2
Chapter 2 A Brief Comparison Between In Vivo DNA Replication
and In Vitro PCR Amplification 9
2.1 Nucleic Acid Targets 9
2.1.1 DNA 9
2.1.2 RNA 11
2.2 Target DNA Strand Separation and Primer Annealing 12
2.3 DNA Dependent DNA Polymerase and Oligonucleotide
Primers 13
2.4 Deoxyribonucleotides and Additional Factors 14
Chapter 3 The PCR in Practice 17
3.1 Brief Overview of PCR Requirements 17
3.1.1 The PCR Reaction Mix 17
3.1.2 The PCR Thermocycling Regime 19
3.1.3 Analysis of PCR Amplification Products 22
3.1.4 Miscellaneous Considerations 22
Chapter 4 The Different Types and Varieties of Nucleic Acid
Target Molecules 25
4.1 General Features 25
4.2 A Brief Description of In Vivo DNA and RNA Targets 26
4.3 DNA Samples 33
4.3.1 DNA Isolation Procedures 34
4.3.2 Comments on Nucleic Acids in Specific
Sample Types 39
4.4 RNA Samples 44
4.4.1 Working Free of RNase Contamination 45
vii
viii Contents
4.4.2 RNA Isolation for RT-PCR 47
4.5 Reverse Transcription and RT-PCR 50
4.5.1 cDNA Synthesis 53
4.5.2 cDNA Synthesis Using RACE 55
4.5.3 RNA Extraction and cDNA
Synthesis Controls 57
Chapter 5 PCR Primers 63
5.1 PCR Primer Design and Quality Requirements 64
5.1.1 Different Primer Species 68
5.2 Primer Hybridisation (Annealing) 76
5.3 Thermodynamic Approach of Tm Calculations 78
5.4 Primer Synthesis 81
5.5 Non-radioactive Primer Labelling 83
5.6 The Effect of Mismatches Between PCR
Primer and Target 86
5.7 Primer Concentration 87
Chapter 6 Deoxynucleotide Triphosphates and Buffer Components 91
6.1 Factors Affecting the Choice of dNTP Concentration 92
6.2 Modified dNTPs and Their Applications 93
6.3 The PCR Buffer 94
6.3.1 Monovalent Ions 98
6.3.2 Magnesium Ions 99
Chapter 7 Taq and Other Thermostable DNA Polymerases 103
7.1 The Advantages and Disadvantages of Taq over
Klenow Fragment DNA Polymerase 104
7.2 Misincorporation of Nucleotides and Fidelity
of DNA Synthesis by Taq Polymerase 107
7.3 Taq DNA Polymerase and Its Modifications 110
7.4 Taq Polymerase Unit Definition and Working
Concentrations 113
7.5 Other Thermostable Polymerases
and Their Applications 113
7.6 Mixtures of Thermostable Polymerases 117
Chapter 8 Important Considerations for Typical, Quantitative
and Real-Time PCR Protocols 119
8.1 The Typical PCR Amplification Protocol 119
8.1.1 Denaturation (Melting) of the Template DNA 121
8.1.2 Annealing (Hybridisation) of PCR Primers 122
8.1.3 Calculating the Primer Annealing
Temperature (Tm) 124
8.1.4 DNA Chain Extension/Elongation 125
Contents ix
8.1.5 PCR Cycle Number 126
8.1.6 The Plateau Phase and Final Stages of PCR
Thermocycling 127
8.1.7 PCR Sensitivity 128
8.2 Quantitative PCR Protocols 128
8.2.1 Quantitative PCR Controls 129
8.3 Real-Time PCR Protocols 133
8.4 RNA Extraction and Treatment 137
Chapter 9 Analysis of PCR Amplification Products 141
9.1 Visualizing PCR Amplification Products 141
9.1.1 Intercalating Chemical Dyes and Silver Ions ...141
9.1.2 Fluorescent or Hapten Labelled Amplimers .... 144
9.2 Post-PCR Electrophoretic Analysis of Amplimers 146
9.2.1 Gel Electrophoresis Methodologies 147
9.2.2 Probe Hybridisation Methodologies 156
9.3 Real-Time Analysis of PCR Amplimers 168
9.3.1 In vitro Analysis Using Intercalating
Chemical Dyes 169
9.3.2 FRET Quenching Assays 170
9.3.3 TaqMan Probes 172
9.3.4 FRET Enhancement Reactions 173
9.4 Nucleic Acid Sequencing 173
9.4.1 DNA Sequencing Using Non-thermostable
DNA Polymerases 177
9.4.2 PCR Sequencing Using Thermostable
DNA Polymerases 178
9.4.3 The Fidelity of PCR Sequencing Reactions 178
Chapter 10 Ensuring PCR Quality - Laboratory Organisation, PCR
Optimization and Controls 183
10.1 The Primary Level of Quality Control - Laboratory
Organization and the Prevention of PCR
Contamination 183
10.1.1 Sources and Routes of Contamination 186
10.1.2 PCR Contamination Issues Within
Individual PCR Laboratories 187
10.1.3 Detecting and Preventing PCR
Contamination 189
10.2 The Secondary Level of PCR Quality Control - PCR
Design and Optimization 192
10.2.1 Extrinsic and Intrinsic Factors 192
10.2.2 The Developmental Steps Needed
to Achieve High Quality PCR Results 194
x Contents
10.2.3 The Use of Positive and Negative Controls
in PCR Quality 199
10.2.4 Causes and Solutions for False Positive
and False Negative PCR Results 201
10.3 Quality Considerations Specific for RT-PCR
Methodologies 207
10.3.1 Problems Likely to Cause False Positive
Results in RT-PCR Assays 208
10.3.2 Problems Likely to Cause False Negative
Results in RT-PCR Assays 209
Chapter 11 Ensuring PCR Quality - Quality Criteria and Quality
Assurance 213
11.1 Quality Control Criteria and PCR 214
11.1.1 Sensitivity and Diagnostic Sensitivity 214
11.1.2 Specificity and Diagnostic Specificity 214
11.1.3 Reference and Threshold Values 217
11.1.4 The Predictive Value 218
11.1.5 Efficiency 219
11.1.6 Error and Accuracy 219
11.1.7 Precision and Correctness 220
11.1.8 Defining the Analytical or Quantification
Range and Sensitivity 222
11.1.9 Recovery, Reproducibility and Quality
Assurance 224
11.2 Quality Assurance and Multicenter Studies 226
Chapter 12 Variants and Adaptations of the Standard PCR Protocol... 231
12.1 Generating Labelled PCR Amplimers for PCR
Product Visualization, DNA Probes and Cloning 231
12.2 Two-Step PCR Protocol 234
12.3 Booster PCR 235
12.4 Hot-Start and Time-Release PCR Protocols 236
12.5 Inverse PCR 240
12.6 Asymmetric PCR 241
12.7 PCR Mediated DNA Sequencing Strategies 243
12.7.1 Generating Single-Stranded DNA
for Sanger Sequencing Reactions 244
12.7.2 Classical Sanger Sequencing
of Single-Stranded PCR Products 244
12.7.3 Direct PCR Sequencing 245
12.7.4 Four-Tube Cycle Sequencing 245
12.7.5 One-Tube Cycle Sequencing 248
12.7.6 Difficult to Sequence Templates 250
Contents xi
12.8 Touchdown and Touch-Up PCR 250
12.9 Multiplex PCR 252
12.10 PCR Using Degenerate Primers 253
12.11 Repeat and Inter-repeat PCR 254
12.11.1 Repeat PCR 255
12.11.2 Inter-repeat PCR and Random Amplification
of Polymorphic DNA (RAPD) 255
12.12 AFLP Fingerprinting 258
12.13 Base Excision Sequence Scanning (BESS-T-Scan)
for Mutation Detection 259
12.14 Differential Display RT-PCR (DD-PCR) 261
12.15 The Protein Truncation Test (PTT) 263
12.16 Methylation Specific PCR and PCR in the Detection
of Mutagens 265
12.17 Breakpoint PCR 266
12.18 Site Directed Mutagenesis by PCR 267
12.19 PCR Amplimers for Cloning and Expression 268
12.20 SAGE 271
12.21 PCR Inhibition by DNA Specific Antibiotics
and Mutagens 273
Chapter 13 In Situ PCR Amplification (ISA) - Major Considerations,
Sample Processing and Applications 277
13.1 Tissue Processing - Nucleic Acid Fixation/Extraction. . 278
13.1.1 Fixation 278
13.1.2 Type of Nucleic Acid 283
13.1.3 Detrimental Effects of Various Fixatives
on Nucleic Acids 284
13.1.4 Effects of Tissue Processing Steps
(Decalcification, Dehydration, Intermedium
Application, Embedding) and Storage
of Paraffin Blocks 286
13.1.5 Effects of Histological and Histochemical
Staining 287
13.2 Differences in Approach for ISH, ISA and
Standard PCR 288
13.2.1 Different Types of Tissue Preparations 288
13.2.2 DNA and RT-PCR on Paraffin-Embedded
Tissue Sections 289
13.2.3 Improvement of PCR Efficiency Using
Fixed Tissue Sections 290
13.3 An Introduction to In Situ Amplification (ISA) 292
13.4 Considerations in the Development of ISA Protocols .. 294
13.4.1 IS-PCR or PCR-ISH 294
13.4.2 Diffusion of Nucleic Acids 295
xii Contents
13.4.3 The Correct Fixative 295
13.4.4 Damage Caused by Paraffin Embedding 296
13.4.5 Detachment of Cells and Tissue Sections 296
13.4.6 Specimen Proteolysis 297
13.4.7 Acetylation and Other Forms of Tissue
Section Pre-treatment 299
13.4.8 Pre-treatment of Preparations for IS-PCR 299
13.4.9 Testing for Loss of Amplimers Due
to Leakage from Their Site of Production. ... 301
13.4.10 Miscellaneous IS-PCR Considerations 301
13.4.11 Mispriming 304
13.4.12 Primer Independent Non-specific
DNA Synthesis 304
13.4.13 Evaporation of Reactants During IS-PCR/
Wet Hot Start Procedure 307
13.4.14 Cell Thickness and ISA 307
13.4.15 Choosing a Hybridisation Control
for Testing Amplimer Specificity 308
13.4.16 Choice of the PCR Processor 309
13.4.17 Choice of the Final Detection Method 309
13.5 ISA Optimisation 309
13.6 ISA Controls 311
Index 319
Color Plates 325
|
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| language | English |
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| physical | XII, 323 S. Ill., graph. Darst. |
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| spelling | Pelt-Verkuil, Elizabeth van Verfasser aut Principles and technical aspects of PCR amplification Elizabeth van Pelt-Verkuil ; Alex van Belkum ; John P. Hays [Dordrecht] Springer 2008 XII, 323 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Polymerase Chain Reaction Laboratory Manuals Polymerase chain reaction Laboratory manuals Polymerase chain reaction Diagnostic use Methode (DE-588)4038971-6 gnd rswk-swf Polymerase-Kettenreaktion (DE-588)4256726-9 gnd rswk-swf Polymerase-Kettenreaktion (DE-588)4256726-9 s Methode (DE-588)4038971-6 s DE-604 Belkum, Alexander F. van Verfasser aut Hays, John P. Verfasser aut HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=017107850&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Pelt-Verkuil, Elizabeth van Belkum, Alexander F. van Hays, John P. Principles and technical aspects of PCR amplification Polymerase Chain Reaction Laboratory Manuals Polymerase chain reaction Laboratory manuals Polymerase chain reaction Diagnostic use Methode (DE-588)4038971-6 gnd Polymerase-Kettenreaktion (DE-588)4256726-9 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4256726-9 |
| title | Principles and technical aspects of PCR amplification |
| title_auth | Principles and technical aspects of PCR amplification |
| title_exact_search | Principles and technical aspects of PCR amplification |
| title_full | Principles and technical aspects of PCR amplification Elizabeth van Pelt-Verkuil ; Alex van Belkum ; John P. Hays |
| title_fullStr | Principles and technical aspects of PCR amplification Elizabeth van Pelt-Verkuil ; Alex van Belkum ; John P. Hays |
| title_full_unstemmed | Principles and technical aspects of PCR amplification Elizabeth van Pelt-Verkuil ; Alex van Belkum ; John P. Hays |
| title_short | Principles and technical aspects of PCR amplification |
| title_sort | principles and technical aspects of pcr amplification |
| topic | Polymerase Chain Reaction Laboratory Manuals Polymerase chain reaction Laboratory manuals Polymerase chain reaction Diagnostic use Methode (DE-588)4038971-6 gnd Polymerase-Kettenreaktion (DE-588)4256726-9 gnd |
| topic_facet | Polymerase Chain Reaction Laboratory Manuals Polymerase chain reaction Laboratory manuals Polymerase chain reaction Diagnostic use Methode Polymerase-Kettenreaktion |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=017107850&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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