Technologies for detection of DNA damage and mutations:
Saved in:
| Format: | Book |
|---|---|
| Language: | English |
| Published: |
New York [u.a.]
Plenum Press
1996
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XXV, 441 S. Ill., graph. Darst. |
| ISBN: | 0306452375 |
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MARC
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| 245 | 1 | 0 | |a Technologies for detection of DNA damage and mutations |c ed. by Gerd P. Pfeifer |
| 264 | 1 | |a New York [u.a.] |b Plenum Press |c 1996 | |
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| _version_ | 1804125520444522496 |
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| adam_text | Technologies for
Detection of DNA Damage
and Mutations
Edited by
Gerd P Pfeifer
Beckman Research Institute of the City of Hope
Duarte, California
Plenum Press • New York and London
Contents
Part I Technologies for Detection of DNA Damage
Chapter 1 Microgel Electrophoresis of DNA from Individual Cells: Principles
and Methodology
Narendra P Singh
1 History 3
2 Basic Mechanism 5
3 Methodology 7
3 1 Neutral Microgel Electrophoresis 8
311 Assay Steps 8
312 Reagents 8
313 General Protocol for Making Microgels on Slides 10
314 Dehydration and DNA Precipitation 10
315 Reexamination of the Slides 10
3 2 Alkaline Microgel Electrophoresis 10
321 Assay Steps 10
322 Alkaline Electrophoretic Buffer 11
3 3 Electrophoretic Conditions for Both Neutral and Alkaline Microgel
Electrophoresis 11
3 4 Data Collection 11
4 Applications 11
4 1 X-rays 12
411 Material^ and Methods 12
412 DNA Single-Strand-Breaks 13
413 DNA Double-Strand Breaks 13
414 Results 14
4 2 Ethanol and Acetaldehyde 17
421 DNA Strand Breaks 17
422 Results 17
4 3 Applications of Microgel Electrophoresis to Other Cell Types 21
431 Brain Cells and Cells from Other Organs of Rats and Mice 22
432 Epithelial Cells 22
433 Cultured Fibroblasts 22
5 Conclusions 22
References 23
xi
xii Contents
Chapter 2 The Cytokinesis-Block Micronucleus Technique
Michael F Fenech
1 Introduction 25
2 Methods 27
2 1 Standard CBMN Assay for Isolated Human Lymphocytes 27
211 Lymphocyte Isolation, Cell Culture, and Cell Harvesting 27
212 Examination of Slides and Assessment of MN Frequency 28
213 Criteria for Scoring MNi in Cytokinesis-Blocked Cells 29
214 Nuclear Division Index 29
215 Adaptation of the Standard CBMN Assay to Whole Blood
Cultures and Murine Lymphocytes 30
2 2 Techniques That Distinguish between Micronuclei Originating from
Whole Chromosomes and Acentric Chromosome Fragments 31
221 Kinetochore Detection in MNi in the CBMN Assay 32
3 Discussion 33
References 34
Chapter 3 Agarose Gel Electrophoresis for DNA Damage Analysis
Regen Drouin, Shuwei Cao, and Gerald P Holmquist
1 Introduction 37
2 Materials and Methods 38
2 1 Alkaline Gel Electrophoresis 38
2 2 Glyoxal Gel Electrophoresis 38
3 Discussion 39
References 42
Chapter 4 32P-Postlabeling for Detection of DNA Adducts
R C Gupta
1 Introduction 45
2 Materials and General Methods 46
2 1 Enzymes 46
2 2 Buffers 47
2 3 [-y-32P]-ATP 47
2 4 Chromatography Supplies 47
2 5 Autoradiography 48
2 6 Handling of 32P-Labeled Materials 48
2 7 Isolation of DNA 48
3 Protocol for a Standard 32P-Postlabeling Assay 50
3 1 Step I: Enzymatic DNA Digestion 50
3 2 Step II: Adduct Enrichment 50
3 3 Step III: 32P-Labeling of Adducts 52
3 4 Step IIIA: 32P-Labeling of Total Nucleotides 52
3 5 Step IV: Chromatography of Adducts 53
3 6 Step IVA: Chromatography of Normal Nucleotides 56
3 7 Step V: Detection and Quantitation of Adducts 57
4 Discussion 58
References 60
Contents xiii
Chapter 5 A Postlabeling Assay for Oxidative Damage
Michael Weinfeld, Michel Liuzzi, and George D D Jones
1 Introduction 63
2 Methods 64
2 1 Materials 64
211 Reagents 64
212 Buffers 65
213 Enzymes 65
2 2 Preparation of Polyacrylamide Gels 66
221 Apparatus 66
222 Preparation 66
2 3 Postlabeling Assay 67
2 4 HPLC Analysis of Radioactive Bands and Nearest-Neighbor Analysis 68
241 Eluting Radioactive Material from Polyacrylamide Pieces 68
242 HPLC Analysis 68
243 Nuclease PI Digestion 68
2 5 Marker Compounds 69
3 Discussion 69
3 1 Notes on the Protocol (Troubleshooting) 69
3 2 Quantitative Aspects of the Assay 70
3 3 General Comments on the Assay (Advantages and Disadvantages) 70
References 70
Chapter 6 Radioimmunoassay of DNA Damaged by Ultraviolet Light
David L Mitchell
1 Introduction 73
2 Methods 74
2 1 Antiserum 74
2 2 Radiolabeled Probe 76
2 3 Sample and Standard Preparation 77
2 4 RIA Protocol 79
3 Discussion 80
References 83
Chapter 7 Monoclonal Antibody-Based Quantification and Repair Analysis of
Specific Alkylation Products in DNA
Jiirgen Thomale, Jorg Engelbergs, Frank Seiler, and Manfred F Rajewsky
1 Introduction 87
2 Immunoanalytical Methodology 88
2 1 HPLC-Competitive Radioimmunoassay 88
2 2 Immuno-Slot-Blot 91
2 3 Sequence (Gene)-Specific Assay 92
2 4 Immunocytological Assay on Individual Cells 96
3 Discussion 98
References 100
xiv Contents
Chapter 8 Detection of Oxidative DNA Base Damages: Immunochemical and
Electrochemical Approaches
Robert J Melamede, Yoke Wah Kow, Ivan A Bespalov, and Susan S Wallace
1 Immunochemical Detection of Oxidative DNA Damages 103
1 1 Introduction 103
1 2 Methods 108
121 Binding of the DNA to Microtiter Plates 108
122 Washing Plates 109
123 Primary Antibody 109
124 Secondary Antibody 109
125 Substrate Development 109
2 HPLC/Electrochemical Detection of Oxidized Bases 110
2 1 Introduction 110
2 2 Methods 110
221 Preparation of DNA Samples 110
222 Digestion of DNA for HPLC Analysis Ill
223 HPLC-EC Detection of DNA Lesions 112
224 Storage of the Glass Electrode 112
225 Buffers 113
References 113
Chapter 9 Strategies for Measuring Damage and Repair in Gene-Sized
Specific DNA Sequences
Charles A Smith and Philip C Hanawalt
1 Introduction 117
2 Removal of Cyclobutane Pyrimidine Dimers—T4 Endonuclease Assays 118
2 1 General Procedure 118
2 2 Choice of Fragments and Lesion Frequencies 119
2 3 Complications Related to DNA Replication 120
2 4 Probes 120
2 5 Amplified Genes 121
2 6 Measuring Hybridization Intensity 122
2 7 Calculations 122
3 Removal of Bulky Adducts—UVRABC Nuclease Assay 123
4 Removal of Bulky Adducts—Other Adduct-Specific Assays 124
5 Removal of Alkylated Bases 125
6 Use of Antibodies to Lesions 125
7 Use of Antibodies to Bromouracil in Repair Patches 126
8 Removal of DNA Cross-Linking 126
References 128
Chapter 10 Methods to Measure the Repair of Genes
Vdhelm A Bohr
1 Introduction 131
2 Methods 131
2 1 Southern Blot Assay 131
2 2 T4 Polymerase Assay 134
Contents xv
2 3 Precipitation with Monoclonal Antibody 135
2 4 Quantitative PCR 136
3 Perspectives 136
References 136
Chapter 11 Mapping and Quantification of Bulky Chemical-Induced DNA
Damage Using UvrABC Nucleases
Moon-shong Tang
1 Introduction 139
2 Methods 140
2 1 Purification of UvrA, UvrB, and UvrC Proteins 140
211 UvrA Purification 140
212 UvrB Purification 141
213 UvrC Purification 142
2 2 Quantification of Bulky DNA Adducts at Defined Sequences Using
UvrABC Incision Analysis and DNA Transfer-Hybridization 143
221 Treatment of Cultured Cells with Chemical Carcinogens or Drugs 143
222 DNA Purification and Preparation 143
223 UvrABC Incision Reaction 144
224 DNA Denaturation 145
225 Separation of Single-Stranded DNA by Electrophoresis 146
226 Southern Blotting and DNA Hybridization 146
227 Quantification 147
2 3 Analysis of Sequence Specificity of Chemical Carcinogen- and
Drug-DNA Binding by Using the UvrABC Nuclease Incision Method 147
231 5 - and 3 -End Labeling the DNA Fragment 149
232 Modification of Radioactively Labeled DNA Fragments with
Carcinogens or Antibiotics 149
233 UvrABC Nuclease Incision 149
234 Denaturing Polyacrylamide Gel Electrophoresis 149
235 Densitometer Scanning 150
3 Discussion 150
References 152
Chapter 12 The Use of DNA Glycosylases to Detect DNA Damage
Timothy R O Connor
1 Introduction 155
2 Methods 156
2 1 Substrates for DNA Glycosylases - 156
211 Substrates for the Analysis of Modified Bases 157
212 Site-Specifically Modified Oligonucleotides 160
213 DNA Fragments Modified by DNA Damaging Agents 160
214 Plasmid Substrates for DNA Glycosylases 161
2 2 DNA Glycosylase Assays 161
221 Assays for DNA Substrates Containing Radiolabeled Modified
Bases 161
222 Oligonucleotide-Based DNA Glycosylase Assays 162
xvi Contents
223 Plasmid-Based DNA Glycosylase Assays 163
2 3 Purification of AlkA, Tag, Fpg, and Nth Proteins 163
231 Production of Recombinant DNA Glycosylases 163
232 Lysis 164
233 Removal of Nucleic Acids—Polyethyleneimine P and/or Ion
Exchange Chromatography 164
234 Phospho Ultrogel A6R Chromatography 164
235 Gel Filtration—AcA 54 Chromatography 164
236 Hydrophobic Interaction—Phenyl Sepharose CL 4B
Chromatography 165
237 Concentration of DNA Glycosylases 165
2 4 Bases Excised by DNA Glycosylases 165
241 Coupled Gas Chromatography—Mass Spectrometry 165
2 5 Detection of Damage in Genomic or Mitochondrial DNA Using DNA
Glycosylases 166
2 6 Detection of Mismatched Bases in DNA—Mismatch Repair Enzyme
Cleavage (MREC) / 167
3 Discussion 167
References 167
Chapter 13 PCR-Based Assays for the Detection and Quantitation of DNA
Damage and Repair
F Michael Yakes, Yiming Chen, and Bennett Van Houten
1 Introduction 171
2 Methods 173
2 1 Cell Culture and Treatment 173
2 2 DNA Isolation and Quantitation 174
2 3 PCR Amplification 175
231 PCR Components and Reagents 175
232 Primer Selection 176
233 Reaction Conditions and Agarose Gel Electrophoresis 176
2 4 Defining QPCR Conditions 176
2 5 Precision of the QPCR Assay 179
2 6 Using QXLPCR to Detect DNA Damage following UV Irradiation 180
3 Discussion 182
3 1 Advantages of the QXLPCR Assay 182
3 2 Technical Limitations 182
3 3 Future Directions 182
References 183
Chapter 14 DNA Damage Analysis Using an Automated DNA Sequencer
Gopaul Kotturi, Wolfgang C Kusser, and Barry W Glickman
1 Introduction 185
1 1 Development of Automated DNA Sequencers 186
1 2 Ancillary Applications of Automated DNA Sequencers 186
2 Methods 187
2 1 Template Generation 187
211 Amplification 187
Contents xvii
212 Template Purification 188
2 2 Internal Standards and Sequencing Reactions 188
2 3 DNA Damage Induction and Fragment Cleavage 189
2 4 Electrophoresis of DNA Samples 189
241 Sample Dilution 189
242 Lane Assignments 190
243 Preparation of the Polyacrylamide Gel 190
244 Electrophoresis Conditions 190
2 5 Data Analysis 191
2 6 Relative Mobility of DNA Fragments 194
3 Discussion 195
3 1 Developing Technologies 195
4 Conclusions 196
References 196
Chapter 15 Ligation-Mediated PCR for Analysis of UV Damage
Silvia Tomaletti and Gerd P Pfeifer
1 Introduction 199
2 Materials and Methods 201
2 1 UV Irradiation 201
2 2 DNA Purification 201
2 3 Cleavage at Cyclobutane Pyrimidine Dimers 202
2 4 Cleavage at (6-4) Photoproducts 202
2 5 Ligation-Mediated Polymerase Chain Reaction (LMPCR) 203
251 Primer Design 203
252 Primer Extension 203
253 Ligation 203
254 Polymerase Chain Reaction 204
255 Gel Electrophoresis 204
256 Electroblotting 204
257 Hybridization 205
258 Preparation of Hybridization Probes 205
3 Example 206
4 Discussion 206
References 208
Chapter 16 Ligation-Mediated PCR for Analysis of Oxidative DNA Damage
Regen Drouin, Henry Rodriguez, Gerald P Holmquist, and Steven A Akman
1 Introduction 211
2 Materials and Methods 215
2 1 Hydrogen Peroxide Treatment of Human Skin Fibroblasts 215
2 2 DNA Purification 215
2 3 DNA Dialysis 216
2 4 Cu/Ascorbate/H,0, Treatment of Purified DNA 216
2 5 Enzyme Digestion 216
2 6 Ligation-Mediated Polymerase Chain Reaction: Primer Extension 217
2 7 LMPCR: Ligation 218
2 8 LMPCR: PCR 218
xviii Contents
2 9 Gel Electrophoresis and Electroblotting 219
2 10 Hybridization 219
2 11 Preparation of Single-Stranded Hybridization Probes 220
3 Discussion 222
References 223
Chapter 17 Single-Strand Ligation PCR for Detection of DNA Adducts
Keith A Grimaldi, Simon R McAdam, and John A Hartley
1 Introduction 227
2 Methods 228
2 1 Buffers and Reagents 228
2 2 DNA Damaging Agent Treatments • 231
221 Treatment of Isolated DNA 231
222 Treatment of Cells 231
2 3 DNA Isolation from Cells 232
2 4 Single-Strand Ligation PCR 232
241 First-Round PCR 232
242 Capture and Ligation 233
243 Second-Round PCR 233
244 Third-Round PCR 233
2 5 Results 233
3 Discussion 237
3 1 Optimization and Troubleshooting 237
311 Biotinylated Primer and Paramagnetic Beads 237
312 Efficiency of Ligation and Ligase Contamination 237
313 PCR Components 237
314 Resolution and Background 237
3 2 Summary 238
References 238
Part II Technologies for Detection of Mutations
Chapter 18 Heteroduplex Analysis
Damjan Glavac and Michael Dean
1 Introduction 241
1 1 HA and SSCP 243
2 Methods 243
2 1 Materials 244
2 2 Gel Preparation 244
2 3 Sample Preparation and Electrophoresis 245
3 Discussion 246
3 1 Structure and Stability of Heteroduplexes 246
3 2 Sensitivity and Reliability of HA 246
3 3 Variations of HA 247
3 4 Conformation-Sensitive Gel Electrophoresis 247
3 5 HA and SSCP 248
References 248
Contents xix
Chapter 19 Mutation Analysis by Denaturing Gradient Gel Electrophoresis
(DGGE)
Riccardo Fodde and Monique Losekoot
1 Introduction 253
1 1 Theory of DGGE 253
1 2 GC-Clamps 255
1 3 Computer Simulation of Melting Behavior 256
1 4 Research and Diagnostic Applications 256
2 Methods 257
2 1 Setting Up DGGE 257
211 Setting Up DGGE: Computer Predictions and Primer Design 257
212 Setting Up DGGE: Perpendicular Denaturing Gradient Gels 257
213 Setting Up DGGE: Parallel Denaturing Gradient Gels 259
2 2 DGGE Protocol 259
221 Gel Apparatus 260
222 Reagents 260
223 Preparation of Denaturing Gradient Gels 260
224 Electrophoresis 261
225 Staining of the Gel 261
3 Discussion 262
3 1 Variations on the Theme 262
3 2 Comparisons with Other Protocols 263
References 263
Chapter 20 Constant Denaturant Gel Electrophoresis (CDGE) in Mutation
Screening
Anne-Lise Bprresen
1 Introduction 267
2 Strategy 270
3 Sensitivity of CDGE 271
4 Sequence-Based CDGE without Sequencing 275
5 Methods 275
5 1 PCR 7T 275
5 2 Denaturing Gel Electrophoresis 276
6 Discussion 276
References 278
Chapter 21 Single-Strand Conformation Polymorphism Analysis
Takao Sekiya
1 Introduction 281
2 Methods 282
2 1 Instruments 282
2 2 Reagents 282
2 3 Buffers and Solutions 284
2 4 Protocol for SSCP Analysis 284
241 Labeling of PCR Products Using the 5 -32P-Labeled Primer 284
xx Contents
242 Labeling the PCR Products Using a Labeled Nucleotide
Substrate 285
2 5 SSCP Analysis 285
2 6 Identification of Mutations 286
261 Separation of Alleles 286
262 Amplification of the Eluted Fragment 286
263 Purification of Amplified DNA Fragments 287
264 Cycle Sequencing 287
265 Preparation of Polyacrylamide Gel for Sequencing 288
266 Polyacrylamide Gel Electrophoresis 288
3 Discussion 289
References 290
Chapter 22 Two-Dimensional Gene Scanning
Daizong Li, Nathalie Van Orsouw, Chris Huang, and Jan Vijg
1 Introduction 291
1 1 Two-Dimensional Gene Scanning: Principles 291
1 2 Test Design: General Aspects 292
1 3 RBI and p53 TDGS Test Designs 293
2 Methods 300
2 1 Equipment 300
2 2 Reagents 301
2 3 PCR Primers 301
2 4 PCR Reactions and Heteroduplexing 301
2 5 Sample Preparation 302
2 6 Two-Dimensional Electrophoresis 302
261 Manual Instruments 302
262 Automatic Instrument 302
3 Discussion 303
References 304
Chapter 23 Ligase Chain Reaction for the Detection of Specific DNA
Sequences and Point Mutations
R Bruce Wallace, Ching-I P Lin, Antonio-A Reyes, Jimmie D Lowery,
and Luis Ugozzoli
1 Introduction 307
2 The Ligase Chain Reaction 308
2 1 Mechanism of LCR 308
2 2 Template-Independent Ligation 310
2 3 Optimization of LCR 311
2 4 Advantages and Disadvantages of LCR 312
3 The Gap Ligase Chain Reaction 312
3 1 Amplification of Specific DNA Sequences Using gLCR 315
3 2 Amplification of Specific Target RNA Sequences Using gLCR 315
3 3 Detecting Mutations in DNA or RNA Using gLCR 316
4 Ligation of Two Oligonucleotides 316
5 Recent Applications of LCR and Related Technologies 317
Contents xxi
6 Postamplification Detection 317
7 Conclusion 320
References 320
Chapter 24 The Protein Truncation Test (PTT) for Rapid Detection of
Translation-Terminating Mutations
Johan T Den Dunnen, Pauline A M Roest, Rob B Van Der Luijt, and Frans B L
Hogervorst
1 Introduction 323
1 1 PTT Principle 324
1 2 Templates 324
1 3 Reverse Transcription and PCR 324
1 4 Gel Analysis of PCR Products 326
1 5 Transcription and Translation 327
1 6 Visualization of Truncated Peptides 327
2 Methods 327
2 1 Collecting Cell Samples 327
211 Preparation of Peripheral Blood Lymphocytes 327
212 Preparation of Cultured Cells 328
2 2 RNA Isolation Using RNAzolB 328
2 3 Analysis of RNA Samples 328
2 4 Reverse Transcription and PCR 328
241 Reverse Transcription 328
242 First PCR 329
243 Second PCR 329
2 5 Gel Analysis of the PCR Products 329
2 6 In Vitro Transcription/Translation 329
2 7 SDS-PAGE 330
2 8 Coomassie Blue Staining and Autoradiography 330
2 9 Solutions and Reagents 331
3 Examples 332
3 1 Using DNA Templates: The APC and BRCA1 Genes 332
3 2 Duchenne Muscular Dystrophy 332
3 3 Hunter Syndrome 334
3 4 PTT and Direct Diagnosis 334
4 Discussion 335
4 1 Advantages of PTT 335
411 Screening Large Regions in One Assay 335
412 No False Positives 336
413 Pinpoints Mutation Site 336
4 2 Limitations and Potential Pitfalls 336
421 Missense Mutations 336
422 Expression of Both Alleles 336
423 Genomic Rearrangements 337
424 N- and C-Terminal Mutations 337
425 Tissue-Specific Expression/Splicing 337
426 Background Translation Products 337
427 Mobility of Translation Products 337
xxii Contents
4 3 Troubleshooting 338
431 RNA Quality 338
432 Reverse Transcription 338
433 Contaminations 338
434 No Amplification of Specific Genes 338
435 Bad Translation 339
436 Spurious Truncated Peptides 339
4 4 Future Developments 339
5 Conclusion 340
References 340
Chapter 25 Single Nucleotide Primer Extension for Analysis of Sequence
Variants
Piroska E Szabo, Gerd P Pfeifer, Jeffrey R Mann, and Judith Singer-Sam
1 Introduction 343
2 The SNuPE Assay for Mutation Detection 344
3 Assay Conditions 346
3 1 PCR 346
3 2 Isolation of Amplified DNA Products 346
3 3 SNuPE 346
4 RNA SNuPE 346
5 Solid-Phase Minisequencing 348
References 349
Chapter 26 Sequencing of PCR Products
Piroska E Szabo, Jeffrey R Mann, and Gerald Forrest
1 Basic Methods 351
1 1 Maxam-Gilbert 351
1 2 Sanger 351
2 Strategies for Sequencing Double-Stranded DNA 352
2 1 Sequenase Strategy 353
211 Sequencing Linear Double-Stranded DNA 354
212 Difficult Templates 356
2 2 Cycle Sequencing 356
3 Primers 357
4 Template Purity 357
4 1 Nonspecific Bands 358
4 2 Contamination by Primers and Nucleotides 358
5 Visualization 358
6 Direct Sequencing versus Subcloning 359
6 1 Cloning of PCR Products 359
611 Blunt-End Ligation 359
612 dT Cloning 361
613 Cloning PCR Fragments with Restriction Sites 362
614 Ligation-Independent Cloning 362
615 In Vivo Cloning 363
Contents xxiii
6 2 Ligation-Mediated Sequencing 363
7 Large-Scale Sequencing Projects 363
7 1 Automatic Fluorescent Sequencing 363
711 Protocol for Automated Direct Sequencing of PCR Products 365
712 Difficult Templates and Automatic Sequencing 366
7 2 Robotization 366
7 3 Multiplex Sequencing 366
References 366
Part III Mammalian Systems for Mutation Analysis
Chapter 27 Detection and Characterization of Mutations in Mammalian Cells
with the pSP189 Shuttle Vector System
Michael M Seidman
1 Introduction 373
1 1 Development of Shuttle Vectors for Studying Mammalian Mutagenesis:
Problems 373
1 2 Solutions 374
1 3 The Use of a Signature Sequence 374
2 Methods 375
2 1 Preparation of Signature Sequences 375
2 2 Plasmid Modification 376
2 3 Cells and Transfection Method 376
2 4 Plasmid Harvest 376
2 5 Electroporation of Competent Bacteria 377
2 6 Thermal Cycle Sequence Analysis of Mutant Plasmids 377
2 7 Choice of Cell Line and Transfection Technology 378
3 Applications of Shuttle Vector Technology 378
References 379
Chapter 28 The HPRT Gene as a Model System for Mutation Analysis
Veronica M Maher and J Justin McCormick
1 Introduction 381
2 Methods 382
2 1 Cells and Cell Culture 382
2 2 Exposing Cells to DNA Damaging Agents 383
2 3 Determining the Cytotoxic Effect of the Agent 384
2 4 Determining the Frequency of Mutations Induced in the HPRT Gene 384
2 5 Achieving Synchronized Populations of Cells 385
2 6 Determining the Spectrum of HPRT Mutations Induced 386
2 7 Determining the Effect of Repair on the Frequency and Spectrum of
Mutations 387
2 8 Determining the Relationship between Site of DNA Damage and Spectra
of Mutations 388
References 389
xxiv Contents
Chapter 29 Bacteriophage Lambda and Plasmid lacZ Transgenic Mice for
Studying Mutations in Vivo
Jan Vijg and George R Douglas
1 Introduction 391
2 Methods 393
2 1 Bacteriophage Lambda System 393
211 Transgenic Animals 393
212E coli Strain 395
213 Reagents 395
214 Tissue Collection 395
215 Isolation of Genomic DNA 396
216 Packaging Lambda Phage 397
217 Determination of Mutant Frequency 397
218 Mutant Characterization: Complementation Assay for Locating
lacZ Mutations 398
219 Mutant Characterization: DNA Sequencing 399
2 2 Plasmid System 402
221 Transgenic Animals 402
222E coli Strain 402
223 Reagents 402
224 Preparation of LacI-LacZ Magnetic Beads 403
225 Tissue Collection 403
226 Extraction of Genomic DNA 404
227 Preparation of Electrocompetent Cells 404
228 Magnetic Bead Rescue of lacZ Plasmid from Mouse Genomic
DNA 404
229 Electroporation, Plating, and Mutant Counting 405
2 2 10 Mutant Characterization 405
3 Discussion 406
References 409
Chapter 30 The Use of lacl Transgenic Mice in Genetic Toxicology
Johan G de Boer, Heather LJZrfle, David Walsh, James Holcroft, and Barry W
Glickman
1 Introduction 411
2 Species-and Tissue-Specific Carcinogenesis 412
3 The Choice of Transgenic System 413
4 The lacl Gene as a Mutational Target 413
5 The Construction of the lacl Transgenic Animal 414
6 Description of the X/LIZ-LacI Mutant Plaque Recovery and Analysis Protocols 414
7 Selective Systems 415
8 DNA Sequencing Using Automated Sequencers 416
9 Limitations of the System 418
10 Dosing Considerations 419
11 Assay Standardization 420
12 Sensitivity 420
13 Mutations in Germ Cells versus Somatic Cells 421
Contents xxv
14 Preliminary Data on the Analysis of Mutagens 421
15 Future Prospects 424
References 424
Chapter 31 Genotypic Mutation Assay (RFLP/PCR)
Fernando Aguilar and Peter Cerutti
1 Introduction 431
2 Methods 432
2 1 HaeIII Restriction of Codon 249 DNA Fragments 433
2 2 Enrichment of Codon 249 Mutants 433
2 3 High-Fidelity Amplification of Codon 249 Mutants 433
2 4 Preparation of Authentic Single-Base-Pair Mutants and Mutant Standard
at Hae III Site 14,072-14,075 of Human p53 Gene 435
2 5 Specific Oligonucleotide Plaque Hybridization of RFLP/PCR Mutants 436
2 6 Calibration of Absolute Mutation Frequencies Using the Mutant Standard 436
3 Discussion 437
References 438
Index 439
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| discipline | Biologie Chemie Chemie-Ingenieurwesen Biotechnologie |
| format | Book |
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| id | DE-604.BV011030149 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T18:02:52Z |
| institution | BVB |
| isbn | 0306452375 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-007385716 |
| oclc_num | 247210427 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR DE-703 DE-20 DE-91G DE-BY-TUM DE-11 |
| owner_facet | DE-355 DE-BY-UBR DE-703 DE-20 DE-91G DE-BY-TUM DE-11 |
| physical | XXV, 441 S. Ill., graph. Darst. |
| publishDate | 1996 |
| publishDateSearch | 1996 |
| publishDateSort | 1996 |
| publisher | Plenum Press |
| record_format | marc |
| spelling | Technologies for detection of DNA damage and mutations ed. by Gerd P. Pfeifer New York [u.a.] Plenum Press 1996 XXV, 441 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier DNS-Schädigung (DE-588)4150350-8 gnd rswk-swf Nachweis (DE-588)4115334-0 gnd rswk-swf DNS (DE-588)4070512-2 gnd rswk-swf Mutation (DE-588)4170883-0 gnd rswk-swf DNS (DE-588)4070512-2 s Mutation (DE-588)4170883-0 s Nachweis (DE-588)4115334-0 s DE-604 DNS-Schädigung (DE-588)4150350-8 s Pfeifer, Gerd Peter Sonstige oth HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=007385716&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Technologies for detection of DNA damage and mutations DNS-Schädigung (DE-588)4150350-8 gnd Nachweis (DE-588)4115334-0 gnd DNS (DE-588)4070512-2 gnd Mutation (DE-588)4170883-0 gnd |
| subject_GND | (DE-588)4150350-8 (DE-588)4115334-0 (DE-588)4070512-2 (DE-588)4170883-0 |
| title | Technologies for detection of DNA damage and mutations |
| title_auth | Technologies for detection of DNA damage and mutations |
| title_exact_search | Technologies for detection of DNA damage and mutations |
| title_full | Technologies for detection of DNA damage and mutations ed. by Gerd P. Pfeifer |
| title_fullStr | Technologies for detection of DNA damage and mutations ed. by Gerd P. Pfeifer |
| title_full_unstemmed | Technologies for detection of DNA damage and mutations ed. by Gerd P. Pfeifer |
| title_short | Technologies for detection of DNA damage and mutations |
| title_sort | technologies for detection of dna damage and mutations |
| topic | DNS-Schädigung (DE-588)4150350-8 gnd Nachweis (DE-588)4115334-0 gnd DNS (DE-588)4070512-2 gnd Mutation (DE-588)4170883-0 gnd |
| topic_facet | DNS-Schädigung Nachweis DNS Mutation |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=007385716&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT pfeifergerdpeter technologiesfordetectionofdnadamageandmutations |