Molecular Biology in Cellular Pathology

John Crocker is the editor of Molecular Biology in Cellular Pathology, published by Wiley. Paul G. Murray is the editor of Molecular Biology in Cellular Pathology, published by Wiley.

Preface xiii

Preface to Molecular Biology in Histopathology xv

List of Contributors xvii

1 Blotting Techniques: Methodology and Applications 1
Fiona Watson and C. Simon Herrington

1.1 Introduction 1

1.2 Blotting techniques 1

1.3 References 15

2 In-situ Hybridisation in Histopathology 19
Gerald Niedobitek and Hermann Herbst

2.1 Introduction 19

2.2 Experimental conditions 20

2.3 Probes and labels 23

2.4 Controls and pitfalls 27

2.5 Double-labelling 29

2.6 Increasing the sensitivity of ISH 31

2.7 What we do in our laboratories 33

2.8 Applications of ISH: examples 35

2.9 Perspective 39

2.10 References 40

3 DNA Flow Cytometry 49
M.G. Ormerod

3.1 Introduction 49

3.2 Definitions and terms 49

3.3 Dye used for DNA analysis 50

3.4 Sample preparation for DNA analysis 52

3.5 Analysis of the DNA histogram 53

3.6 Quality control 53

3.7 Computer analysis of the DNA histogram 55

3.8 Multiparametric measurement 57

3.9 Acknowledgements 59

3.10 References 59

4 Interphase Cytogenetics 61
Sara A. Dyer and Jonathan J. Waters

4.1 Introduction 61

4.2 Interphase cytogenetics 62

4.3 Applications 67

4.4 Conclusion 76

4.5 References 77

5 Oncogenes 79
Fiona Macdonald

5.1 Introduction 79

5.2 Identification of the oncogenes 79

5.3 Functions of the proto-oncogenes 80

5.4 Mechanism of oncogene activation 89

5.5 Oncogenes in colorectal cancer 91

5.6 Oncogenes in breast cancer 94

5.7 Oncogenes in lung cancer 95

5.8 Oncogenes in haematological malignancies 96

5.9 Other cancers 99

5.10 Conclusion 100

5.11 References 100

6 Molecular and Immunological Aspects of Cell Proliferation 105
Karl Baumforth and John Crocker

6.1 The cell cycle and its importance in clinical pathology 105

6.2 Molecular control of the cell cycle 108

6.3 Cell cycle control 111

6.4 The cell cycle and cancer 112

6.5 Immunocytochemical markers of proliferating cells 115

6.6 References 133

6.7 Further Reading 135

7 Interphase Nucleolar Organiser Regions in Tumour Pathology 137
Massimo Derenzini, Davide Treré, Marie-Françoise O’Donohue and Dominique Ploton

7.1 Introduction 137

7.2 The AgNORs 138

7.3 NOR silver-staining 142

7.4 Quantitative AgNOR analysis 145

7.5 AgNORs as a parameter of the level of cell proliferation 146

7.6 Application of the AgNOR technique to tumour pathology 147

7.7 What future for AgNORs in tumour pathology? 151

7.8 References 152

8 Apoptosis and Cell Senescence 153
Lee B. Jordan and David J. Harrison

8.1 Introduction 153

8.2 Apoptosis 153

8.3 Cell senescence 174

8.4 Summary 178

8.5 References 179

9 The Polymerase Chain Reaction 193
Timothy Diss

9.1 Introduction 193

9.2 Principles 194

9.3 Analysis of products 197

9.4 Rt-pcr 199

9.5 Quantitative PCR 200

9.6 DNA and RNA extraction 200

9.7 Correlation of the PCR with morphology 201

9.8 Problems 202

9.9 Applications 202

9.10 Diagnostic applications 203

9.11 Infectious diseases 209

9.12 Identity 209

9.13 The future 210

9.14 References 210

9.15 Online information 212

10 Laser Capture Microdissection: Techniques and Applications in the Molecular Analysis of the Cancer Cell 213
Amanda Dutton, Victor Lopes and Paul G. Murray

10.1 Introduction 213

10.2 The principle of LCM 214

10.3 Technical considerations 216

10.4 Advantages and disadvantages of LCM 217

10.5 Applications of LCM 222

10.6 Future perspectives 229

10.7 Acknowledgements 229

10.8 References 229

11 The In-situ Polymerase Chain Reaction 233
John J. O’Leary, Cara Martin and Orla Sheils

11.1 Introduction 233

11.2 Overview of the methodology 234

11.3 In-cell PCR technologies 235

11.4 In-cell amplification of DNA 238

11.5 Detection of amplicons 242

11.6 Reaction, tissue and detection controls for use with in-cell DNA PCR assays 243

11.7 In-cell RNA amplification 244

11.8 Problems encountered with in-cell PCR amplification 246

11.9 Amplicon diffusion and back diffusion 247

11.10 Future work with in-cell PCR-based assays 247

11.11 References 249

12 TaqMan® Technology and Real-Time Polymerase Chain Reaction 251
John J. O’Leary, Orla Sheils, Cara Martin, and Aoife Crowley

12.1 Introduction 251

12.2 Probe technologies 252

12.3 TaqMan® probe and chemistry (first generation) 254

12.4 Second generation TaqMan® probes 256

12.5 Hybridisation 258

12.6 TaqMan® PCR conditions 259

12.7 Standards for quantitative PCR 260

12.8 Interpretation of results 261

12.9 End-point detection 262

12.10 Real-time detection 263

12.11 Relative quantitation 263

12.12 Reference genes 264

12.13 Specific TaqMan® PCR applications 265

12.14 References 268

13 Gene Expression Analysis Using Microarrays 269
Sophie E. Wildsmith and Fiona J. Spence

13.1 Introduction 269

13.2 Microarray experiments 269

13.3 Data analysis 273

13.4 Recent examples of microarray applications 284

13.5 Conclusions 284

13.6 Acknowledgements 284

13.7 References 284

13.8 Further Reading 286

13.9 Useful websites 286

14 Comparative Genomic Hybridisation in Pathology 287
Marjan M. Weiss, Mario A.J.A. Hermsen, Antoine Snijders, Horst Buerger, Werner Boecker, Ernst J. Kuipers, Paul J. van Diest andGerritA.Meijer

14.1 Introduction 287

14.2 Technique 289

14.3 Data analysis 292

14.4 Applications 293

14.5 Clinical applications 299

14.6 Screening for chromosomal abnormalities in fetal and neonatal genomes 299

14.7 Future perspectives 300

14.8 Acknowledgements 301

14.9 References 301

15 DNA Sequencing and the Human Genome Project 307
Philip Bennett

15.1 Introduction 307

15.2 DNA sequencing: the basics 308

15.3 Applications of DNA sequencing 318

15.4 The Human Genome Project 320

15.5 References 327

15.6 Further Reading 327

15.7 Useful websites 328

16 Monoclonal Antibodies: The Generation and Application of ‘Tools of the Trade’ Within Biomedical Science 329
Paul N. Nelson, S. Jane Astley and Philip Warren

16.1 Introduction 329

16.2 Antibodies and antigens 331

16.3 Polyclonal antibodies 332

16.4 Monoclonal antibody development 333

16.5 Monoclonal antibody variants 338

16.6 Monoclonal antibody applications 341

16.7 Therapy 345

16.8 Specific applications 346

16.9 Conclusions 347

16.10 Acknowledgements 347

16.11 References 347

17 Proteomics 351
Kathryn Lilley, Azam Razzaq and Michael J. Deery

17.1 Introduction 351

17.2 Definitions and applications 352

17.3 Stages in proteome analysis 352

17.4 Future directions 368

17.5 References 368

Index 371