MOLECULAR BIOLOGY IN CELLULAR PATHOLOGY
CONTENTS 1 BLOTTING TECHNIQUES: METHODOLOGY AND APPLICATIONS 1 1 INTRODUCTION 1 2 BLOTTING TECHNIQUES 1 3 REFERENCES 2 IN-SITU HYBRIDISATION IN HISTOPATHOLOGY 2 1 INTRODUCTION 2 2 EXPERIMENTAL CONDITIONS 2 3 PROBES AND LABELS 2 4 CONTROLS AND PITFALLS 2 5 DOUBLE-LABELLING 2 6 INCREASING THE SENSITIVITY OF ISH 2 7 WHAT WE DO IN OUR LABORATORIES 2 8 APPLICATIONS OF ISH: EXAMPLES 2 9 PERSPECTIVE 2 10 REFERENCES 3 DNA FLOW CYTOMETRY 3 1 INTRODUCTION 3 2 DE?NITIONS AND TERMS 3 3 DYE USED FOR DNA ANALYSIS 3 4 SAMPLE PREPARATION FOR DNA ANALYSIS 3 5 ANALYSIS OF THE DNA HISTOGRAM 3 6 QUALITY CONTROL 3 7 COMPUTER ANALYSIS OF THE DNA HISTOGRAM 3 8 MULTIPARAMETRIC MEASUREMENT 3 9 ACKNOWLEDGEMENTS 3 10 REFERENCES 4 INTERPHASE CYTOGENETICS 4 1 INTRODUCTION 4 2 INTERPHASE CYTOGENETICS 4 3 APPLICATIONS 4 4 CONCLUSION 4 5 REFERENCES 5 ONCOGENES 5 1 INTRODUCTION 5 2 IDENTI?CATION OF THE ONCOGENES 5 3 FUNCTIONS OF THE PROTO-ONCOGENES 5 4 MECHANISM OF ONCOGENE ACTIVATION 5 5 ONCOGENES IN COLORECTAL CANCER 5 6 ONCOGENES IN BREAST CANCER 5 7 ONCOGENES IN LUNG CANCER 5 8 ONCOGENES IN HAEMATOLOGICAL MALIGNANCIES 5 9 OTHER CANCERS 5 10 CONCLUSION 5 11 REFERENCES 6 MOLECULAR AND IMMUNOLOGICAL ASPECTS OF CELL PROLIFERATION 6 1 THE CELL CYCLE AND ITS IMPORTANCE IN CLINICAL PATHOLOGY 6 2 MOLECULAR CONTROL OF THE CELL CYCLE 6 3 CELL CYCLE CONTROL 6 4 THE CELL CYCLE AND CANCER 6 5 IMMUNOCYTOCHEMICAL MARKERS OF PROLIFERATING CELLS 6 6 REFERENCES 6 7 FURTHER READING 7 INTERPHASE NUCLEOLAR ORGANISER REGIONS IN TUMOUR PATHOLOGY 7 1 INTRODUCTION 7 2 THE AGNORS 7 3 NOR SILVER-STAINING 7 4 QUANTITATIVE AGNOR ANALYSIS 7 5 AGNORS AS A PARAMETER OF THE LEVEL OF CELL PROLIFERATION 7 6 APPLICATION OF THE AGNOR TECHNIQUE TO TUMOUR PATHOLOGY 7 7 WHAT FUTURE FOR AGNORS IN TUMOUR PATHOLOGY? 7 8 REFERENCES 8 APOPTOSIS AND CELL SENESCENCE 8 1 INTRODUCTION 8 2 APOPTOSIS 8 3 CELL SENESCENCE 8 4 SUMMARY 8 5 REFERENCES 9 THE POLYMERASE CHAIN REACTION 9 1 INTRODUCTION 9 2 PRINCIPLES 9 3 ANALYSIS OF PRODUCTS 9 4 RT-PCR 9 5 QUANTITATIVE PCR 9 6 DNA AND RNA EXTRACTION 9 7 CORRELATION OF THE PCR WITH MORPHOLOGY 9 8 PROBLEMS 9 9 APPLICATIONS 9 10 DIAGNOSTIC APPLICATIONS 9 11 INFECTIOUS DISEASES 9 12 IDENTITY 9 13 THE FUTURE 9 14 REFERENCES 9 15 ONLINE INFORMATION 10 LASER CAPTURE MICRODISSECTION: TECHNIQUES AND APPLICATIONS IN THE MOLECULAR ANALYSIS OF THE CANCER CELL 10 1 INTRODUCTION 10 2 THE PRINCIPLE OF LCM 10 3 TECHNICAL CONSIDERATIONS 10 4 ADVANTAGES AND DISADVANTAGES OF LCM 10 5 APPLICATIONS OF LCM 10 6 FUTURE PERSPECTIVES 10 7 ACKNOWLEDGEMENTS 10 8 REFERENCES 11 THE IN-SITU POLYMERASE CHAIN REACTION 11 1 INTRODUCTION 11 2 OVERVIEW OF THE METHODOLOGY 11 3 IN-CELL PCR TECHNOLOGIES 11 4 IN-CELL AMPLI?CATION OF DNA 11 5 DETECTION OF AMPLICONS 11 6 REACTION, TISSUE AND DETECTION CONTROLS FOR USE WITH IN-CELL DNA PCR ASSAYS 11 7 IN-CELL RNA AMPLI?CATION 11 8 PROBLEMS ENCOUNTERED WITH IN-CELL PCR AMPLI?CATION 11 9 AMPLICON DIFFUSION AND BACK DIFFUSION 11 10 FUTURE WORK WITH IN-CELL PCR-BASED ASSAYS 11 11 REFERENCES 12 TAQMAN?TECHNOLOGY AND REAL-TIME POLYMERASE CHAIN REACTION 12 1 INTRODUCTION 12 2 PROBE TECHNOLOGIES 12 3 TAQMAN?PROBE AND CHEMISTRY (?RST GENERATION) 12 4 SECOND GENERATION TAQMAN?PROBES 12 5 HYBRIDISATION 12 6 TAQMAN?PCR CONDITIONS 12 7 STANDARDS FOR QUANTITATIVE PCR 12 8 INTERPRETATION OF RESULTS 12 9 END-POINT DETECTION 12 10 REAL-TIME DETECTION 12 11 RELATIVE QUANTITATION 12 12 REFERENCE GENES 12 13 SPECI?C TAQMAN?PCR APPLICATIONS 12 14 REFERENCES 13 GENE EXPRESSION ANALYSIS USING MICROARRAYS 13 1 INTRODUCTION 13 2 MICROARRAY EXPERIMENTS 13 3 DATA ANALYSIS 13 4 RECENT EXAMPLES OF MICROARRAY APPLICATIONS 13 5 CONCLUSIONS 13 6 ACKNOWLEDGEMENTS 13 7 REFERENCES 13 8 FURTHER READING 13 9 USEFUL WEBSITES 14 COMPARATIVE GENOMIC HYBRIDISATION IN PATHOLOGY 14 1 INTRODUCTION 14 2 TECHNIQUE 14 3 DATA ANALYSIS 14 4 APPLICATIONS 14 5 CLINICAL APPLICATIONS 14 6 SCREENING FOR CHROMOSOMAL ABNORMALITIES IN FETAL AND NEONATAL GENOMES 14 7 FUTURE PERSPECTIVES 14 8 ACKNOWLEDGEMENTS 14 9 REFERENCES 15 DNA SEQUENCING AND THE HUMAN GENOME PROJECT 15 1 INTRODUCTION 15 2 DNA SEQUENCING: THE BASICS 15 3 APPLICATIONS OF DNA SEQUENCING 15 4 THE HUMAN GENOME PROJECT 15 5 REFERENCES 15 6 FURTHER READING 15 7 USEFUL WEBSITES 16 MONOCLONAL ANTIBODIES: THE GENERATION AND APPLICATION OF ‘TOOLS OF THE TRADE’ WITHIN BIOMEDICAL SCIENCE 16 1 INTRODUCTION 16 2 ANTIBODIES AND ANTIGENS 16 3 POLYCLONAL ANTIBODIES 16 4 MONOCLONAL ANTIBODY DEVELOPMENT 16 5 MONOCLONAL ANTIBODY VARIANTS 16 6 MONOCLONAL ANTIBODY APPLICATIONS 16 7 THERAPY 16 8 SPECI?C APPLICATIONS 16 9 CONCLUSIONS 16 10 ACKNOWLEDGEMENTS 16 11 REFERENCES 17 PROTEOMICS 17 1 INTRODUCTION 17 2 DE?NITIONS AND APPLICATIONS 17 3 STAGES IN PROTEOME ANALYSIS 17 4 FUTURE DIRECTIONS 17 5 REFERENCES