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1．INTRODUCTION 1

2．TISSUE CULTURE TECHNIQUES AND CHROMOSOME PREPARATION 6

Basics of Tissue Culture 6

Blood 8

Bone Marrow 14

Prenatal Diagnosis 16

Skin and Other Tissues 27

Solid Tumors 29

Preparation and Manipulation of Somatic Cell Hybnds 31

Cell Cultures:Maintenance and Storage 42

Meiotic Techniques 48

Human Sperm Chromosome Karyotypes 56

Bibliography 63

3．BANDING TECHNIQUES 72

Differential Staining Techniques 72

Selective Staining Techniques 77

Fluorochromes and Counterstaining Techniques 90

Staining Techniques Using Antibodies 109

Restriction Endonuclease/Giemsa Banding 113

Bibliography 127

4．SPECIALIZED TECHNIQUES 134

High-Resolution Banding 134

Chromosomal Fragile Sites 137

Sister Chromatid Differentiation 143

Replication Pattems 151

Lateral Asymmetry 156

Chromatin Undercondensation Techniques 158

X-and Y-Chromatin 164

Bibliography 166

5．CHROMOSOME ANALYSIS BY FLOW CYTOMETRY 172

Instrumentation 175

Chromosome Preparation 177

Flow Sorting Chromosomes for Library Construction 179

Generation of Chromosome Paints 180

Bibliography 182

6．FLUORESCENCE IN SITU HYBRIDIZATION TECHNIQUES 184

Metaphase Chromosomes,Interphase Nuclei,and Extended Chromatin Fibers 184

Comparative Genomic Hybridization 191

Multiple-Colored Chromosomes by Fluorescence In Situ Hybridization 210

Multicolor Primed In Situ Labeling 218

Fluorescence In Situ Hybridization in Human Sperm 223

Bibliography 227

7．IMMUNOCYTOGENETIC TECHNIQUES 232

Antibodies Against DNA or Base Analogs Incorporated Into DNA 232

Antibodies Against Chromosomal Proteins 240

Immunocytogenetics and In Situ Hybridization 257

Immunophenotyping of Mitotic Cells 260

Bibliography 264

8．MICRODISSECTION AND DIRECT PCR AMPLIFICATION OF HUMAN CHROMOSOMES 271

Technical Considerations 271

Microdissection Protocols 275

Bibliography 278

9．IN SITU HYBRIDIZATION 280

Slide Preparation 281

Slide Pretreatment 282

Probe Preparation 282

Hybridization 285

Chromosomal DNA Denaturation 286

Posthybridization Washing 286

Hybrid Detection 287

G-Banding with Wright Stain 288

Appendix：Stock Solutions 289

Bibliography 289

10．SOUTHERN AND NORTHERN BLOTTING TECHNIQUES:PRINCIPLES AND APPLICATIONS 292

Southern Blotting 292

Northern Blotting 298

Preparation of Radioactively Labeled Nucleic Acid Probe 301

Hybridization of Filter or Membrane with a Nucleic Acid Probe 303

Detection of Signals 306

Applications of Blotting Techniques 307

Bibliography 309

Appendix：Buffers and Solutions for Protocols 311

11．THE POLYMERASE CHAIN REACTION:APPLICATIONS IN GENOMIC ANALYSIS 312

Historical Perspective 312

Scope 312

The Polymerase Chain Reaction:Principles and Basic Enzymology 313

Setting Up PCR in the Laboratory:Basic Laboratory Organization 319

PCR Reactions:Basic Methods 321

Applications of PCR Technology to Chromosome Analysis 330

Bibliography 337

12．KARYOTYPE INTERPRErATION 345

Historical Account 345

Classification of Unbanded Chromosomes 345

Nomenclature of the Banded Mitotic Chromosomes 346

High-Resolution Bands of Chromosomes 348

Cancer Cytogenetics 349

Chromosomal Aberrations 355

Evaluation of Banding Pattems 358

Nomenclature for Human Meiotic Chromosomes 358

Bibliography 360

13．CHROMOSOMES IN CLINICAL MEDICINE 362

Indications for Referral 362

Classical Chromosomal Syndromes 363

Cytogenetics of Spontaneous Abortion 363

Chromosomal Anomalies in Prenatal Diagnosis 363

Chromosome Anomalies in Mental Retardation 363

Chromosomes of Human Neoplasia 364

Chromosomal Basis of Oncogenesis 370

Probing the Human Genome 373

Bibliography 406

Index 411