Cytogenetics is the study of chromosome morphology, structure, pathology, function, and behavior. The field has evolved to embrace molecular cytogenetic changes, now termed cytogenomics. Cytogeneticists utilize an assortment of procedures to investigate the full complement of chromosomes and/or a targeted region within a specific chromosome in metaphase or interphase. Tools include routine analysis of G-banded chromosomes, specialized stains that address specific chromosomal structures, and molecular probes, such as fluorescence in situ hybridization (FISH) and chromosome microarray analysis, which employ a variety of methods to highlight a region as small as a single, specific genetic sequence under investigation. The AGT Cytogenetics Laboratory Manual, Fourth Edition offers a comprehensive description of the diagnostic tests offered by the clinical laboratory and explains the science behind them. One of the most valuable assets is its rich compilation of laboratory-tested protocols currently being used in leading laboratories, along with practical advice for nearly every area of interest to cytogeneticists. In addition to covering essential topics that have been the backbone of cytogenetics for over 60 years, such as the basic components of a cell, use of a microscope, human tissue processing for cytogenetic analysis (prenatal, constitutional, and neoplastic), laboratory safety, and the mechanisms behind chromosome rearrangement and aneuploidy, this edition introduces new and expanded chapters by experts in the field. Some of these new topics include a unique collection of chromosome heteromorphisms; clinical examples of genomic imprinting; an example-driven overview of chromosomal microarray; mathematics specifically geared for the cytogeneticist; usage of ISCN's cytogenetic language to describe chromosome changes; tips for laboratory management; examples of laboratory information systems; a collection of internet and library resources; and a special chapter on animal chromosomes for the research and zoo cytogeneticist. The range of topics is thus broad yet comprehensive, offering the student a resource that teaches the procedures performed in the cytogenetics laboratory environment, and the laboratory professional with a peer-reviewed reference that explores the basis of each of these procedures. This makes it a useful resource for researchers, clinicians, and lab professionals, as well as students in a university or medical school setting.
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- Verlag: John Wiley & Sons
- Seitenzahl: 1218
- Erscheinungstermin: 03.03.2017
- ISBN-13: 9781119061175
- Artikelnr.: 52556137
Contributing authors xxvii Preface xxix Acknowledgments xxxi 1 The cell and cell division 1 Margaret J. Barch and Helen J. Lawce 1.1 The cell 1 1.2 The cell cycle 14 1.3 Recombinant DNA techniques 19 1.4 The human genome 21 References 22 2 Cytogenetics: an overview 25 Helen J. Lawce and Michael G. Brown 2.1 Introduction 25 2.2 History of human cytogenetics 25 2.3 Cytogenetics methods 29 2.4 Slide-making 49 2.5 Chromosome staining 58 2.6 Chromosome microscopy/analysis 59 2.7 Laboratory procedure manual 69 References 70 Contributed protocols 75 Protocol 2.1 Slide-making 75 Protocol 2.2 Slide-making 76 Protocol 2.3 Making wet slides for chromosome analysis 78 Protocol 2.4 Slide-making 82 Protocol 2.5 Slide preparation 82 Protocol 2.6 Slide preparation procedure 84 3 Peripheral blood cytogenetic methods 87 Helen J. Lawce and Michael G. Brown 3.1 Using peripheral blood for cytogenetic analysis 87 3.2 Special uses of peripheral blood cultures 88 3.3 Peripheral blood constituents 89 3.4 Specimen handling 91 3.5 Cell culture equipment and supplies 93 3.6 Harvesting peripheral blood cultures 95 3.7 Chromosome analysis of peripheral blood 95 3.8 Storage of fixed specimens 95 Acknowledgments 95 References 95 Contributed protocols 98 Protocol 3.1 Blood culture and harvest procedure 98 Protocol 3.2 High-resolution peripheral blood method 100 Protocol 3.3 Constitutional cytogenetic studies on peripheral blood 108 Protocol 3.4 Blood culture and harvest procedure for microarray confirmation studies 115 4 General cell culture principles and fibroblast culture 119 Debra F. Saxe, Kristin M. May and Jean H. Priest 4.1 Definitions of a culture 119 4.2 Basic considerations in cell culture 121 4.3 Fibroblast culture 128 4.4 Lymphoblastoid cell lines 132 Glossary 132 Reference 133 Additional readings 133 Contributed protocols section 134 Protocol 4.1 Solid tissue collection for establishing cultures 134 Protocol 4.2 Solid tissue transport and sendout media 135 Protocol 4.3 Tissue culture reagents 138 Protocol 4.4 Phosphate buffer solution deficient in Ca2+ and Mg2+ 141 Protocol 4.5 Solid tissue and fibroblast culture setup 141 Protocol 4.6 Solid tissue setup and processing 142 Protocol 4.7 Flask and coverslip setup for POC/fibroblast cultures 145 Protocol 4.8 Coverslip setup for solid tissue biopsy specimens 147 Protocol 4.9 Solid tissue (fibroblast) culturing and harvesting 150 Protocol 4.10 Fibroblast culture maintenance: media feeding and changing 154 Protocol 4.11 Routine subculture of fibroblast cultures 155 Protocol 4.12 Manual harvest for flasks 157 Protocol 4.13 Treated media for contamination 158 Protocol 4.14 Fungizone-mycostatin solution for treatment of fungus/yeast contaminated cultures 158 Protocol 4.15 Mycoplasma testing 159 Protocol 4.16 Plating efficiency of serum 160 Protocol 4.17 Routine replication plating for human diploid cells 160 Protocol 4.18 Cell counting chamber method 161 Protocol 4.19 Cell viability by dye exclusion 161 Protocol 4.20 Mitotic index 161 Protocol 4.21 Growth rate-estimation of mean population doubling time during logarithmic growth 162 Protocol 4.22 Maintenance of fibroblast cultures as non-mitotic population 163 Protocol 4.23 Synchronization at S-phase with BrdU 163 Protocol 4.24 Making direct FISH preparations from abortus tissue 164 Protocol 4.25 Cryopreservation 165 Protocol 4.26 Cryopreservation with Nalgene cryogenic container 166 Protocol 4.27 Lymphoblastoid lines 167 Protocol 4.28 Freezing tissue cultures (cryopreservation) 171 5 Prenatal chromosome diagnosis 173 Kristin M. May, Debra F. Saxe and Jean H. Priest 5.1 Introduction 173 5.2 Amniotic fluid 173 5.3 Culture of amniotic fluid 175 5.4 Analysis of amniotic fluid 178 5.5 Chorionic villus sampling
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