1 Karyotyping Biotechnology IPowerpoint modified from powerpoint provided by NIH (Mark Nardone and Roland Nardone, authors)
2 Essential Question How do we use human cell cultures to look for abnormalities in chromosome structure?
3 Human Cells Have 23 pairs of chromosomes or 46 totalOne of the pair donated by the biological mother The other donated by the biological father
4 Karyotyping-DefinitionPhysical Characterization of chromosomes Examine diploid chromosomes for Size Shape Look for structural change which reveals abnormality As part of physical characterization take a photomicrograph of the chromosomes either digitally or physically
5 Karyotyping The photomicrograph is cutChromosome pairs are lined up 1-23 Look for abnormalities
6 Purpose of KaryotypingServes many areas of basic biology and medicine including Species identification Gene mapping- where genes are located on the chromosome Genetic disease analysis
7 Species IdentificationEach species has a characteristic karyotype Humans Mouse
8 Gene Mapping Genes are mapped to specific chromosomesEach chromosome has a unique banding pattern based of staining more or less condensed portion of DNA Banding patterns are species specific
9 Inherited Genetic DiseasesMany arise from changes in chromosomes Changes can be Number of chromosomes- Ex Down’s Syndrome – 3 of chromosome 21
12 Sources of Cultured Cells for Karyotyping.S Source Purpose Lymphocytes from peripheral blood Clinical Diagnosis Cultured fibroblasts collected by amniocentesis or chorionic villus sampling Prenatal Diagnosis Established cell lines from human or rodent: IMR 90 Human Lung Fibroblast Chinese Hamster Ovary Cells T3 Mouse Prostate Cells Research
13 Cells in Lab today Human tumor cell line –HeLa cellsOriginated from cervical cancer cells of Henrietta Lacks Because cells are of tumor origin, divide continuously Do not contain the normal diploid number of chromosomes Greater than 23 pairs
14 Cell Cycle in EukaryotesAlternates between two phases Interphase=non-dividing phase Cell grow Cell metabolically active-synthesize macromolecule During this phase chromosomes duplicated in preparation for division Represents 90% of life cycle M-phase=dividing phase Shortest part of life cycle. Two events occur Mitosis – nuclear division Duplicated chromosomes separate Cytokinesis-cytoplasm division
15 Cell Cycle in EukaryotesEnd result two cells with same number of chromosomes that are genetically identical
16 M Phase Mitosis-nuclear division Duplicated chromosomes separateDivided into five stages Prophase, prometaphase, metaphase, anaphase, telophase (PPMAT) For karyotyping freeze the cells in metaphase where chromosomes highly condensed and visible
17 Metaphase Blockade Purpose is to increase the number of cells in metaphase Accomplished by adding the drug, colchicine Colchicine block cells from going from metaphase to anaphase
18 Hypotonic Treatment of Metaphase Blocked CellsHypotonic salt solution (0.075M KCl). Causes cell swelling, cells are more fragile, rupture easily when splattered onto slides.
19 Application of Fixed Cells to SlideDispense cell suspension, dropwise, from a fine bore pipette onto a clean slide. The pipette tip should be at least 12 inches (3 feet is best!) above the slide. The slide should be propped up at a 45O angle. Allow slide to air dry while blowing over it. Spreading of chromosomes is enhanced by rapid evaporation of the fixative.
20 Staining the Chromosome SpreadsAfter cells are fixed on the slide, they will be stained with stain 1 followed by stain 2 A 2 drops of paramount will be applied to stained area A glass coverslip will be placed on top of paramount Gently apply pressure to the coverslip to spread the paramount evenly under coverslip May want to place two coverslips side by side to view entire microscope slide
21 Search for Ideal Chromosome Spread Key CharacteristicsSearch for Ideal Chromosome Spread Key Characteristics No overlapping chromosomes Chromosomes easily counted Arms of chromosomes readily visualized Make photographs of chromosome spread
22 Human Chromosome Spread