GENERAL BIOLOGY                            HDL 121               CYTOGENETIC TECHNIQUEPREPARED BY:MANEGASCHOOL OF MLTFACUL...
CYTOGENETIC TECHNIQUELearning Outcomes    After completing this lecture, students will be able to:       (a) List few tech...
CYTOGENETIC TECHNIQUETopic Outlines           1.1. Cytogenetic technique           1.1.1 Example           1.1.2 Purpose  ...
CYTOGENETIC TECHNIQUE1.1. Cytogenetic techniqueIntroduction    Cytogenetic: a branch of genetics that is concerned     wi...
CYTOGENETIC TECHNIQUE1.1. Cytogenetic techniqueTechniques used in cytogenetic technique   Routine analysis of G-Banded ch...
CYTOGENETIC TECHNIQUECell culture   The process by which prokaryotic or eukaryotic cells    are grown under controlled co...
CYTOGENETIC TECHNIQUE    General procedures     (a) Isolation of cells     (b) Maintaining cells in culture     (c) Manip...
CYTOGENETIC TECHNIQUE   Purpose:    - manufacture of viral vaccines & many products of    biotechnology (recombinant DNA,...
CYTOGENETIC TECHNIQUECytogenetic technique   Investigation into human karyotype (new method)    (a) Using cells in cultur...
CYTOGENETIC TECHNIQUE Routine Chromosome Analysis   Refers to analysis of metaphase chromsomes which    have been banded ...
CYTOGENETIC TECHNIQUESlide Preparation   Cells from bone marrow, blood, amniotic fluid, cord    blood, tumour & tissues (...
CYTOGENETIC TECHNIQUE   The cells are then centrifuged & media & mitotic inhibitor    is removed, & replaced with a hypot...
CYTOGENETIC TECHNIQUE   The cells are generally fixed repeatedly to remove any    debris or remaining red blood cells.   ...
CYTOGENETIC TECHNIQUEKaryotyping & Analysis   Analysis of banded chromosomes is done at a    microscope by a clinical lab...
CYTOGENETIC TECHNIQUEFluorescence in situ hybridisation (FISH)   Uses fluorescent molecules to vividly paint genes    or ...
CYTOGENETIC TECHNIQUE   FISH can be used to identify chromosomal    rearrangements, by detecting specific DNA    sequence...
CYTOGENETIC TECHNIQUE                        DML 202 General Biology & Human   11/16/2011             Genetics         (Ch...
CYTOGENETIC TECHNIQUE      A metaphase cell positive for the bcr/abl rearrangement using FISH. The     chromosomes can be ...
CYTOGENETIC TECHNIQUE How does FISH work?   FISH involves the preparation of short sequences of    single-stranded DNA, c...
CYTOGENETIC TECHNIQUE   Unlike most other techniques used to study    chromosomes, which require that the cells be active...
CYTOGENETIC TECHNIQUE                        DML 202 General Biology & Human   11/16/2011             Genetics         (Ch...
CYTOGENETIC TECHNIQUE                        Slide 22 of 10                            Topics
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Chapt 09

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Chapt 09

  1. 1. GENERAL BIOLOGY HDL 121 CYTOGENETIC TECHNIQUEPREPARED BY:MANEGASCHOOL OF MLTFACULTY OF HEALTH SCIENCE
  2. 2. CYTOGENETIC TECHNIQUELearning Outcomes After completing this lecture, students will be able to: (a) List few techniques used in cytogenetic field (b) Know & understand - Cell culture technique - Harvesting & chromosome spread - Karyotyping & analysis Slide 2 of 10 Topics © 2010 Cosmopoint
  3. 3. CYTOGENETIC TECHNIQUETopic Outlines 1.1. Cytogenetic technique 1.1.1 Example 1.1.2 Purpose 1.2. Cell culture 1.2.1 General procedure 1.2.2 Condition & Purpose 1.3. Harvesting & chromosome spread 1.3.1 General procedure & purpose 1.4. Karyotyping & analysis 1.4.1 General procedure & purpose Slide 3 of 10 © 2010 Cosmopoint
  4. 4. CYTOGENETIC TECHNIQUE1.1. Cytogenetic techniqueIntroduction Cytogenetic: a branch of genetics that is concerned with the study of structure & function of cell, esp. chromosomes & cell division Cytogenetics focuses on the microscopic examination of genetic components of the cell, including chromosomes, genes & gene products Older cytogenetic techniques involve placing cells in paraffin wax, slicing thin sections, & preparing them for microscopic study 11/16/2011 4 Slide 4 of 10
  5. 5. CYTOGENETIC TECHNIQUE1.1. Cytogenetic techniqueTechniques used in cytogenetic technique Routine analysis of G-Banded chromosome Molecular cytogenetics (a) Fluorescent in situ hybridisation (FISH) (b) Comparative genomic hybridisation (CGH) DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 5 Slide 5 of 10 Cytogenetic Technique)
  6. 6. CYTOGENETIC TECHNIQUECell culture The process by which prokaryotic or eukaryotic cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multi-cellular eukaryotes, especially animal cells. Epithelial cells in culture, stained for keratin (red) & DNA (green) > DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 6 Slide 6 of 10 Cytogenetic Technique)
  7. 7. CYTOGENETIC TECHNIQUE  General procedures (a) Isolation of cells (b) Maintaining cells in culture (c) Manipulation of cultured cells - Media changes, passaging cells & transfecting + tranducting cells Transfecting – introduction of foreign DNA to cause cells to express a protein of interest Transduction – DNA is inserted into cell using viruses DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 7 Slide 7 of 10 Cytogenetic Technique)
  8. 8. CYTOGENETIC TECHNIQUE Purpose: - manufacture of viral vaccines & many products of biotechnology (recombinant DNA, rDNA) - eg. Enzymes, synthetic hormones, monoclonal antibodies, interleukins & anticancer agents DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 8 Slide 8 of 10 Cytogenetic Technique)
  9. 9. CYTOGENETIC TECHNIQUECytogenetic technique Investigation into human karyotype (new method) (a) Using cells in culture (b) Pre-treating cells in a hypotonic solution which swells them & spreads the chromosomes (c) Arresting mitosis in metaphase by a solution of colchicine (d) Squashing the preparation on the slide forcing the chromosomes into a single plane (e) Cutting up a photomicrograph & arranging the result into an indisputable karyogram DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 9 Slide 9 of 10 Cytogenetic Technique)
  10. 10. CYTOGENETIC TECHNIQUE Routine Chromosome Analysis Refers to analysis of metaphase chromsomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two This creates unique banding patterns on the chromosomes The molecular mechanism & reason for these patterns is unknown, although it likely related to replication timing & chromatin packing DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 10 Slide 10 of 10 Cytogenetic Technique)
  11. 11. CYTOGENETIC TECHNIQUESlide Preparation Cells from bone marrow, blood, amniotic fluid, cord blood, tumour & tissues (including skin, umbilical cord, liver & many other organs) can be cultured using standard cell culture techniques in order to increase their number A mitotic inhibitor (colchicine, colcemid) is then added to the culture This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 11 Slide 11 of 10 Cytogenetic Technique)
  12. 12. CYTOGENETIC TECHNIQUE The cells are then centrifuged & media & mitotic inhibitor is removed, & replaced with a hypotonic solution. This causes the cells to swell so that the chromosomes will spread when added to a slide. After the cells have been allowed to sit in hypotonic, Carnoy’s fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells, lyses the red blood cells & hardens the nuclei of the remaining white blood cells. DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 12 Slide 12 of 10 Cytogenetic Technique)
  13. 13. CYTOGENETIC TECHNIQUE The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging, the slides in an oven or waiting a few days they are ready for banding & analysis. DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 13 Slide 13 of 10 Cytogenetic Technique)
  14. 14. CYTOGENETIC TECHNIQUEKaryotyping & Analysis Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analysed which is enough to rule out mosaicism to an acceptable level. The results are summarized & given to a board- certified medical geneticist & a pathologist taking into account the patients previous history & other clinical findings DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 14 Slide 14 of 10 Cytogenetic Technique)
  15. 15. CYTOGENETIC TECHNIQUEFluorescence in situ hybridisation (FISH) Uses fluorescent molecules to vividly paint genes or chromosomes This technique is particularly useful for gene mapping & for identifying chromosomal abnormalities Have become invaluable tools for the diagnosis & identification of the numerous chromosomal aberrations that are associated with neoplastic disease, including both haematological malignancies & solid tumours. DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 15 Slide 15 of 10 Cytogenetic Technique)
  16. 16. CYTOGENETIC TECHNIQUE FISH can be used to identify chromosomal rearrangements, by detecting specific DNA sequences with fluorescently labelled DNA probes. The technique of comparative genomic hybridisation (CGH) involves two-colour FISH. It can be used to establish ratios of fluorescence intensity values between tumour DNA & control DNA along normal reference metaphase chromosomes, & thereby to detect DNA copy-number changes eg. gains & losses of specific chromosomal regions & gene amplifications. DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 16 Slide 16 of 10 Cytogenetic Technique)
  17. 17. CYTOGENETIC TECHNIQUE DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 17 Slide 17 of 10 Cytogenetic Technique)
  18. 18. CYTOGENETIC TECHNIQUE A metaphase cell positive for the bcr/abl rearrangement using FISH. The chromosomes can be seen in blue. The chromosome that is labelled with green and red spots (up left) is the one where the wrong rearrangement is present DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 18 Slide 18 of 10 Cytogenetic Technique)
  19. 19. CYTOGENETIC TECHNIQUE How does FISH work? FISH involves the preparation of short sequences of single-stranded DNA, called probes, which are complementary to the DNA sequences the researchers wish to paint & examine. These probes hybridize, or bind, to the complementary DNA &, because they are labelled with fluorescent tags, allow researchers to see the location of those sequences of DNA. DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 19 Slide 19 of 10 Cytogenetic Technique)
  20. 20. CYTOGENETIC TECHNIQUE Unlike most other techniques used to study chromosomes, which require that the cells be actively dividing, FISH can also be performed on non-dividing cells, making it a highly versatile procedure. DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 20 Slide 20 of 10 Cytogenetic Technique)
  21. 21. CYTOGENETIC TECHNIQUE DML 202 General Biology & Human 11/16/2011 Genetics (Chapter 15: 21 Slide 21 of 10 Cytogenetic Technique)
  22. 22. CYTOGENETIC TECHNIQUE Slide 22 of 10 Topics

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