Chapt 09
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Transcript of Chapt 09
GENERAL BIOLOGY
SCHOOL OF MLTFACULTY OF HEALTH SCIENCE
PREPARED BY:MANEGA
HDL 121CYTOGENETIC TECHNIQUE
CYTOGENETIC TECHNIQUE
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Learning 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
Topics© 2010 Cosmopoint
CYTOGENETIC TECHNIQUE
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Topic Outlines
1.1. Cytogenetic technique1.1.1 Example1.1.2 Purpose
1.2. Cell culture1.2.1 General procedure1.2.2 Condition & Purpose
1.3. Harvesting & chromosome spread1.3.1 General procedure & purpose
1.4. Karyotyping & analysis1.4.1 General procedure & purpose
© 2010 Cosmopoint
CYTOGENETIC TECHNIQUE
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Introduction
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
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1.1. Cytogenetic technique
CYTOGENETIC TECHNIQUE
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Techniques used in cytogenetic technique
Routine analysis of G-Banded chromosomeMolecular cytogenetics
(a) Fluorescent in situ hybridisation (FISH)
(b) Comparative genomic hybridisation (CGH)
04/10/2023DML 202 General Biology & Human
Genetics (Chapter 15: Cytogenetic Technique)
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1.1. Cytogenetic technique
CYTOGENETIC TECHNIQUE
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Cell 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) >
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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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
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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Purpose:
- manufacture of viral vaccines & many products of biotechnology (recombinant DNA, rDNA)
- eg. Enzymes, synthetic hormones, monoclonal antibodies, interleukins & anticancer agents
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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Cytogenetic 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
04/10/2023DML 202 General Biology & Human
Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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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
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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Slide 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.
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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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.
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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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.
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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Karyotyping & 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
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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Fluorescence 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.
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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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.
04/10/2023DML 202 General Biology & Human
Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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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
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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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.
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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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.
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Genetics (Chapter 15: Cytogenetic Technique)
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CYTOGENETIC TECHNIQUE
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