This document discusses cytogenetic techniques used in biology. It begins by outlining the learning objectives which are to list cytogenetic techniques and understand cell culture, harvesting chromosomes, and karyotyping. It then describes various techniques including cell culture, harvesting and spreading chromosomes, karyotyping, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH). The purpose of these techniques is to examine genetic components of cells including chromosomes to study genetic abnormalities.

1. GENERAL BIOLOGY
HDL 121
CYTOGENETIC TECHNIQUE
PREPARED BY:
MANEGA
SCHOOL OF MLT
FACULTY OF HEALTH SCIENCE

2. CYTOGENETIC TECHNIQUE
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
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Topics
© 2010 Cosmopoint

3. CYTOGENETIC TECHNIQUE
Topic 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
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© 2010 Cosmopoint

4. CYTOGENETIC TECHNIQUE
1.1. Cytogenetic technique
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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5. CYTOGENETIC TECHNIQUE
1.1. Cytogenetic technique
Techniques used in cytogenetic technique
Routine analysis of G-Banded chromosome
Molecular cytogenetics
(a) Fluorescent in situ hybridisation (FISH)
(b) Comparative genomic hybridisation (CGH)
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11/16/2011 Genetics (Chapter 15: 5
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6. CYTOGENETIC TECHNIQUE
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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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
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8. CYTOGENETIC TECHNIQUE
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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9. CYTOGENETIC TECHNIQUE
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
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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
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11. CYTOGENETIC TECHNIQUE
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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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.
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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.
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14. CYTOGENETIC TECHNIQUE
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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11/16/2011 Genetics (Chapter 15: 14
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15. CYTOGENETIC TECHNIQUE
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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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.
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17. CYTOGENETIC TECHNIQUE
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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
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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.
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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.
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22. CYTOGENETIC TECHNIQUE
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