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


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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


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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


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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


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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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http://en.wikipedia.org/wiki/File:Epithelial-cells.jpg

http://en.wikipedia.org/wiki/File:Epithelial-cells.jpg


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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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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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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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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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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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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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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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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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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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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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DML 202 General Biology & Human Genetics (Chapter 15:

Cytogenetic Technique)17

http://en.wikipedia.org/wiki/File:FISH_(technique).gif


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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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http://en.wikipedia.org/wiki/File:Bcrablmet.jpg


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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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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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DML 202 General Biology & Human Genetics (Chapter 15:

Cytogenetic Technique)21

http://upload.wikimedia.org/wikipedia/commons/5/57/FISH_(Fluorescent_In_Situ_Hybridization).jpg


Slide 22 of 10Topics

THANK YOU

Chapt 09

Technology

Transcript of Chapt 09