Dr. Attya Bhatti

CytogeneticsGeneral Genetics

Cytogenetics

Is the study of the structure and properties of

chromosomes, chromosomal behaviour during mitosis

and meiosis, chromosomal influence on the phenotype

and the factors that cause chromosomal changes.

Related to disease status caused by abnormal

chromosome number and/or structure.

Methods for chromosomal analysis: Karyotyping and banding

The collection of all the chromosomes is referred to as a

Karyotype.

The method used to analyze the chromosome constitution of

an individual, known as chromosome banding.

Chromosomes are displayed as a karyogram.

Cell source:

Blood cells

Skin fibroblasts

Amniotic cells / chorionic villi

Increasing the mitotic index

- proportion of cells in mitosis using colcemid

Synchronizing cells to analyze prometaphase

chromosomes

Obtaining and preparing cells forchromosome analysis

Key procedure

In the case of peripheral (venous) blood

A sample is added to a small volume of nutrient medium containing

phytoheamagglutinin, which stimulates T lymphocytes to divide.

The cells are cultured under sterile conditions at 37C for about 3

days, during which they divide, and colchicine is then added to

each culture.

This drug has the extremely useful property of preventing formation

of the spindle, thereby arresting cell division during metaphase, the

time when the chromosomes are maximally condensed and

therefore most visible.

Hypotonic saline is then added, which causes the red blood cells

to lyze and results in spreading of the chromosomes, which are then

fixed , mounted on a slide and stained ready for analysis

PREPARATION OF CHROMOSOMES

Following Steps are involved; Counting the number of cells, sometimes referred as

metaphase spread Analysis of the banding pattern of each individual

chromosome in selected cells. Total chr. Count is determined in 10-15 cells, but if

mosaicism is suspected then 30 or more cell count will be undertaken.

Detailed analysis of the banding pattern of the individual chromosomes is carried out in approx. 3-5 metaphase spread, which shows high quality banding.

The banding pattern of each chromosome is specific and shown in the form of Idiogram.

Karyotype Analysis

MITOTIC CHROMOSOMAL SPREAD

Chromosome Banding

Chromosome banding is developed based on the

presence of heterochromatin and euchromatin.

Heterochromatin is darkly stained whereas

euchromatin is lightly stained during chromosome

staining.

oEuchromatin, which undergoes the normal process of

condensation and decondensation in the cell cycle, and

oHeterochromatin, which remains in a highly condensed state

throughout the cell cycle, even during interphase.

Euchromatin Exist in extended state, dispersed through the nucleus and staining diffusely.

Early-replicating and GC rich region.

In prokaryotes, euchromatin is the only form of chromatin present.

Genes may oy may not expressed

Heterochromatindarkly stained two types

1.Constitutive ; always inactive an condensed.

Consist of repetitive DNA

Late replicating and AT rich region

Present at identical positions on all chromosomes in all cell types of an organism.

Genes poorly expressed.

Human chromosomes 1, 9, 16, and the Y chromosome contain large regions of constitutive heterochromatin.

Occurs around the centromere and near telomeres.

2. Facultative;

Genetically active(decondensed) and inactive (condensed)

Variable in its expression. It varies with the cell type and may be manifested as condensed, or heavily stained.

Types of chromosome banding

G-banding

C-banding

Q-banding

R-banding T-banding

Chromosomal Banding G-banding, gives dark bands

C-banding: C-banding stains the constitutive heterochromatin, which usually lies near the centromere.

Q-banding: Q-banding is a fluorescent pattern obtained using quinacrine for staining. The pattern of bands is very similar to that seen in G-banding.

R-banding: reverse of G-banding (the R stands for "reverse").

Dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).

T-banding: Identifies a subset of the R bands which are especially concentrated at the telomeres.

G-Banding

۩- G-banding is obtained with Giemsa stain following digestion of

chromosomes with enzyme trypsin.

۩- Giemsa stain, named after Gustav Giemsa, an early malariologist, is

used for the histopathological diagnosis of malaria and other parasites.

۩- It is a mixture of methylene blue and eosin.

۩- It is specific for the phosphate groups of DNA and attaches itself to

regions of DNA where there are high amounts of adenine-thymine

bonding.

۩- it yields a series of lightly and darkly stained bands – the dark regions

tend to be heterochromatic, late-replicating and AT rich.

۩- The light regions tend to be euchromatic, early-replicating

and GC rich .

G- Banding

Key procedure

In the case of peripheral (venous) blood

A sample is added to a small volume of nutrient medium containing

phytoheamagglutinin, which stimulates T lymphocytes to divide.

The cells are cultured under sterile conditions at 37C for about 3

days, during which they divide, and colchicine is then added to

each culture.

This drug has the extremely useful property of preventing formation

of the spindle, thereby arresting cell division during metaphase, the

time when the chromosomes are maximally condensed and

therefore most visible.

Hypotonic saline is then added, which causes the red blood cells

to lyze and results in spreading of the chromosomes, which are then

fixed , mounted on a slide and stained ready for analysis

PREPARATION OF CHROMOSOMES

Molecular Methods for chromosomal analysis Molecular Cytogenetics

Fluorescent in situ Hybridization (FISH)

Chromosome painting

Comparative Genomic Hybridization

(CGH)

Molecular karyotyping and Multiplex

FISH(M-FISH)

Spectral Karyotyping

Array CGH

Key points Cytogenetic analysis usually focuses on chromosomes in

dividing cells.

Dyes such as Quinacrine and Giemsa create banding patterns that’s are useful in identifying individual chromosomes within a cell.

A karyotype shows the photographed chromosomes of a cell arranged for cytogenetic analysis.

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