“These are how the G bands look like.
There are brighter areas
CYTOGENETIC TECHNIQUE
OUTLINE
I Banding
A Types of Banding Technique
B Other Types of Banding Technique
II Karyotyping
A Steps involved in Karyotyping
B Conditions diagnosed with karyotype Test
III. Fish Method
A. Types of probes for Fish Method
BANDING
● A band is a part of a chromosome which is clearly
distinguishable from its adjacent segments by appearing
darker or brighter.
● The chromosomes are visualized as consisting of a
continuous series of bright and dark bands.
● Chromosomes in metaphase can be identified using
certain staining techniques.
● The cells are stopped in metaphase to maximize the
number of suitable cells.
● They are spread on a slide, stained with suitable dye, and
visualized under the microscope.
TYPES OF BANDING TECHNIQUE
GTL BANDING (G BANDING)
● This is also known as the “Giemsa staining method”
because it uses a Giemsa dye.
● It is used to identify individual chromosome and their
structural anomalies given the resulting banding pattern.
● G banding is widely used in clinical practice because it
provides distinct permanent bands that allow the
identification of all human chromosomes and accurate
characterization of numeric and structural anomalies.
● G banding allows each chromosome to be identified by its
characteristic banding pattern.
● Different genes also have different banding patterns.
● This banding pattern can distinguish chromosomal
abnormalities or structural rearrangements (translocations,
deletions, insertions, and inversions.)
● G banding has been divided into regions, bands, and sub
bands.
● G banding is a commonly used technique, it took its name
from the “Giemsa dye” but it still can be produced with
other dyes.
● In G bands, the darker regions tend to be heterochromatic,
the brighter regions are euchromatic.
➔ heterochromatic – tightly packed form of DNA
➔ euchromatic – composed of a loosely packed from of
chromatin
in darker bands. This is basically the output or result of G
banding.”
QFQ BANDING (Q BANDING)
● This fluorescent staining method uses “quinacrine”
which is used to identify chromosomes and their
structural anomalies.
● The characteristic binding pattern can be used to
identify each chromosome accurately.
● Staining of chromosomes gives bands that fluoresce
on exposure to UV light.
● The patterns of the Q banding can be correlated to
the G bands.
● This allows the precision identification of different
chromosome pairs and also the identification of
structural chromosome rearrangements.
● Q banding is technically among the simplest banding
technique.
● It can be recognized by a yellow fluorescence of
differing intensities resulting after treatment of the
chromosomes with quinacrine mustard (QM) or
quinacrine fluorochromes.
● Quinacrine mustard is an alkaline agent which gives
highly specific banding patterns, particularly in human
chromosomes.
● A disadvantage of this technique is that the
fluorescent intensity fades rapidly, observations and
photographs must be made within few minutes of
staining.
C BANDING
● This staining method is used to analyze the normal
and abnormal structural variations in chromosomes
by locating centromeric heterochromatin.
● This is a specialized Giemsa technique that primarily
stains chromosomes at the centromere, which have
large amounts of ATrich satellite DNA.
● C bands are present in centromeric regions of
chromosomes of analyzed
species.
This is an example of a C band. They are
centromeric. Meaning they focus on the
centromeres."
 LEC 11 CYTOGENETIC TECHNIQUE

● Examining chromosomes through karyotyping allows

CLASSIFICATION OF CHROMOSOME BANDS the doctor to determine whether there are any
abnormalities or structural problems within the
HETEROCHROMATIC They are demonstrated by C patient's chromosome.
BANDS Banding techniques, as well as ● An unusual number of chromosomes or malformed
various methods of chromosomes can all be signs of genetic condition
fluorochrome staining. (e.g. Down syndrome, Turner syndrome)
● Babies can be karyotype tested before they are born
EUCHROMATIC BANDS They form a pattern of to diagnose genetic abnormalities that indicate
alternating positively and serious birth defects (e.g. Klinefelter syndrome)
negatively stained (or
fluorescently) bands STEPS INVOLVED IN KARYOTYPING
throughout the length of the 1. SAMPLE COLLECTION
chromosome. - They are ● In newborns, a blood sample containing red blood
demonstrated by G bands, R cells, white blood cells, serum, and other fluids is
bands, Q bands, and certain collected.
fluorochromes. ● A karyotype will be done on the white blood cells
which are actively dividing which is a state known as
NUCLEOLUS They are segments of mitosis.
ORGANIZER REGIONS chromosomes that contain ● During pregnancy, the sample can either be amniotic
genes for ribosomal RNA, fluid collected during an amniocentesis or a piece of
which gives rise to the the placenta collected during a chorionic villi sample
interphase nucleoli. - They can test (CVS).
be stained with Ag-NOR (silver ● The amniotic fluid contains fetal skin cells which are
nucleolus organizer region) used to generate a karyotype
staining 2. TRANSPORTATION TO THE LABORATORY
● A specific type of laboratory is used for the
KINETOCHORES They are centromeric performance of karyotypes which is called the
structures through which “cytogenetics lab.”
mitotic and meiotic 3. SEPARATION OF CELLS
chromosomes are attached to ● In order to analyze chromosomes, the sample must
the spindle microtubules and contain cells that are actively dividing.
are generally labeled using ● In blood, the white blood cells actively divide. Most
autoimmune CREST area. fetal cells actively divide as well.
● Once the sample reaches the cytogenetics lab, the
non-dividing cells are separated from the dividing
OTHER TYPES OF BANDING TECHNIQUES
cells using special chemicals.
DISTAMYCIN / DAPI STAINING
4. GROWING OF CELLS
● The staining method identifies heterochromatic
● In order to have enough cells to analyze, the dividing
regions found on chromosomes 1, 9, 15, 16 and Y.
cells are grown in special media or a cell culture.
AGNOR STAINING FOR SATELLITE REGIONS
● This media contains chemicals and hormones that
● This is used to locate nucleolar organizer regions on
enable the cells to divide and multiply.
chromosomes. This technique is also useful for
● This process of culturing can take three to four days
studies of chromosomes with double satellites,
for blood cells, and up to a week for fetal cells.
chromosome polymorphism, and structural
5. SYNCHRONIZING OF CELLS
abnormalities.
● Chromosomes are a long string of human DNA. In
NON-BANDING
order to see chromosomes under a microscope,
● This chromosome banding technique uses
chromosomes have to be in their most compact form
nucleoprotein. Typically 20 metaphases are stained
in a phase of cell division (MITOSIS) known as
by non-banding and analyzed for:
metaphase.
➔ Minor anomalies (chromatid breaks and gaps) o Major
● In order to get all the cells to this specific stage of cell
anomalies (acentric fragments and dicentric
division, the cells are treated with a chemical which
chromosomes)
stops cell division at the point where the
➔ Radial configurations (tri radialis and quadra radialis)
chromosomes are the most compact.
➔ Complex radial formations
6. RELEASING OF CHROMOSOMES FROM THEIR
CELLS
KARYOTYPING ● In order to see these compact chromosomes under a
● This is a laboratory procedure where it allows the microscope, the chromosomes have to be out of the
doctor to examine a set of chromosomes. white blood cells.
● A ‘karyotype’ refers to the actual collection of ● This is done by treating the white blood cells with a
chromosomes being examined. special solution that causes them to burst.
● A Karyotype test examines the dividing cells. The ● This is done while the cells are on a microscope slide.
pairs of chromosomes are arranged by their size and The leftover debris from the white blood cells is
appearance. washed away, leaving the chromosomes stuck to the
● Karyotyping can be used to detect a variety of genetic side.
disorders.

2D – GRP 4 2
 LEC 11 CYTOGENETIC TECHNIQUE
7. STAINING OF CHROMOSOMES
● Chromosomes are naturally colorless. In order to tell
one chromosome from another, a special dye called
“Giemsa dye” is applied to the slide.
● Giemsa dye stains regions of chromosomes that are
rich in the bases adenine and thymine. When stained,
the chromosomes look like strings with light and dark
bands.
● Each chromosome has a specific pattern of light and
dark bands which enable the cytogeneticist to tell one
chromosome from another. Each dark or light band
encompasses hundreds of different genes.
8. ANALYSIS OF CHROMOSOMES
● Once chromosomes are stained, the slide is put under
the microscope for analysis. A picture is then taken of
the chromosomes. By the end of the analysis, the
total number of chromosomes will be determined, and
the chromosomes are arranged by size.
9. COUNTING OF THE CHROMOSOMES
● The first step of the analysis is counting the
chromosomes.
● Most humans have 46 chromosomes. People with
Down syndrome have 47 chromosomes. It is possible
for people to have missing chromosomes, more than
one extra chromosome, or a portion of a chromosome
that is either missing or duplicated.
● By looking at just the number of chromosomes, it is
possible to diagnose different conditions including
Down syndrome
10. CHECKING THE STRUCTURE OF THE
CHROMOSOMES
● After determining the number of chromosomes, the
cytogeneticist will start sorting the chromosomes.
● To sort the chromosomes, a cytogeneticist will
compare chromosome length, the placement of
centromeres (the areas where the two chromatids are
joined), and the location and sizes of G bands.
● The chromosome spares are numbered from largest
(1) to smallest (22).
● There are 22 pairs of chromosomes, called
“autosomes”, which match up exactly.
● There are also the sex chromosomes, females have
two X chromosomes while males have an X & a Y.
11. THE FINAL RESULT OF KARYOTYPING
● In the end, the final karyotype shows the total number
of chromosomes, the sex, and any structural
abnormalities with individual chromosomes.
● A digital picture of the chromosomes is generated
with all of the chromosomes arranged by number
CONDITIONS DIAGNOSED WITH A KARYOTYPE
TEST
● Karyotypes can be used to screen for and confirm
chromosomal abnormalities such as Down’s
syndrome and Cat Eye Syndrome, and there are
several different types of abnormalities which may be
detected.
Chromosomal abnormalities:
➔ TRISOMIES – in which there are 3 copies of one of
the chromosomes rather than 2.
Examples include:
➢ Down syndrome (trisonomy 21)
➢ Edward syndrome (trisonomy 18)
➢ Patau syndrome (trisonomy 13)
2D – GRP 4
➢ Kinefelter’s syndrome (XXY and other
variations) – occurs in 1 in 500 newborn
males.
➢ Triple X syndrome (XXX)
➔ MONOSOMIES – in which only one copy (instead of
2) is present.
Examples include:
➢ Turner syndrome (X0) or monosomy X –
Roughly 10% of first trimester miscarriages
are due to Turner syndrome, but this
monosomy is present in only around 1 in
2,500 live female births.
➔ CHROMOSOME DELETIONS – in which a part of
one chromosome is missing.
Examples include:
➢ Cri-du-chat syndrome (missing chromosome
5)
➢ Williams syndrome (missing chromosome 7)
➔ CHROMOSOME TRANSLOCATIONS – in which a
part of one chromosome is attached to another
chromosome.
Examples include:
➢ Translocation Down syndrome
➢ Robertsonian translocations – fairly
common, occurring in roughly 1 in 1000
people.
➢ Mosaicism – is a condition in which some
cells in the body have a chromosomal
abnormality while others do not. For
example, mosaic Down syndrome or mosaic
trisomy 9. Full trisomy 9 is not compatible
with life, but mosaic trisomy 9 may result in a
live birth.
FISH METHOD
●
Fluorescence In Situ Hybridization (FISH) method
●
A kind of cytogenetic technique which uses
fluorescent probes binding parts of the chromosome
to show a high degree of sequence complementarity.
● Fluorescence microscopy can be used to find out
where the fluorescent probes are bound to the
chromosome.
● This technique provides a novel way for researchers
to visualize and map the genetic material in an
individual cell, including specific genes or portions of
genes.
● It is an important tool for understanding a variety of
chromosomal abnormalities and other genetic
mutations.
● Different from most other techniques used for
chromosomes study, FISH has no need to be
performed on cells that are actively dividing, which
makes it a very versatile procedure.
HOW DOES FISH WORK?
● FISH is useful to help a researcher identify where a
particular gene falls within an individual’s
chromosomes.
1 Make a probe complementary to the known
sequence
2 When making the probe, label it with a fluorescent
marker, e.g. fluorescein, by incorporating nucleotides
that have the marker attached to them.
3
 LEC 11 CYTOGENETIC TECHNIQUE

WHOLE CHROMOSOME PROBES

3 Put the chromosomes on a microscope slide and
denature them.
4 Denature the probe and add it to the microscope
slide, allowing the probe to hybridize to its
complementary site.
5 Wash off the excess probe and observe the
chromosomes under a fluorescent microscope. The
probe will show us one or more fluorescent signals in
the microscope, depending on how many sites it can
hybridize to.
● Are actually collections of smaller probes, each of
which binds to a different sequence along the length
of a given chromosome.
● Using multiple probes labeled with a mixture of
different fluorescent dyes, scientists are able to label
each chromosome in its own unique color.
● The resulting full color map of the chromosome is
known as a “spectral karyotype”.
● Whole chromosome probes are particularly useful for
examining chromosomal abnormalities, for example,
when a piece of 1 chromosome is attached to the end
of another chromosome

WHAT IS FISH USED FOR?

● FISH is widely used for several diagnostic
applications:
➢ Identification of numerical and structural
abnormalities
➢ Characterization of marker chromosomes
➢ Monitoring the effects of therapy
➢ Detection of minimal residual disease
➢ Tracking the origin of cells after bone marrow
transplantation
➢ Identification of regions of deletion or
amplification
➢ Detection of chromosome abnormalities in
non-dividing or terminally differentiated cells.
➢ Determination of lineage involvement of
clonal cells.
● FISH is also used to compare the genomes of two
biological species due to the deduced evolutionary
relationships.

TYPES OF PROBES FOR FISH METHOD

LOCUS SPECIFIC PROBES
● Bind to a particular region of a chromosome.
● This type of probe is useful when researchers have
isolated a small portion of a gene and want to
determine on which chromosome the gene is located.
ALPHOID OR CENTROMERIC REPEAT PROBES
● Are generated from repetitive sequences found in the
middle of each chromosome.
● Researchers use these probes to determine whether
an individual has the correct number of
chromosomes.
● These probes can also be used in combination with
“local specific probes” to determine whether an
individual is missing a genetic material from a
particular chromosome.

2D – GRP 4 4