1st SEMESTER | 2022-2023 NOVEMBER 7, 2022

LESSON 11: CYTOGENETIC TECHNIQUES

LECTURER: Ms. Jamie Therese Go

- This banding pattern can distinguish

TOPIC OUTLINE chromosomal abnormalities or
structural rearrangements
BANDING (translocations, deletions, insertions,
KARYOTYPING and inversions.)
- G banding has been divided into
FISH METHOD 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
BANDING
produced with other dyes.
➢ A band is a part of a chromosome which - In G bands, the darker regions tend
is clearly distinguishable from its to be heterochromatic, the brighter
adjacent segments by appearing regions are euchromatic.
darker or brighter. *heterochromatic – tightly packed form of DNA
➢ The chromosomes are visualized as *euchromatic – composed of a loosely packed
consisting of a continuous series of from of chromatin
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
“These are how the G bands look like. There are
microscope.
brighter areas in darker bands. This is basically
the output or result of G banding.”
TYPES OF BANDING TECHNIQUE
1. GTL BANDING (G Banding)
2. QFQ BANDING (Q Banding)
- This is also known as the “Giemsa
- This fluorescent staining method
staining method” because it uses a
uses “quinacrine” which is used to
Giemsa dye.
identify chromosomes and their
- It is used to identify individual
structural anomalies.
chromosome and their structural
- The characteristic binding pattern
anomalies given the resulting
can be used to identify each
banding pattern.
chromosome accurately.
- G banding is widely used in clinical
- Staining of chromosomes gives
practice because it provides
bands that fluoresce on exposure to
distinct permanent bands that allow
UV light.
the identification of all human
- The patterns of the Q banding can
chromosomes and accurate
be correlated to the G bands.
characterization of numeric and
- This allows the precision
structural anomalies.
identification of different
- G banding allows each
chromosome pairs and also the
chromosome to be identified by its
identification of structural
characteristic banding pattern.
chromosome rearrangements.
- Different genes also have different
- Q banding is technically among the
banding patterns.
simplest banding technique.

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 - It can be recognized by a yellow “This is an example of C band. They are
fluorescence of differing intensities centromeric. Meaning they focus on the
resulting after treatment of the centromeres."
chromosomes with quinacrine
mustard (QM) or quinacrine CLASSIFICATION OF CHROMOSOME BANDS
fluorochromes. HETEROCHROMATIC They are
- Quinacrine mustard is an alkaline BANDS demonstrated by C-
agent which gives highly specific banding techniques,
banding patterns, particularly in as well as various
human chromosomes. methods of
- A disadvantage of this technique is fluorochrome
that the fluorescent intensity fades staining.
rapidly, observations and EUCHROMATIC - They form a pattern
photographs must be made within BANDS of alternating
few minutes of staining. positively and
negatively stained (or
fluorescently) bands
throughout the
length of the
chromosome.
- They are
demonstrated by G
bands, R bands, Q
bands, and certain
fluorochromes.
3. C BANDING
NUCLEOLUS - They are segments
- This staining method is used to
ORGANIZER REGIONS of chromosomes that
analyze the normal and abnormal
contains genes for
structural variations in
ribosomal RNA,
chromosomes by locating
which gives rise to the
centromeric heterochromatin.
interphase nucleoli.
- This is a specialized Giemsa
- They can be stained
technique that primarily stains
with Ag-NOR (silver-
chromosomes at the centromere,
nucleoli organizer
which have large amounts of AT-
region) staining.
rich satellite DNA.
KINTETOCHORES They are centromeric
- C bands are present in centromeric
structures through
regions of chromosomes of
which mitotic and
analyzed species.
meiotic
chromosomes are
attached to the
spindle microtubules
and are generally
labeled using auto-
immune CREST area.

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 OTHER TYPES OF BANDING TECHNIQUES signs of genetic condition (e.g. Down
4. DISTAMYCIN / DAPI STAINING syndrome, Turner syndrome)
- The staining method identifies ➢ Babies can be karyotype tested before
heterochromatic regions found on they are born to diagnose genetic
chromosomes 1, 9, 15, 16 and Y. abnormalities that indicate serious birth
5. AGNOR STAINING FOR SATELLITE defects (e.g. Klinefelter syndrome)
REGIONS
- This is used to locate nucleolar STEPS INVOLVED IN KARYOTYPING
organizer regions on chromosomes. 1 SAMPLE COLLECTION
This technique is also useful for - In newborns, a blood
studies of chromosomes with sample containing red
double satellites, chromosome blood cells, white blood
polymorphism, and structural cells, serum, and other fluids
abnormalities. is collected.
6. NON-BANDING - A karyotype will be done on
- This chromosome banding the white blood cells which
technique uses nucleoprotein. are actively dividing which is
Typically 20 metaphases are stained a state known as mitosis.
by non-banding and analyzed for: - During pregnancy, the
o Minor anomalies sample can either be
(chromatid breaks and amniotic fluid collected
gaps) during an amniocentesis or
o Major anomalies (acentric a piece of the placenta
fragments and dicentric collected during a chorionic
chromosomes) villi sample test (CVS).
o Radial configurations - The amniotic fluid contains
(triradialis and fetal skin cells which are
quadraradialis) used to generate a
o Complex radial formations karyotype.
2 TRANSPORTATION TO THE LABORATORY
KARYOTYPING - A specific type of laboratory
is used for the performance
➢ This is a laboratory procedure where it of karyotypes which is called
allows the doctor to examine a set of the “cytogenetics lab.”
chromosomes. 3 SEPARATION OF CELLS
➢ A ‘karyotype’ refers to the actual - In order to analyze
collection of chromosomes being chromosomes, the sample
examined. must contain cells that are
➢ A Karyotype test examines the dividing actively dividing.
cells. The pairs of chromosomes are - In blood, the white blood
arranged by their size and appearance. cells actively divide. Most
➢ Karyotyping can be used to detect a fetal cells actively divide as
variety of genetic disorders. well.
➢ Examining chromosome through - Once the sample reaches
karyotyping allows the doctor to the cytogenetics lab, the
determine whether there are any non-dividing cells are
abnormalities or structural problem separated from the dividing
within the patient's’ chromosome. cells using special
➢ An unusual number of chromosomes or chemicals.
malformed chromosomes can all be

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4 GROWING OF CELLS
- In order to have enough
cells to analyze, the dividing
cells are grown in special
media or a cell culture.
- This media contains
chemicals and hormones
that enable the cells to
divide and multiply.
- This process of culturing can
take three to four days for
blood cells, and up to a
week for fetal cells.
5 SYNCHRONIZING OF CELLS
- Chromosomes are a long
string of human DNA. In
order to see chromosomes
under a microscope,
chromosomes have to be in
their most compact form in a
phase of cell division
(MITOSIS) known as
metaphase.
- In order to get all the cells to
this specific stage of cell
division, the cells are treated
with a chemical which stops
cell division at the point
where the chromosomes
are the most compact.
6 RELEASING OF CHROMOSOMES FROM
THEIR CELLS
- In order to see these
compact chromosomes
under a microscope, the
chromosomes have to be
out of the white blood cells.
- This is done by treating the
white blood cells with a
special solution that causes
them to burst.
- This is done while the cells
are on a microscope slide.
The leftover debris from the
white blood cells is washed
away, leaving the
chromosomes stuck to the
side.
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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 CHROMSOMES
- 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 chromosomes rather than 2.
including Down syndrome. Examples include:
10 CHECKING THE STRUCTURE OF THE o Down syndrome
CHROMOSOMES (trisonomy 21)
- After determining the o Edward syndrome
number of chromosomes, (trisonomy 18)
the cytogeneticist will start o Patau syndrome
sorting the chromosomes. (trisonomy 13)
- To sort the chromosomes, a o Kinefelter’s syndrome
cytogeneticist will compare (XXY and other
chromosome length, the variations) – occurs in 1
placement of centromeres in 500 newborn males.
(the areas where the two o Triple X syndrome
chromatids are joined), and (XXX)
the location and sizes of G • MONOSOMIES – in which only
bands. one copy (instead of 2) is
- The chromosome spares are present. Examples include:
numbered from largest (1) to o Turner syndrome (X0)
smallest (22). or monosomy X –
- There are 22 pairs of Roughly 10% of first
chromosomes, called trimester miscarriages
“autosomes”, which match are due to Turner
up exactly. syndrome, but this
- There are also the sex monosomy is present in
chromosomes, females only around 1 in 2,500
have two X chromosomes live female births.
while males have an X & a Y. • CHROMSOME DELETIONS – in
11 THE FINAL RESULT OF KARYOTYPING which a part of one
- In the end, the final chromosome is missing.
karyotype shows the total Examples include:
number of chromosomes, o Cri-du-chat syndrome
the sex, and any structural (missing chromosome
abnormalities with individual 5)
chromosomes. o Williams syndrome
- A digital picture of the (missing chromosome
chromosomes is generated 7)
with all of the chromosomes • CHROMOSOME
arranged by number TRANSLOCATIONS – in which a
part of one chromosome is
CONDITIONS DIAGNOSED WITH A KARYOTYPE attached to another
TEST chromosome. Examples
» Karyotypes can be used to screen for include:
and confirm chromosomal o Translocation Down
abnormalities such as Down’s syndrome syndrome
and Cat Eye Syndrome, and there are o Robertsonian
several different types of abnormalities translocations – fairly
which may be detected. common, occurring in
» Chromosomal abnormalities: roughly 1 in 1000
• TRISONOMIES – in which there people.
are 3 copies of one of the o Mosaicism – is a
condition in which

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 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.
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.

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
bound to the chromosome.
➢ This technique provides a novel way for
WHAT IS FISH USED FOR?
researchers to visualize and map the
➢ FISH is widely used for several diagnostic
genetic material in an individual cell,
applications:
including specific genes or portions of
✓ Identification of numerical and
genes.
structural abnormalities
➢ It is an important tool for understanding
✓ Characterization of marker
a variety of chromosomal abnormalities
chromosomes
and other genetic mutations.
✓ Monitoring the effects of therapy
➢ Different from most other techniques
✓ Detection of minimal residual
used for chromosomes study, FISH has
disease
no need to be performed on cells that
✓ Tracking the origin of cells after
are actively dividing, which makes it a
bone marrow transplantation
very versatile procedure.
✓ Identification of regions of deletion
or amplification
HOW DOES FISH WORK?
✓ Detection of chromosome
➢ FISH is useful to help a researcher identify
abnormalities in non-dividing or
where a particular gene falls within an
terminally differentiated cells.
individual’s chromosomes.
✓ Determination of lineage
1 Make a probe complementary to the
involvement of clonal cells.
known sequence.
➢ FISH is also used to compare the
2 When making the probe, label it with a genomes of two biological species due
fluorescent marker, e.g. fluorescein, by to the deduce evolutionary
incorporating nucleotides that have the relationships.
marker attached to them.
3 Put the chromosomes on a microscope
slide and denature them.

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 TYPES OF PROBES FOR FISH METHOD
1. 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.
2. 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.
3. WHOLE CHROMOSOME PROBES
- 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.

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