CYTOGENETICS LEC

BSMT 2 - 2ND SEMESTER

CYTOGENETIC TECHNIQUES

Lesson 11: CYTOGENETIC TECHNIQUES QFQ Banding (Q Banding)

Mrs. Jamie Go

THREE (3) CYTOGENETIC TECHNIQUES

● Banding
● Karyotyping
● FISH Method

BANDING TECHNIQUES

● A band is a part of a chromosome which is clearly distinguishable

from its adjacent segments by appearing darker or brighter.
● The chromosome are visualized as consisting of a continuous
series of bright and dark bands
● Chromosomes in metaphase can be identified using certain
staining techniques
● 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
GTL Banding (G banding)
This is how G bands or their results would look like.
There are brighter areas and darker bands.
● This is also known as the Giemsa staining method.
● 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 have different banding pattern.
● 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 can be produced with other dyes
● In G bands, the darker regions tend to be heterochromatic, the
brighter regions are euchromatic.
○ Heterochromatic- they are tightly pack form of
DNA in the nucleus
○ Euchromatic- composed of loosely packed form of
chromatin.
● 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.
○ When it is exposed to UV light, the bands will show up.
● The patterns can be correlated to G bands
● This allows the precise identification of different chromosome
pairs and also the identification of structural chromosome
rearrangements
● Q banding is technically among the simplest banding technique
● Fluorescent Q banding 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 (QM), is an alkaline agent which gives highly
specific banding patterns, particularly human chromosomes
● A disadvantage of this technique is that the fluorescent
intensity fades rapidly, thus observations and photographs must
be made within few minutes of staining after staining
○ They flouresce. The fluorescence will fade after a minutes
of doing the procedure. That’s why the laboratory
technician must take photographs and make observations
directly after how many minutes of staining because it
fades after a period of time.
C Banding
Example of C-band.
It focuses primarily on the centromeric samples of chromosomes
● This staining method is used to analyze the normal and abnormal
structural variations in chromosomes by locating centromeric
heterochromatin.

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● It is called C Banding because it locates the centromeric
heterochromatin.
● This is a specialized Giemsa technique that primarily stains
chromosomes at the centromere, which have large amounts of
AT-rich satellite DNA.
● C bands are present in centromeric regions of chromosomes of
analyzed species
CLASSIFICATION OF CHROMOSOME BANDS
● They are demonstrated by C-Banding
HETEROCHROM
Techniques, as well as various methods of
ATIC BANDS
fluorochrome staining
● The form a pattern of alternating positively and
negatively stained (or fluorescently) bands
EUCHROMATIC
through the length of the chromosome
BANDS
● They are demonstrated by G bands, R bands,
Q bands, and certain fluorochromes
● They are segments of chromosomes that
NUCLEOLUS contains genes for ribosomal RNA, which gives
ORGANIZER rise to the interphase nucleoli.
REGIONS ● They can be stained with Ag-NOR staining
(Silver Nucleolus Organizer Region Staining)
● They are centromeric structures through which
mitotic and meiotic chromosomes are
KINETOCHORES attached to the spindle microtubules, and are
generally labeled using auto-immune crest
area.
OTHER TYPES OF BANDING TECHNIQUES
DISTAMYCIN-A ● This staining method identifies heterochromatic
or DAPI regions found on chromosomes 1, 9, 15, 16,
Staining and Y.
● Silver Staining of Nuclear Organizer Regions
● This is used to locate nucleolar organizer regions
AgNOR
on chromosomes.
Staining for
● This technique is also useful for studies of
Satellite
chromosomes with double satellites,
Regions
chromosome polymorphism, and structural
abnormalities.
● This chromosome banding technique uses
nucleoprotein.
● Typically 20 metaphases are stained by non
banding and analyzed for
○ Minor anomalies such as chromatid breaks
Non-Banding
and gaps,
○ Major anomalies such as, acentric fragments
and dicentric chromosomes,
○ Radial configurations such as, triradialis,
quaradialis, and complex radial formations.
KARYOTYPING
● A laboratory procedure where it allows the doctor to examine a
set of chromosomes.
● A “karyotype” refers to the actual collection of chromosomes
being examined.
● A karyotype test examines the dividing cells. The pairs of
chromosomes are arranged by their size and appearance
● Karyotyping can be used to detect a variety of genetic disorders
● Examining chromosome through karyotyping allows the doctor to
determine whether there are abnormalities or structural problem
within the chromosomes
● An unusual number of chromosomes or malformed
chromosomes can all be signs of genetic condition
○ e.g. Down Syndrome & Turner Syndrome
● Babies can be karyotype tested before they are born to diagnose
genetic abnormalities that indicate serious birth defects
○ e.g. Klinefelter Syndrome
Fenequito E., Gonzaga R.
STEPS INVOLVED IN KARYOTYPING
SAMPLE COLLECTION
● In newborns, a blood sample containing red blood cells,
white blood cells, serum, and other fluids is collected.
● A karyotype will be done on the white blood cells which
are actively dividing (a state known as mitosis).
1 ● During pregnancy, the sample can either be amniotic
fluid collected during an amniocentesis or a place of
the placenta collected during a chorionic villi sampling
test (CVS).
● The amniotic fluid contains fetal skin cells which are
used to generate a karyotype
TRANSPORTATION TO THE LABORATORY
2 ● Karyotypes are performed in a specific laboratory called a
cytogenetics lab
SEPARATION OF THE CELLS
● In order to analyze chromosomes, the sample must
contain cells that are actively dividing.
● In blood, the white blood cells actively divide. Most fetal
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cells actively divide as well.
● Once the sample reaches the cytogenetics lab, the
non-dividing cells are separated from the dividing
cells using special chemicals.
GROWING OF CELLS
● In order to have enough cells to analyze, the dividing cells
are grown in special media or a cell culture.
4 ● 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.
SYNCHRONIZATION 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
5 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.
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.
6 ● 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 microscopic slide.
● The leftover debris from the white blood cells is washed
away, leaving the chromosomes stuck to the slide
STAINING OF THE 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 (A) and thymine (T). When
7 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. so unique jud each chromosome.
ANALYSIS OF THE CHROMOSOMES
● Once chromosomes are stained, the slide is put under the
microscope for analysis.
8 ● A picture of the chromosomes is then taken. By the end of
the analysis, the total number of chromosomes will be
determined and the chromosomes arranged by size
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 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
9 is also 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.
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
10 location and sizes of G-bands.
● The chromosomes pairs are numbered from largest
(number 1) to smallest (number 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 and 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 chromosome arranged by number.
CONDITIONS THAT CAN BE DIAGNOSED WITH A KARYOTYPE
TEST
● Karyotype 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
3 copies of one of the chromosomes rather than 2.
Example:
● Down syndrome (trisomy 21)
● Edward syndrome (trisomy 18)
Trisonomies ● Patau syndrome (trisomy 13)
● Klinefelter’s syndrome (XXY and other
variations)
○ Occurs in 1 in 500 newborn males
● Triple X syndrome (XXX)
Only one copy (instead of 2) is present
Example:
● Turner syndrome (XO) or monosomy X
Monosomies ○ Roughly 10% of the first trimester
miscarriage are due to Turner’s
syndrome, but this monosomy is
present in only around 1 in 2,500 live
female births
Part of chromosome is missing
Chromosome Example:
Deletions ● Cri-du-chat syndrome (missing chromosome 5)
● Williams syndrome (missing chromosome 7)
Part of one chromosome is attached to another
chromosome
Example:
Chromosome
● There are many examples of down syndrome
translocations including translocation down syndrome
● Robertsonian translocations are fairly
common, occurring in roughly 1 in 1000
people
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● Mosaicism is a condition in which some
cells in the body have chromosomal
abnormality while other 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
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 probe 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.
○ So di na kailangan ng undergo mitosis ang chromosomes
for FISH
HOW DOES FISH WORK?
● FISH is useful to help a researcher identify where a particular gene
falls within an individual’s chromosomes. Here’s how it works
STEPS INVOLVED IN KARYOTYPING
1 Make a probe complementary to the known sequence
When making the probe, label it with a fluorescent marker (e.g.
2 fluorescein) by incorporating nucleotides that have the marker
attached to them
3 Put the chromosomes on a microscope slide and denature them
Denature the probe and add it to the microscope slide, allowing
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the probe hybridize to its complementary site
Wash off the excess probe and observe the chromosomes under
a fluorescent microscope.
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The probe will show as one or more fluorescent signals in the
microscope, depending on how many sites it can hybridize to.
After unmasking of DNA, Probe and target denaturation happens. After
denaturation, Probe-target DNA hybridization follows. Next will be
detection which will bind to the fluorochrome (fluorescein). After, image
analysis on the microscope shoulds be done kay mu-fluoresce mana
ang sample so mas makita clearly sa laboratory technician.
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 WHAT IS FISH USED FOR?

● FISH is widely used for several diagnostic applications:

○ Identification of numerical 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 of the
chromosomes
○ 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 deduce evolutionary relationships

TYPES OF PROBES FOR FISH METHOD

CHROMOSOMAL ABNORMALITIES
● Bind to a particular region of a chromosome.
● This type of probe is useful when researchers
Locus Specific
have isolated a small portion of a gene and
Probes
want to determine on which chromosome the
gene is located.
● Are generated from repetitive sequences
found in the middle of each chromosome.
● Researchers use these probes to determine
Alphoid or whether an individual has the correct number
Centromeric of chromosomes.
Repeat Probes ● These probes can also be used in combination
with “locus-specific” probes to determine
whether an individual is missing genetic
material from a particular chromosome
● 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
Whole unique color.
Chromosome ● The resulting full-color map of the chromosome
Probes is known as spectral karyotype.
● Whole chromosome probes are particularly
useful for examining chromosomal
abnormalities
○ For example, when a piece of one
chromosome is attached to the end of
another chromosome

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