CYTOGENETIC TECHNIQUES

QFQ BANDING (Q Banding)

BSMT 2 | S.Y. 2021-2022 Second Semester – FINALS • This fluorescent staining method uses quinacrine
which is used to identify chromosomes and their
Lecture Outline
structural anomalies
• Banding
• The characteristic binding pattern can be used to identify
• Karyotyping
each chromosome accurately
• FISH (Fluorescence in situ hybridization) Method
• Staining of chromosomes gives bands that fluoresce on
exposure to UV light
BANDING TECHNIQUES
a. A band is a part of a chromosome which is clearly • The patterns can be correlated to G bands
distinguishable from the adjacent segments by appearing • This allows the precise identification of different
darker or brighter chromosome pairs and also the identification of structural
b. The chromosome is visualized as consisting of a chromosome rearrangements
continuous series of bright and dark bands • Q banding is technically among the simplest banding
c. Chromosomes in metaphase can be identified using technique
certain staining techniques • Fluorescent Q banding can be recognized by a yellow
d. Cells are stopped in metaphase to maximize the number fluorescence of differing intensities resulting after
of suitable cells treatment of the chromosomes with quinacrine mustard
e. They are spread on a slide, stained with suitable dye and (QM) or quinacrine fluorochromes
visualized under the microscope • QM, is an alkaline agent which gives highly specific
banding patterns, particularly human chromosomes
TYPES OF BANDING TECHNIQUES • A disadvantage of this technique is that the fluorescent
intensity fades rapidly, thus observation and photographs
GTL BANDING (G Banding) must be made within few minutes of staining
• This is also known as the Giemsa staining method (It o That is why the laboratory technicians take
uses Giemsa dye) photographs and make observations directly
• It is used to identify individual chromosome and their after how many minutes of staining
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
o Different genes have different banding patterns
• This banding pattern can distinguish chromosomal
abnormalities or structural rearrangements C BANDING
(translocations, deletions, insertions, and inversions)
• This staining method is used to analyze the normal and
• G banding has been divided into regions, bands, and
abnormal structural variations in chromosomes by
sub bands
locating centromeric heterochromatin
• G banding is a commonly used technique, it took its name
• This is named as C banding because it locates
from the 'Giemsa dye,' but it can be produced with other
centromeric heterochromatin
dyes
• This is a specialized Giemsa technique that primarily
• In G bands, the darker regions tend to be
stains chromosomes at the centromere, which have large
heterochromatic
amounts of AT-rich satellite DNA
o Heterochromatic: tightly packed form of DNA
• C bands are present in centromeric regions of
and nucleus
chromosomes of analyzed species
• The brighter regions are euchromatic
o Euchromatic: composed of a loosely pack form
of chromatin

This is how G bands look like. There is brighter areas and darker
bands. The pic represents the output or result of G banding.
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CLASSIFICATION OF CHROMOSOME BANDS f. An unusual number of chromosomes or malformed
chromosomes can all be signs of genetic condition
a. HETEROCHROMATIC BANDS
o (e.g., Down syndrome, Turner Syndrome)
o They are demonstrated by C-Banding techniques,
g. Babies can be karyotype tested before they are born to
as well as various methods of fluorochrome
diagnose genetic abnormalities that indicate serious birth
staining
defects
o (e.g., Klinefelter Syndrome)
b. EUCHROMATIC BANDS
o They form a pattern of alternating positively and
STEPS INVOLVED IN KARYOTYPING
negatively stained (or fluorescently) bands
throughout the length of the chromosome
o They are demonstrated by G bands, R bands, Q 1. Sample Collection 7.Staining of the
bands, and certain fluorochromes 2.Transportation to the chromosomes
Laboratory 8.Analysis of the
c. NUCLEOLUS ORGANIZER REGIONS 3. Separation of Cells chromosomes
o They are segments of chromosomes that contains 4. Growing of Cells 9.Counting of the
genes for ribosomal RNA, which gives rise to the 5. Synchronizing of Cells Chromosomes
interphase nucleoli 6.Releasing of 10.Checking the structure of
o They can be stained with Ag-NOR staining (silver chromosomes from their the chromosomes
nucleolus organized region) cells 11.Final result of
Karyotyping
d. KINETOCHORES
o They are centromeric structures through which 1. Sample Collection
mitotic and meiotic chromosomes are attached to a. In newborns, a blood sample containing red blood
the spindle microtubules and are generally labeled cells, white blood cells, serum, and other fluids is
using auto-immune CREST area collected
b. A karyotype will be done on the white blood cells
OTHER BANDING TYPE TECHNIQUES which are actively dividing (a state known as mitosis)
c. During pregnancy, the sample can either be
a. DISTAMYCIN-A/ DAPI STAINING
amniotic fluid collected during an amniocentesis or a
o This staining method identifies heterochromatic
piece of the placenta collected during a chorionic villi
regions found on chromosomes 1, 9, 15, 16 and Y
sampling test (CVS)
d. The amniotic fluid contains fetal skin cells which are
b. AGNOR STAINING FOR SATELLITE REGIONS
used to generate a karyotype
(SILVER STAINING OF NUCLEAR ORGANIZER
REGIONS)
2. Transportation to the Laboratory
o This is used to locate nucleolar organizer regions
a. Karyotypes are performed in a specific laboratory
on chromosomes This technique is also useful for
called a cytogenetics lab
studies of chromosomes with double satellites,
chromosome polymorphism, and structural
3. Separation of the cells
abnormalities
a. In order to analyze chromosomes, the samples must
contain cells that are actively dividing
c. NON-BANDING
b. In blood, the white blood cells actively divide. Most
o This chromosome banding technique uses
fetal cells actively divide as well
nucleoprotein. Typically, 20 metaphases are
c. Once the sample reaches the cytogenetics lab, the
stained by non-banding and analyzed for minor
non-dividing cells are separated from the dividing
anomalies such as chromatid breaks and gaps,
cells using special chemicals
major anomalies such as, acentric fragments and
dicentric chromosomes, and radial configurations
4. Growing of cells
such as, triradialis, quaradialis, and complex radial
a. In order to have enough cells to analyze, the dividing
formations
cells are grown in a special media or a cell culture
b. This media contains chemicals and hormones that
KARYOTYPING
enable the cells to divide and multiply
a. A laboratory procedure where it allows the doctor to
c. This process of culturing can take three or four days
examine a set of chromosomes
for blood cells, and up to a week for fetal cells
b. A ‘karyotype’ refers to the actual collection of
chromosomes being examined
5. Synchronizing of cells
c. A karyotype test examines the dividing cells. The pairs of
a. Chromosomes are a long string of human DNA. In
chromosomes are arranged by their size and appearance
order to see chromosomes under a microscope,
d. Karyotyping can be used to detect a variety of genetic
chromosomes have to be in their most compact form
disorders
in a phase cell division (mitosis) known as
e. Examining chromosomes through karyotyping allows the
metaphase
doctor to determine whether there are any abnormalities
or structural problems within a patient’s chromosomes
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6. Releasing of chromosomes from their cells 11. The final result of karyotyping
a. In order to see these compact chromosomes under a. In the end, the final karyotype shows the total
a microscope, the chromosomes have to be out of number of chromosomes, the sex, and any structural
the white blood cells abnormalities with individual chromosomes
b. This is done by treating the white blood cells with a b. A digital picture of the chromosomes is generated
special solution that causes them to burst with all of the chromosomes arranged by number
c. This is done while the cells are on a microscopic slide
d. The leftover debris from the white blood cells is CONDITIONS DIAGNOSED WITH A KARYOTYPE TEST
washed away, leaving the chromosomes stuck to the a. Karyotypes can be used to screen for and confirm
slide chromosomal abnormalities such as Down’s syndrome
and Cat Eye Syndrome, and there are several different
7. Staining of the chromosomes types of abnormalities which may be detected
a. Chromosomes are naturally colorless. In order to tell b. Chromosomal abnormalities
one chromosome from another, a special dye called • TRISONOMIES in which there are 3 copies of one of
Giemsa dye is applied to the slide the chromosomes rather than 2.
b. Giemsa dye stains regions of chromosomes that are o Examples of trisonomies include:
rich in the bases adenine (A) and thymine (T). When ▪ Down Syndrome (trisomy 21)
stained, the chromosomes look like strings with light ▪ Edward Syndrome (trisomy 18)
and dark bands ▪ Patau Syndrome (trisomy 13)
c. Each chromosome has a specific pattern of light and ▪ Klinefelter’s Syndrome (XXY and
dark bands which enable the cytogeneticist to tell other variations) Klinefelter’s
one chromosome from another. Each dark or light Syndrome occurs in 1 in 500
band encompasses hundreds of different genes newborn males
▪ Triple X Syndrome (XXX)
8. Analysis of the chromosomes • MONOSOMIES in which only one copy (instead of 2)
a. Once chromosomes are stained, the slide is put is present
under the microscope for analysis o Examples of monosomies include:
b. A picture is then taken of the chromosomes. ▪ Turner Syndrome (Xo) or
c. By the end of the analysis, the total number of monosomy X – Roughy 10% of
chromosomes will be determined and the first trimester miscarriage are due
chromosomes will be arranged by size to the Turner syndrome, but this
monosomy is present in only
9. Counting of the chromosomes around 1 in 2,500 live female
a. The first step of the analysis is the counting of the births
chromosomes • CHROMOSOME DELETIONS in which a part of one
b. Most humans have 46 chromosomes chromosome is missing
c. People with down syndrome have 47 o Examples of chromosomal deletions
chromosomes include:
d. It is also possible for people to have missing ▪ Cri-du-cha syndrome (missing
chromosomes, more than one extra chromosome, or chromosome 5)
a portion of a chromosome that is either missing or ▪ Williams syndrome (missing
duplicated (Chromosomal Abnormalities) chromosome 7)
e. By looking at just the number of chromosomes, it is
• CHROMOSOME TRANSLOCATIONS in which a
possible to diagnose different conditions including
part of one chromosome is attached to another
Down syndrome
chromosome
o Translocations:
10. Checking the structure of the chromosomes
▪ There are many examples of
a. After determining the number of chromosomes, the
translocation including
cytogeneticist will start sorting the chromosomes
translocation Down Syndrome
b. To sort the chromosomes, a cytogeneticist will
o Robertsonian translocation are fairly
compare chromosome length, the placement of
common, occurring in roughly 1 in 1000
centromeres (the areas where the two chromatids
people
are joined), and the location and sizes of G-bands
o Mosaicism is a condition in which some
c. The chromosome pairs are numbered from largest
cells in the body have a chromosomal
(number 1) to smallest (number 22)
abnormality while others do not.
d. There are 22 pairs of chromosomes, called
▪ For example, mosaic Down
autosomes, which match up exactly
syndrome or mosaic trisomy 9.
e. There are also the sex chromosomes, females have
▪ Full trisomy 9 is not compatible
two X chromosomes while males have an X and a Y
with life, but Mosaic trisomy 9
may result in a live birth.

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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.
o The chromosomes for FISH don’t need to
undergo mitosis which makes it versatile.
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:
a. Make a probe complementary to the known
sequence.
b. When making the probe, label it with a fluorescent
marker, e.g., fluorescein, by incorporating
nucleotides that have the marker attached to them.
c. Put the chromosomes on a microscope slide and
denature them.
d. Denature the probe and add it to the microscope
slide, allowing the probe hybridize to its
complementary site.
e. Wash off the excess probe and observe the
chromosomes under a fluorescent microscope. The
probe will show as one or more fluorescent signals
in the microscope, depending on how many sites it
can hybridize to.
KENNY. LOPEZ. PANOREL. PISCADERO. ROLLENAS. ROMA. SANICO. SO. SULAPAS. TAMAYO. VERGARA. VILLAROYA
This image shows us how the FISH method works.
1. DNA unmasking
2. Probe and target denaturation
3. Probe-target DNA hybridization
4. Detection – it binds with the fluorochrome antibody
or fluorescein.
5. Image analysis on fluorescent microscope –
because the sample fluoresces, the laboratory
technician can see it clearly.
WHAT IS FISH USED FOR?
a. FISH is widely used for several diagnostic applications:
o Identification of numerical and structural
abnormalities
o Characterization of marker chromosomes
o Monitoring the effects of therapy
o Detection of minimal residual disease
o Tracking the origin of cells after bone marrow
transplantation
o Identification of regions of deletion or amplification
o Detection of chromosome abnormalities in non-
dividing or terminally differentiated cells
o Determination of lineage involvement of clonal
cells
b. FISH is also used to compare the genomes of two
biological species due to deduce evolutionary
relationships
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.
• Centromeric = middle
• Researchers use these probes to determine whether
an individual has the correct number of
chromosomes.
• 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.
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 one chromosome is
attached to the end of another chromosome.
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