The document outlines various cytogenetic staining techniques essential for visualizing chromosomes and identifying abnormalities, including Giemsa staining (g-band), reverse banding (r-band), C-banding, fluorescence staining (FISH), silver staining, Q-banding, and DAPI staining, each with distinct methodologies and applications. It also discusses the process of protein synthesis, gene regulation mechanisms, the significance of molecular biology in fields like medicine and agriculture, and the nature of mutations along with their types and effects on genetic material. Additionally, it details the differences between germline and somatic mutations and various mutation types and their consequences.

1. Garmin Technical Vocational Institute
Department of Medical Laboratories Technology
Lecture -3
2024-2025
Cytogenetic

2. Staining is a foundational technique in practical cytogenetics, used to visualize
chromosomes, identify structural abnormalities, and detect chromosomal disorders.
Classification of chromosome banding
1. Giemsa Staining (G-banding)
Purpose: The most widely used technique to create a distinct banding pattern on
chromosomes, which aids in identifying individual chromosomes and detecting
structural abnormalities.
Method: Chromosomes are treated with trypsin (to digest proteins partially) and stained
with Giemsa dye, producing a pattern of dark and light bands unique to each
chromosome.
Applications: Karyotyping, detecting deletions, duplications, translocations, and
aneuploidies

3. 2. Reverse Banding (R-banding)
Purpose: Produces banding patterns that are essentially the reverse of G-
banding, helpful in identifying telomeric regions and for chromosomes with
difficult-to-distinguish bands.
Method: Chromosomes are heat-treated before staining with Giemsa,
resulting in a banding pattern that highlights the regions not visualized in G-
banding.
Applications: Used for fine analysis of chromosomal ends, detecting sub-
microscopic deletions, and verifying certain chromosomal abnormalities.

4. 3. C-banding
Purpose: Stains the centromeric and heterochromatic regions of
chromosomes, useful in identifying centromere position and polymorphisms
in heterochromatic regions.
Method: Chromosomes are treated with alkali, which denatures the DNA
in non-centromeric regions, then stained with Giemsa.
Applications: Identifying centromeric regions, studying chromosomal
polymorphisms, and detecting large-scale structural variations.

5. 4. Fluorescence Staining and FISH (Fluorescence In Situ Hybridization)
Purpose: Allows for visualization of specific DNA sequences on
chromosomes using fluorescent probes, enabling high precision in identifying
genetic abnormalities.
Method: Fluorescently labelled DNA probes bind to specific chromosomal
regions or genes under microscopic observation.
Applications: Detecting microdeletions, translocations, aneuploidy, and
gene-specific abnormalities in prenatal and cancer diagnostics.

6. 5. Silver Staining for Nucleolar Organizing Regions (NOR-staining)
Purpose: Stains nucleolar organizing regions (NORs) on chromosomes,
highlighting regions associated with ribosomal RNA (rRNA) synthesis.
Method: Chromosomes are treated with silver nitrate, which binds to
NOR-associated proteins, staining NORs as black or dark brown regions.
Applications: Studying NORs in various species, differentiating
acrocentric chromosomes, and identifying specific rRNA synthesis regions.

7. 6. Q-banding (Quinacrine Staining)
Purpose: One of the earliest banding techniques, uses fluorescent dyes to
stain chromosomes and is used in identifying certain chromosomal regions.
Method: Quinacrine dye binds to AT-rich regions of DNA, which
fluoresces under UV light, giving each chromosome a unique banding
pattern.
Applications: Used in early cytogenetic studies, particularly helpful in
identifying Y chromosomes due to its distinct fluorescence pattern.

8. 7. DAPI Staining
Purpose: DAPI is a fluorescent stain that binds strongly to A-T rich
regions of DNA and is widely used to visualize nuclear material.
Method: Cells are treated with DAPI, which fluoresces under UV light,
making it easy to visualize chromosomes in interphase and metaphase.
Applications: Often used in combination with FISH, as a counterstain in
karyotyping and cell cycle studies.

9. protein
The process of protein synthesis involves two main stages: transcription and translation. During
transcription, DNA is converted into mRNA, which is then translated into a polypeptide chain that
folds into a functional protein.
Proteins are polymers of amino acids. peptide bonds link these amino acids together to form
peptides and later these peptides are modified to form proteins.
•Proteins are widely used in cells to serve diverse functions. Some proteins provide the structural
support for cells while others act as enzymes to catalyze certain reactions.

10. Gene Regulation Mechanisms
Gene regulation is essential for controlling gene expression. Mechanisms such as promoters,
enhancers, and repressors interact with DNA to regulate when and how much of a gene is expressed,
ensuring proper cell function.
Modern molecular biology employs various techniques such as PCR, gel electrophoresis, and
CRISPR for genetic manipulation. These techniques allow scientists to analyze and modify DNA and
genes, leading to significant advancements in biotechnology.
Applications of Molecular Biology
Molecular biology has vast applications in fields such as medicine, agriculture, and forensics. It aids
in the development of gene therapies, genetically modified organisms, and techniques for DNA
analysis in criminal investigations.

11. MUTATION
 Mutation is a change in genetic material.
 Mutation are the result of error during the DNA replication process/ error during DNA repair.
Some types of mutations are known to be caused by certain chemicals and ionizing radiation UV.
 Induced mutations caused by mutagens; Radiation, Chemicals, or Viruses.
 Spontaneous mutations that arise naturally and not as a result of exposure to mutagens.
Small-scale mutation: (Point mutation): Insertion or Deletion.
Large-scale mutation: Amplifications or Deletions of large chromosomal regions.
Germline mutation is a heritable change in the DNA.
It occurs in a germ cell and is incorporated in every cell of the body.
Can be transmitted to the next generation (genetic diseases)
Somatic mutation: Occurs in any of the cells of the body except germ cell (Can not transmitted to
the next generation).

12. Gain of function mutations (Often called a neomorphic mutation) gains a new and abnormal
function .
Loss of function mutations
Point Mutation:
Transition: the replacement of a base by the
other base of the same chemical category
(purine/purine; pyrimidine/ pyrimidine
Transversion: the replacement of a base
of one chemical category by a base of the other
(pyrimidine/ purine or purine/ pyrimidine)

13. Effect of Point Mutation
 Silent mutation: Single substitution mutation when
the change in the DNA base sequence results
in a new codon still coding for the same ammo acid.
 Missense mutation: One triplet codon altered,
results in one wrong codon and one wrong ammo acid.
1. Acceptable missense: this occurs when a single base
change results replacement of one A.A by another
With rather the same function
2. Nonacceptable missense: this occurs when a single
base change results in the replacement of one ammo acid
with another with a completely different function
 Nonsense mutation: Change a codon that specifies an
 ammo acid into a termination codon.