2. Comet assay
• The Comet Assay, also known as single-cell gel
electrophoresis,
• is a versatile and sensitive technique used to assess DNA
damage in individual cells.
• Developed by Swedish scientist Per E. Olsson in 1984, the
assay has revolutionized genotoxicity and DNA damage
research.
• The Comet Assay's applications span diverse scientific
domains, contributing to our understanding of DNA damage and
its implications across various contexts.
• Its adaptability continues to make it a valuable tool for research,
regulation, and risk assessment.
3. Comet assay principle
• DNA Strand Breaks and
Migration:
• When DNA is damaged, such as
by genotoxic agents or other
factors, it can result in single-
strand breaks (SSBs) or double-
strand breaks (DSBs).
• Damaged DNA fragments,
especially if they have lost their
normal structural integrity,
become more susceptible to
migration during
electrophoresis.
• damaged DNA fragments
migrate away from the nucleus
during electrophoresis, creating
a distinctive pattern resembling
a comet.
4. Comet assay types
• 1- Neutral Comet Assay:
• Performed under neutral conditions (pH 7-8), preserving DNA strand
integrity.
• Primarily detects double-strand breaks and cross-links.
• 2- Alkaline Comet Assay (pH > 13):
• This is the most widely used variation of the Comet Assay.
• Detects a wide range of DNA damage, including single-strand breaks,
double-strand breaks, and alkali-labile sites.
• Alkaline conditions cause DNA strands to unwind and reveal damage
more effectively.
• Offers sensitivity to a variety of genotoxic agents, including ionizing
radiation, chemicals, and environmental pollutants.
5. Comet assay application
1. Genotoxicity Assessment
•Evaluate the potential of chemicals, drugs, and environmental agents to induce DNA
damage and mutations.
•Used in regulatory toxicology for safety assessment of new compounds.
2. Environmental Monitoring
•Assess the impact of pollutants, radiation, and contaminants on DNA integrity in organisms
and ecosystems.
•Provides insights into the genotoxic effects of pollutants on aquatic and terrestrial
organisms.
3. Clinical Research
•Monitor DNA damage in patient samples to evaluate disease progression and treatment
response.
•Aid in cancer research by assessing DNA damage in tumor cells and predicting therapeutic
outcomes.
4. Personalized Medicine
•Assess an individual's DNA damage susceptibility and response to treatments.
•Guide treatment strategies by identifying patients with higher risk of treatment-related DNA
damage.
6. Comet assay application
5. Occupational Health
•Evaluate DNA damage in workers exposed to potentially hazardous
substances.
•Assist in identifying workplace environments that may pose genotoxic risks to
employees.
6. Radiation Studies
•Analyze DNA damage caused by ionizing and non-ionizing radiation.
•Assess radiation-induced genotoxicity and its implications for health.
7. Pharmaceutical Development
•Screen potential drug candidates for genotoxic effects during preclinical stages.
•Optimize drug formulations to minimize DNA damage risk in patients.
8. Food Safety
•Assess the genotoxic potential of food additives, contaminants, and processing
methods.
•Contribute to ensuring the safety of the food supply chain.
9. Ecotoxicology
•Study the impact of pollutants on DNA integrity in wildlife and organisms in their
natural habitats.
•Provide insights into environmental health and conservation efforts.
7. Procedure outline
• 1- cells suspension
• 2- slide precoating
• 3-Embedding in
Agarose Gel:
• 4- Cell Lysis:
• 5- Electrophoresis
• 6- Staining and
Visualization
• 7- Analysis
• 8- Quantification of
DNA Damage:
8. 1- Free cells preparation
• WBC can be prepared from blood by RBC lysis
• bone marrow are ready to use
• Cultured cells are ready to use
• Cells in tissues have to dispersed mechanically or by enzyme treatment
• Count the cells
• Cell count have to be around 10^5/ ml
9. 2- slides pre-coating
1- clean the slide with ethanol
( optional )
2- Melt 1% ordinary agarose in
water by microwave
3- Prepare agarose-precoated
slides by dipping the slides into
molten 1% agarose and wiping
one side clean.
4- Allow agarose to air-dry to a
thin film. Slides can be prepared
ahead of time and stored with
desiccant
10. 3- Cell on slide
adjust cell density to about
2 × 104 cells/ml in phosphate-
buffered saline lacking divalent
cations.
mix 0.4 ml of cells with 1.2 ml
1% low-gelling-temperature
agarose at 40 °C.
Mix and rapidly pipet 1.2 ml of
cell suspension onto pre-
coated slide.
Allow agarose to gel for about
2 min
11. 4- Cell lysis
• Alkaline lysis : submerge slides
in a covered dish containing lysis
solution. Handle slides gently
• alkaline lysis solution
• 1.2 M NaCl,
• 100 mM Na2EDTA,
• 0.1% sodium lauryl
sarcosinate, ( or tritonX100
1% )
• 10% dimethyl sulfoxide
• 0.26 M NaOH (pH > 13);
• equilibrate at 4 °C.
• ▲ CRITICAL Prepare fresh
on day of experiment.
• Lyse samples overnight (18−20
h) at 4 °C or 1 h in the dark.
12. 5- Electrophoresis
- After 1-h or overnight lysis, carefully remove
slides and submerge in room temperature (18−25
°C) in electrophoresis solution for 20 min.
- Repeat two times to ensure removal of salt and
detergent. Take care not to allow DNA to
renature even briefly
• After these three rinses, submerge slides in
electrophoresis tank consistent volume of
electrophoresis buffer that is about 1–2 mm above
the top of the agarose
• Electrophoresis buffer
• 0.03 M NaOH and 2 mM Na2EDTA,
• pH 12.3,
• To prepare 250 ml
• 0.3 g naoh
• 0.168 g na2edta
• Conduct electrophoresis 25 min at a voltage of
0.6 V/cm.
• The current should be about 40 mA if using20 V.
13. 6- Slide Staining
• Remove slides from electrophoresis chamber and rinse and neutralize in
400 ml of distilled water.
• Place slides for 20 min in staining solution containing 2.5 µg/ml of :
• propidium iodide.
• acridine orange.
• Sybr green
• Eva green
• Ethidium bromide
• Rinse slides with 400 ml distilled water to remove excess stain.
• Let the slide dry at room temperature
14. Image analysis and Scoring
• Image analysis involves
capturing digital images (50
comet )
• using specialized flourescnese
microscopy techniques.
• Software tools are then
employed to quantify the
extent of DNA damage. (comet
score )
• Key parameters include the
comet tail length (distance of
migrated DNA), tail intensity
(fluorescence intensity in the
tail)
• Comet tail DNA % ; which
represent the ratio of DNA in
the tail comparing to the DNA
ratio in head
15. Scoring
• Scoring typically
involves categorizing
comets into different
classes based on their
characteristics, ranging
from undamaged (no
tail) to highly damaged
(long tail).
