Eukaryotic DNA Isolation: Principle, Materials, Procedure and Results

What is DNA Extraction?

Deoxyribonucleic acid (DNA) extraction is the process by which DNA is separated from proteins, membranes, and other cellular components present inside a cell from which it is recovered.

In eukaryotic cells (such as human and plant cells), DNA is organized into chromosomes and enclosed within a membrane-bound organelle called the nucleus. These cells also possess:

• A lipid bilayer outer membrane
• Cytoplasm containing proteins, sugars, lipids, and inorganic ions
• Other membrane-enclosed organelles

Therefore, isolating DNA from eukaryotic cells requires breaking multiple protective barriers.

Basic Steps of Eukaryotic DNA Isolation

The isolation of DNA from eukaryotic cells involves three essential steps:

1. Lysis (breaking open the cell and nucleus)
2. Precipitation (separating DNA from other biomolecules)
3. Purification (obtaining clean DNA free from contaminants)

Principle of Eukaryotic DNA Isolation

The first and most crucial step in DNA isolation is cell lysis, during which the cell membrane and nuclear membrane are disrupted to release DNA.

Methods of Cell Lysis

Cell lysis can be achieved by:

Mechanical disruption
(using a tissue homogenizer (small blender), mortar and pestle, or cutting tissue into small pieces)

Chemical and enzymatic lysis
(using detergents and enzymes like Proteinase K (enzyme that digests proteins))

Extraction Buffer Used

An extraction buffer is commonly used and contains:

• 50 mM Tris (pH 8)
• 25 mM EDTA
• 200 mM NaCl
• 1% SDS

Each component has a specific role:

Tris buffer
Maintains a stable pH, which is essential for DNA stability.

EDTA (Ethylenediaminetetraacetic acid)
Binds divalent metal ions (Ca²⁺, Mg²⁺, Mn²⁺) (metal ions needed by DNases), preventing:

• DNA degradation by DNases (DNA-cutting enzymes)
• Formation of salts with the *phosphate (PO₄³⁻) group of DNA
• DNA precipitation It also destabilizes the cell membrane.

NaCl (Sodium chloride)
Helps:

• Loosen the cell wall
• Increase DNA solubility and stability
• Shield the negative phosphate groups of DNA, allowing DNA strands to come closer and precipitate efficiently in alcohol

SDS (Sodium dodecyl sulfate)
An anionic detergent that:

Disrupts ionic interactions between proteins

Breaks down cell membranes by emulsifying lipids and proteins

Causes proteins and lipids to precipitate out of solution

DNA Precipitation

• DNA is soluble in detergent solution
• DNA is insoluble in alcohol
• Adding cold absolute ethanol or isopropanol causes DNA to precipitate, while other components remain dissolved
• DNA appears as white thread-like strands

Materials and Reagents Required for Eukaryotic DNA Isolation

1. 5 mL microcentrifuge tubes
2. Water bath (80°C)
3. Isopropanol (room temperature)
4. 70% ethanol (room temperature)
5. Lysis solution
6. RNase solution (removes RNA)
7. Protein precipitation solution
8. Absorbent paper

Procedure of Eukaryotic DNA Isolation

1. Depending on the sample (animal cell or plant cell), perform cell lysis
2. Add 3 µL of RNase solution to the lysate
3. Invert the tube 2–5 times to mix
4. Incubate at 37°C for 15–30 minutes
5. Cool to room temperature for 5 minutes
6. Add 200 µL of protein precipitation solution
7. Vortex at high speed for 20 seconds
8. Chill the sample for 5 minutes
9. Centrifuge at 13,000–16,000 × g for 4 minutes
10. A white protein pellet forms; carefully remove the supernatant
11. Transfer the supernatant to a 1.5 mL microcentrifuge tube
12. Add 600 µL of room temperature isopropanol
13. Gently invert the tube to mix
14. White thread-like DNA strands become visible
15. Centrifuge at 13,000–16,000 × g for 1 minute
16. Decant the supernatant after a small white DNA pellet forms
17. Add 100 µL of 70% ethanol
18. Invert several times to wash the DNA
19. Centrifuge at 13,000–16,000 × g for 1 minute
20. Aspirate the ethanol carefully
21. Invert the tube on clean absorbent paper
22. Air-dry the DNA pellet for 10–15 minutes
23. Add 100 µL of DNA rehydration solution
24. Incubate at 65°C for 1 hour (rehydration of DNA)
25. Store DNA at 2–8°C

DNA Quantification by Spectrophotometry

• DNA concentration is determined by measuring absorbance using a spectrophotometer
• DNA absorbs UV light at 260 nm
• Proteins absorb UV light at 280 nm
• Purity of DNA is assessed using the A260/A280 ratio

Interpretation

• Pure DNA → 1.8
• DNA contaminated with protein → < 1.8

Expected Results of Eukaryotic DNA Isolation

• White thread-like strands or a white DNA pellet
• Clear separation of DNA from proteins and other cellular debris