DNA can be isolated from cells using various techniques. The basic steps involve lysing the cells to release DNA, separating DNA from other biomolecules using chemicals like chloroform, precipitating purified DNA using ethanol, washing away impurities, and resuspending the DNA. Assessment of DNA quantity and quality is done using gel electrophoresis and spectrophotometry. Isolated DNA has many applications including genetic studies, identifying genes for important traits, and disease diagnosis.

1. DNA Isolation Techniques: Basics &
Applications

2. DNA isolation
• DNA isolation means extracting out DNA from the cell under
laboratory conditions
• Involves procedure/ steps
• Chemicals (Buffers and solutions)
• Equipments
DNA was isolated for the first time by Friedrich Miescher in 1989.
Isolation techniques vary from samples to sample (plants, animals, microbes,
viruses)
Protocols are usually named after scientists who first described the procedure or
on the name of principle chemical component
Eg. Dellaporta method, CTAB method, SDS method

3. • Sampling (sample collection and sample preparation)
• Sample processing (DNA isolation)
• Assessment of quantity & quality of DNA
Basic steps in DNA isolation
Samples from distant sites need to be
collected in ice/ gel packs

4. Four steps are used to extract out DNA from the rest of the cell:
1. Lysis- to break up the cells
2. Separation- separating out DNA from other biomolecules
3. Precipitation- obtaining separated DNA
4. Wash- cleaning DNA
5. Resuspension- Dissolving

5. 1. Lysis
• Lysis means disruption of tissue and cells to dissociate cellular
components- chemical+ mechanical force
DNA
• Manual
• Machine aided
CTAB extraction buffer is widely used for lysis
of plant tissues
Mortar and pestle
Tissue lyzer

6. 2. Phase separation
• Involves separating DNA from proteins and carbohydrates and other
cellular debris
• Reagents such as Chloroform and iso-amyl alcohol mixture (24: 1) are
added to the cell extract and centrifuged at high speed
• It separates lipids and polysaccharides in the lower organic phase
while DNA in the upper aqueous phase
aqueous phase containing DNA
organic phase
inter phase

7. • The clear aqueuoes phase liquid is transferred to new micro tube
• Chilled ethanol is added, mixed gently and kept in freezer for precipitation
3. DNA precipitation
Centrifuged at high speed
to obtain DNA pellet at the
bottom

8. 5. Resuspension or dissolving DNA
• Isolated DNA is dissolved in TE buffer/ sterile water
• TE- Tris & EDTA, pH-8.0. Tris maintains the pH of the DNA solution while EDTA
protects it from degradation by nuleases.
• This DNA can be stored from days to months to several years under appropriate
conditions.
• The precipitated DNA is washed with 70% alcohol, dried and dissolved in buffer.
• This DNA can be stored for further use
4. Washing
Pure DNA
looks like this
Not like this

9. Buffers & Solutions
• Lysis buffer/ Extraction buffer
• Chloroform isoamyl alcohol mixture
• Alcohol (Isopropanol/ ethanol)
• TE buffer

10. • Determination of quantity
• Determination of quality (purity)
• Both quantity and quality can be assessed by agarose gel
electrophoresis
• Spectrophotometer gives more accurate readings.
• Quantity in terms of nanogram/ microliter
• Quality in terms of absorbance ratio
• (absorbance ratio of 1.8 at 260/280 nm wavelength indicates good
quality DNA)
Good
Bad
Assessment of DNA

11. In summary
Manual isolation
technique
Kit based isolation-
• Use kit components & follow
manufacturer’s instruction
• Fast (usually takes 2-2.5 hours)
• Simple
• Costly

12. Applications:
• DNA isolation is the starting point for number of genetic
studies
• To study genetic diversity
• To identify and study genes responsible for economically important
traits
• Diagnostic
• To understand genetic basis of disease and insect resistance

13. Thanks for your attention