The document outlines the principles and methods of DNA extraction, highlighting techniques such as phenol-chloroform extraction and salting out, which is noted for its cost-effectiveness and minimal contamination. It details the procedures involved, including cell lysis, purification, and the subsequent detection of DNA through gel electrophoresis, along with applications ranging from medical diagnostics to forensic science. Additionally, it provides specific methodologies and necessary reagents used in the extraction process, with references for further reading.

1. Presenter
TUBA NAFEES
M.Sc. In Biotechnology, University of Karachi
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2. PRINCIPLE OF DNA EXTRACTION
1. CELL LYSIS
2. SEPARATION OF DNA FROM
CELL DEBRIS
3. PURIFICATION OF DNA
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3. METHODS OF DNA EXTRACTION
1. Phenol-chloroform extraction
2. Magnetic bead separation
3. Precipitation chemistry (or “Salting out”).
When the ionic strength of a protein solution is increased
by adding salt, the solubility decreases, and protein
precipitates
Results in little-to-no contamination, gives high DNA yields
and is cost-effective.
ADVANTAGES
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Salt
molecules Protein
molecules

4. REAGENT SYSTEM & COLUMN
SYSTEM
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5. DNA EXTRACTION BY (Salting Out/
Precipitation Chemistry)
1. Cell lysis (To expose
DNA)
• Buffer (To maintain pH),
Detergents (To break lipids)
• PROTEASE (To break proteins)
• RNase (To degrade RNA)
CELL LYSIS
BUFFER
Use of high concentration of NaCl ( 6
M). After protein precipitation DNA
floats freely in the supernatant and is
separated from clumped cell debris
2. Precipitation of cell debris
4. Precipitation/Purification of
DNA
Isoproanol/ Ethanol precipitation,
minicolumn binding.
ADDIOTIONAL
REAGENTS
Cell lysis buffer uses NaCl (in low conc) to
precipitate cell debris ( detergents,
broken proteins, RNA , lipids etc)
3. Precipitation of Protein
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6. MATERIALS & METHOD
CHEMICALS
• CELL LYSIS BUFFER ( pH 7.6)
Tris HCl , Triton-X, Mg Cl2 , KCl , NaCl (low conc),
EDTA( Ethylenediaminetetraacetic acid).
• SDS Solution
• NaCl ( High concentration)
• Isopropanol ( Ice cold)
• 70 % Ethanol
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7. METHODOLOGY
EDTA
EDTA acts as an
“Anticoagulant” ( It
prevents blood
clotting)and is the
best choice for blood
DNA extraction than
heparin and sodium
citrate.
SAMPLE STORAGE
Within 3 days =
Refrigerate at 4 ͦC
For longer use =
Store at -80 ͦC
SAMPLE PREPARATION
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8. Blood sample
invert mixing
Cell lysis
Buffer
Centrifugation at 4000 rpm
for 10 min
Discard
supernatant
in a new
microfuge
tube
Addition of
SDS
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Cell debris

9. Incubate at
50 °C for 30
min
Addition of
NaCl
Collection of
Supernatant
Centrifugation at 4500
rpm for 15 min
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DNA
Precipitation of
Proteins

10. Addition of 95 %
ice- cold
isopropanol
Centrifugation at
12000 rpm for 4 min 10

11. Wash the pellet
with 70 %
ethanol
Centrifugation at
12000 rpm for 4 min
Air dry and
Resuspend in TE
buffer ( pH 8 )
/Pure water
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12. DETECTION OF DNA
BY GEL ELECTROPHORESIS
• The gel consists of a permeable matrix, a
bit like a sieve, through which molecules
can travel when an electric current is
passed across it.
• Smaller molecules migrate through the gel
more faster than larger fragments
• To visualise the DNA, the gel is stained
with a fluorescent dye (ethidium bromide)
that binds to the DNA, and is placed on an
ultraviolet transilluminator which will
show up the stained DNA as orange bands.
• The length of the DNA fragments is
compared to a marker containing
fragments of known length.
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DNA
MARKER
Sample DNA
BANDS
Smaller
fragment
Larger fragment

13. PURITY OF DNA
• Take absorbance of DNA at two different
wavelengths i.e. 260nm and 280nm and then
finding their ratio. DNA absorbs UV light both at
260 and 280 nm while proteins absorbs UV at
only 280nm.
• Pure DNA with no protein contamination will give
a ratio of 1.8 at 260/280. On the other hand, a
DNA with protein impurities will give lower value
than 1.8.
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14. APPLICATIONS OF DNA
EXTRACTION
Medical
Diagnostics
Genome
Sequencing
Forensic
Science
Detection of
viruses and
bacteria
Pharmaceutical
Products
Genetic
Engineering
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16. REFERENCES
• Sugna et al. (2014). DNA Isolation from Human Whole Blood
Samples by Non-Enzymatic Salting Out Method.
International Journal of Pharmacy and Pharmaceutical
Sciences, Vol 6, 198-199.
• Shahid. (2019). Genomic DNA Extraction – Principle, Steps
and Functions of Reagents. How Biotech. Retrieved from
http://howbiotech.com/genomic-dna-extraction-principle-
steps-and-functions-of-reagents
• Miller, S.A., Dykes, D.D., Polesky, H.F. (1998). A Simple
Salting Out Procedure for Extracting DNA from Human
Nucleated. Nucleic Acids Research,16(3), 1215
• Rice G.DNA Extraction. Educational Resources, Microbial
Life. Montana State University. Retrieved 17 February 2017.
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