DNA: Extraction

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DNA Extraction Purification
By
Amr S. Moustafa, M.D. Ph.D.
Assistant Prof. Consultant, Medical
Biochemistry Dept. College of Medicine,
KSU amrsm_at_hotmail.com
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Objectives

• The principle of standard method

• The comparison of the standard Vs other methods

• The experimental protocol for DNA purification

• The evaluation of the purification procedure

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DNA Extraction Purification Key Steps
Lysis of the cells
Removal of contaminants Proteins RNA Other
macromolecules
Concentration of purified DNA (if required)
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DNA Extraction PurificationThe Standard
Method-1
(1) Lysis of cells Lysis buffer SDS and/or 8.0
M urea
(2) Removal of contaminants Proteinase
K Phenolchloroform extraction
(3) Concentration of DNA Ethanol precipitation
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DNA Extraction PurificationThe Standard
Method-2
Phenolchloroformisoamyl alcohol mixture
Phenol
Denatures proteins Solubilizes denatured proteins
Chloroform
Denatures proteins Stabilizes the aqueous/organic
boundary
Isoamyl alcohol Separation of
aqueous/organic phases Prevents foaming upon
vortexing
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DNA Extraction PurificationThe Standard
Method-3
DNA is found in the upper aqueous layer
Exceptions high salt content, the aqueous
phase forms lower layer If phenol is not
equilibrated, DNA in the organic phase
Tris-buffered, redistilled phenol, pH 8.0
Oxidation products can damage DNA chains
Addition of 8-hydroxyquinolone antioxidant
yellow color of phenol
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DNA Extraction PurificationThe Standard
Method-4
Ethanol precipitation Concentration DNA
solutions Removal of residual organic
solvents DNA-free solutes

• Ethanol induces a structural transition in DNA
• precipitation
• High monovalent cation conc. (0.1 0.5 M)
• Most salts are soluble in 70 ethanol,
  precipitation

and washing desalt DNA
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DNA Extraction PurificationThe Standard
Method-5
Disadvantages

• Time-consuming
• Hazardous organic solvents
• Residual amounts of organic solvents interfere
• with accurate measurement of DNA conc.
• with enzymatic manipulations of DNA

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DNA Extraction PurificationThe Standard Vs
other Methods
Key Differences
Sample Type Quantity/volume Requirement Ti
me for extraction Equipments and
reagents Cost/sample DNA Yield
conc. Purity
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DNA Extraction PurificationQIAamp DNA mini
kits-1
(1) Lysis of cells (2) Spin column DNA
adsorption to silica-gel membrane (3)
Two-different wash steps Removal of
contaminants (4) Elution Pure and concentrated
DNA
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DNA Extraction PurificationQIAamp DNA mini
kits-2
Advantages

• Sample versatile
• Simple and rapid
• No use of organic solvents
• Pure and concentrated DNA

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DNA Extraction PurificationEvaluation
DNA purity A260/A280 ratio 1.7 1.9
DNA concentration (µg/ml) A260 X 50
DNA yield DNA conc. X Total volume of DNA
solution