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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
[email protected]
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
DNA Extraction & Purification:
Key Steps
Lysis of the cells
Removal of contaminants
Proteins
RNA
Other macromolecules
Concentration of purified DNA (if required)
DNA Extraction & Purification:
The Standard Method-1
(1) Lysis of cells:
Lysis buffer: SDS and/or 8.0 M urea
(2) Removal of contaminants:
Proteinase K
Phenol:chloroform extraction
(3) Concentration of DNA:
Ethanol precipitation
DNA Extraction & Purification:
The Standard Method-2
Phenol:chloroform:isoamyl 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
DNA Extraction & Purification:
The 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
DNA Extraction & Purification:
The 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
DNA Extraction & Purification:
The 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
DNA Extraction & Purification:
The Standard Vs other Methods
Key Differences:
Sample:
Type
Quantity/volume
Requirement:
Time for extraction
Equipments and reagents
Cost/sample
DNA:
Yield & conc.
Purity
DNA Extraction & Purification:
QIAamp 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
DNA Extraction & Purification:
QIAamp DNA mini kits-2
Advantages
 Sample: versatile
 Simple and rapid
 No use of organic solvents
 Pure and concentrated DNA
DNA Extraction & Purification:
Evaluation
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