DNA: Extraction & Purification By Amr S. Moustafa, M.D.; Ph.D. Assistant Prof. & Consultant, Medical Biochemistry Dept. College of Medicine, KSU

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DNA Purification & Quantitation

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Presentation transcript:

DNA: Extraction & Purification By Amr S. Moustafa, M.D.; Ph.D. Assistant Prof. & Consultant, Medical Biochemistry Dept. College of Medicine, KSU

Objectives  The principle of standard method  The comparison of the standard Vs other methods  The evaluation of the purification procedure  The experimental protocol for DNA purification

DNA Extraction & Purification: Key Steps Removal of contaminants Proteins RNA Other macromolecules Lysis of the cells 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: Denatures proteins Solubilizes denatured proteins Denatures proteins Stabilizes the aqueous/organic boundary Isoamyl alcohol: Separation of aqueous/organic phases Prevents foaming upon vortexing Phenol: Chloroform:

DNA Extraction & Purification: The Standard Method-3 Tris-buffered, redistilled phenol, pH 8.0: Oxidation products can damage DNA chains Addition of 8-hydroxyquinolone:antioxidant yellow color of phenol 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

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

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