dna: extraction & purification by amr s. moustafa, m.d.; ph.d. assistant prof. & consultant,...
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 contaminantsProteinsRNAOther 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 KPhenol:chloroform extraction
(3) Concentration of DNA:Ethanol precipitation
DNA Extraction & Purification:
The Standard Method-2Phenol:chloroform:isoamyl alcohol mixture:
Denatures proteinsSolubilizes denatured proteins
Denatures proteinsStabilizes the aqueous/organic boundary
Isoamyl alcohol:Separation of aqueous/organic phasesPrevents 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-4Ethanol precipitation:
Concentration DNA solutionsRemoval of residual organic solventsDNA-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, precipitationand 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 MethodsKey Differences:
Sample: Type Quantity/volume
Requirement:Time for extractionEquipments and reagentsCost/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-2Advantages
Sample: versatile
Simple and rapid
No use of organic solvents
Pure and concentrated DNA
DNA Extraction & Purification:Evaluation
DNA purity: A260/A280 ratio1.7 – 1.9
DNA concentration (μg/ml):A260 X 50
DNA yield:DNA conc. X Total volume of DNA solution