DNA Extraction And Purification BY Dr Naglaa Fathy

DNA Extraction And Purification BY Dr. Naglaa Fathy Lecturer of Biochemistry and Molecular Biology , faculty of medicine , Benha university 2008

Structure of the cell • Plasma membrane and membranes of organelles nuclear envelope included • DNA located in nucleus • A lot of proteins around • Mitochondrial DNA

Extraction of genomic DNA Ø Cell collection Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents Ø Digest sample with protease to degrade proteins Ø Precipitate DNA with cold alcohol in high salt Ø

Lysis buffer 50 m. M Tris-HCI, p. H 8. 0 to maintain the p. H of the solution at a level where DNA is stable Ø 1% SDS to break open the cell and nuclear membranes, allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins, making them more susceptible to protease cleavage) Ø

Why add protease? Protease destroys nuclear proteins that bind DNA and cytoplasmic enzymes that breakdown and destroy DNA Ø Protease treatment increases the amount of intact DNA that is extracted Ø

Adding salt Ø Ø Ø • The addition of Na. CI allows the DNA molecules to come together instead of repelling each other, thus making it easier for DNA to precipitate out of solution when alcohol is added • Na+ ions bind to the phosphate groups of DNA molecules, neutralizing the electric charge of the DNA molecules • Our protease solution already contains salt

Precipitation of DNA does not dissolve in alcohol. Addition of cold alcohol makes the DNA clump together and precipitate out of solution Ø Ø Precipitated DNA molecules appear as long pieces of fluffy, stringy, web-like strands. Ø Microscopic oxygen bubbles “aggregate”together, as the DNA precipitates. Ø The larger, visible air bubbles “lift” the DNA out of solution, from the aqueous into the organic phase Ø The DNA in the glass vial can last foryears

DNA Extraction & Purification: Key Steps Ø Lysis of the cells Ø Removal of contaminants Proteins RNA Other macromolecules Concentration of purified DNA (if required)

Standard Protocol for DNA Extraction Organic solvent Ø Salting out Ø Cation Exchange Resins Ø

The Standard Method-1 Ø Lysis of cells: Lysis buffer: SDS and/or 8. 0 M urea Ø Removal of contaminants: Proteinase K Phenol: chloroform extraction Ø Concentration of DNA: Ethanol precipitation

Phenol: chloroform: isoamyl alcohol mixture: Phenol : - Denatures proteins - Solubilizes denatured proteins Chloroform -Denatures proteins -Stabilizes the aqueous/organic boundary Alcohol -Separation of aqueous/organic - phases Prevents foaming upon vortex

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, p. H 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 Ø Ethanol induces a structural transition in DNA precipitation Ø High monovalent cation conc. (0. 1 – 0. 5 M) Ø Most salts are soluble in 70% ethanol, precipitation and washing desalt DNA

DNA Extraction & Purification: The Standard Method-5 Disadvantages: ØTime-consuming ØHazardous organic solvents ØResidual 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: QIA amp 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: QIA amp DNA mini kits-2 Advantages • Simple and rapid • No use of organic solvents • Sample: versatile • Pure and concentrated DNA

DNA Extraction & Purification: Evaluation Ø DNA purity: A 260/A 280 ratio 1. 7 – 1. 9 Ø DNA concentration (μg/ml): A 260 X 50 Ø DNA yield: DNA conc. X Total volume of DNA solution