2. DNA Extraction And Purification BY Dr. Naglaa Fathy Lecturer of Biochemistry and Molecular Biology, faculty of medicine, Benha university Benha university2008

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

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

5. Lysis buffer  Lysis buffer 50 mM Tris-HCI, pH 8.0 to maintain the pH of the solution at a level where DNA is stable 50 mM Tris-HCI, pH 8.0 to maintain the pH of the solution at a level where DNA is stable  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)

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

7. Adding salt  The addition of NaCI 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

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

9. DNA Extraction & Purification: Key Steps  Lysis of the cells  Removal of contaminants ProteinsRNA Other macromolecules Concentration of purified DNA (if required)

10. Standard Protocol for DNA Extraction  Organic solvent  Salting out  Cation Exchange Resins

11. The Standard Method-1  Lysis of cells: Lysis buffer: SDS and/or 8.0 M urea  Removal of contaminants: Proteinase K Phenol:chloroform extraction  Concentration of DNA: Ethanol precipitation

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

13. 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

14. 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

15. 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:

16. 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

17. 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

18. DNA Extraction & Purification: QIA amp DNA mini kits-2 Advantages Simple and rapid No use of organic solvents Sample: versatile Pure and concentrated DNA

19. 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