1. DNA extraction

2. DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis.

3. Structure of the cell

4. Physical Characteristics of DNA
DNA absorbs UV light at 260 nm Allows quantitation
DNA is water soluble
DNA precipitates in alcohols
DNA carries a net negative charge
DNA subject to shear forces
DNA has characteristic melting & annealing temperatures

5. 1-Breaking the cells open, commonly referred to as cell disruption, to expose the DNA within.
2-Removing membrane lipids by adding a detergent.

6. Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents
• 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)

7. Precipitating the DNA with an alcohol — usually ethanol or isopropanol
Precipitating the DNA with an alcohol — usually ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation.

8. Nucleic Acid Preparation Sample Source?
Amniocytes or amniotic fluid
Dried blood spots
Fresh or frozen tissue (biopsy material)
Sputum, urine, CSF, or other body fluids
Fixed or paraffin-embedded tissue
Whole blood
Buffy coat
Serum or plasma
Bone material
Buccal cells
Cultured cells

9. DNA extraction – the basic concept
Process
Common procedure
Cell lysis
Protein removal
DNA precipitation
SDS,
CTAB
Proteinase K
Freezing
Grinding
Chemical
Enzymatic
Mechanic
Phenol
chloroform
Sodium chloride
Sodium acetate
Membrane
Beads
Organic solvents
Salt
DNA binding
Alcohol
Ethanol
Iso-propanol

10. Cells
Extract
HOW?
Organic extraction
Brown p.28
Pure DNA

11. Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample

12. DNA Isolation Methods Liquid Phase Organic Extraction
Phenol :chloroform/isoamyl alcohol
Since phenol and water are immiscible, two phases form - a water phase and a phenol phase.
The phases are then mixed thoroughly. This forces the phenol into the water layer where it forms an emulsion of droplets throughout. The proteins in the water phase are denatured and partition into the phenol, while the DNA stays in the water.

13. The mixture is then centrifuged and the phases separate.
The DNA-containing water phase can now be pipetted off, and the phenol/protein solution is discarded.

14. Ethanol precipitation

15. Disadvantages:
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere

16. DNA extraction kit 􀂄 Two main advantages: Saves time and
makes the process of DNA purification a
relatively easy and straightforward process.
Can handle up to 100 μg of DNA

17. Chelex Extraction Method
• More rapid than organic extraction method
• Involves few steps and fewer opportunities for contamination

18. Chelex extraction Put sample in tube Add 5% Chelex® beads; vortex
Boil at 100°C
Supernatant can be used directly for quantitation/PCR

20. Evaluation of Nucleic Acids
Spectrophotometrically
• quantity
• quality

21. Assessment of DNA quality
• gel electrophoresis
Assessment of DNA quality