1. DNA extraction

2. DNA is building block of the life for all living creatures.
Every thing from bacteria to human has DNA in their cellular stucture.
Every thing living contain DNA
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 has characteristic melting & annealing temperatures

5. Why extract DNA?
The isolation of the DNA from biological sample is an essential step in the DNA technology (PCR - RFLP- cloning - hyberdization all this approaches require DNA as template
• disease diagnosis
• DNA sequencing
• genetically modified organisms (GMO) - agriculture,
pharmaceutical

6. 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

7. How to isolate DNA?
They are many types of methods to isolate DNA depending on the type of the sample and the purpose from extraction (organic method (phenol – chloroform )-chlex –methanol - commercial kit (FTA)
In genral all type of methods are following three coral steps
1-lyses cell wall by lyseis buffer
2-precipitation of proteins
3- precipitation of DNA

8. 1-Breaking the cells open, commonly referred to as cell disruption, to expose the DNA within.
2-Removing membrane lipids by adding a detergent.

9. 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)

10. 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.

11. 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

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

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

14. 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.

15. 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.

16. Ethanol precipitation

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

18. 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

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

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

22. Evaluation of Nucleic Acids
Spectrophotometrically
• quantity
• quality

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