Download DNA extraction PRESENTAION

DNA extraction
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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.
Structure of the cell
Physical Characteristics of DNA
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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
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 
Nucleic Acid Preparation
Sample Source?
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Whole blood
Buffy coat
Serum or plasma
Bone material
Buccal cells
Cultured cells
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Amniocytes or
amniotic fluid
Dried blood spots
Fresh or frozen tissue
(biopsy material)
Sputum, urine, CSF, or
other body fluids
Fixed or paraffinembedded tissue
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
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1-Breaking the cells open, commonly referred
to as cell disruption, to expose the DNA
within.
2-Removing
detergent.
membrane lipids by adding a
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Add Lysis buffer to cells to break open cell
and nuclear membranes and release nuclear
contents
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Lysis buffer
• 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)
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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.
DNA extraction – the basic concept
Process
Common procedure
Chemical
Cell lysis
Enzymatic
Mechanic
•SDS,
•CTAB
•Proteinase K
•Freezing
•Grinding
Organic solvents
•Phenol
•chloroform
Salt
•Sodium chloride
•Sodium acetate
DNA binding
•Membrane
•Beads
Protein removal
DNA precipitation
Alcohol
•Ethanol
•Iso-propanol
Cells
Extract
HOW?
Organic
extraction
Pure DNA
Phenol extraction of DNA samples
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Phenol extraction is a common technique
used to purify a DNA sample
DNA Isolation Methods
Liquid Phase Organic Extraction
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Phenol :chloroform/isoamyl alcohol
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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.
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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.
Ethanol precipitation
Disadvantages:
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
DNA extraction kit
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􀂄 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
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Chelex Extraction Method
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• More rapid than organic extraction method
• Involves few steps and fewer opportunities
for contamination
Chelex extraction
1. Put sample in tube
2. Add 5% Chelex® beads;
vortex
3. Boil at 100°C
Supernatant can be used directly
for quantitation/PCR
Evaluation of Nucleic Acids
Spectrophotometrically
 • quantity
 • quality
•gel electrophoresis
Assessment of DNA quality