Transcript DNA (isolate

Isolating DNA
In order to Study DNA, it must be isolated (extracted)
DNA is in all living tissues
“If there are cells, there is DNA.”
DNA can be obtained from dead cells if they are not too damaged.
Forensic pathology techniques are improving so that it is possible
to get useable DNA from many types of post-mortem tissues.
Examples: Bone marrow and tooth pulp
Dried cells are likely to have DNA that is not degraded.
Example:
Skin cells from a cigarette butt
There are many protocols for DNA extraction
Method depends on the type of tissue
For example, plants have cells walls
Some plant tissue is woody - requires extra steps
All methods include 3 basic steps.
Sometimes extra steps are added for further purification during
one or more of these steps.
STEP 1 - Cell Lysis
To lyse - to break open
Once the cell is lysed, the contents are called the cell lysate.
• Since the cell membrane is a lipid bilayer, a detergent will
dissolve the Non-polar lipid part.
• Other chemicals that break down protein will dissolve the
membrane because proteins exist in the membrane.
Cell Membrane
Outside of Cell
Hydrophilic
(Polar)
Lipid Bilayer
Hydrophobic
(Non-polar)
Polar
Proteins
Inside of Cell
(Also called Sodium dodecyl sulfate (SDS))
The detergent
dissolves
the membrane by
replacing the lipid
bilayer
with the hydrophobic
portions of the
detergent molecule.
We’ll use SDS
STEP 2 - Protein Denaturation
Of the hundreds of different types of proteins in a cell, the ones we need to
Denature are the DNAses.
DNAses are enzymes that function to degrade DNA during programmed
cell death.
In a cell that is intact (not lysed) DNAses are tightly regulated.
In a cell lysate, there is anarchy; the DNAses would chop up
any DNA they come in contact with.
How do we denature the DNAse enzymes?
One of Three methods is commonly used (all of these methods
break hydrogen bonds so that the protein’s shape is changed,
thus making it non-functional):
1) A different type of enzyme called a protease, since enzymes
are a type or protein (proteases don’t break themselves
down.)
2) Heat (we cook food to denature the proteins.)
3) Change the pH – use acid or base
We’ll use HEAT
STEP 3 – Precipitate the DNA
The DNA must be separated from the hundreds of other
types of molecules in the lysate.
Since DNA is insoluble in alcohol, adding alcohol will
allow the DNA to precipitate out of solution.
Ethanol works, isopropanol works.
We’ll use Ethanol – ice cold to decrease DNA’s solubility
At this point you can “spool’ the DNA onto a rod, or spin it in a
centrifuge so it forms a pellet of DNA at the bottom of a tube.
You can discard the supernatant liquid leaving the DNA.
(Sometimes this alcohol precipitation step is done
twice for cleaner DNA.)
Review- 3 steps
1) cell lysis
2) denature DNAses
3) precipitate DNA (isolate it from the
lysate)
We’ll use a rod to spool it up out of the solution
DNA
Ethanol
Alcohol/Aqueous
interface
Wheat Germ
lysate