In order to Study DNA, it must be isolated (extracted)

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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
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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.
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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
Inside of Cell
Proteins
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(Also called Sodium dodecyl sulfate (SDS))
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The detergent dissolves the membrane by replacing
the lipid bilayer with the hydrophobic portions
of the detergent molecule.
Well use SDS
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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.
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How do we denature the DNAse enzymes?
One of Three methods is commonly used (all of
these methods break hydrogen bonds so that the
proteins shape is changed, thus making it
non-functional)

• A different type of enzyme called a protease,
  since enzymes are a type or protein (proteases
  dont break themselves down.)
• Heat (we cook food to denature the proteins.)
• 3) Change the pH use acid or base

Well use HEAT
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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.
Well use Ethanol ice cold to decrease DNAs
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.)
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Review- 3 steps

• 1) cell lysis
• 2) denature DNAses
• 3) precipitate DNA (isolate it from the lysate)

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Well use a rod to spool it up out of the solution
DNA
Ethanol
Alcohol/Aqueous interface
Wheat Germ lysate