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

DNA is in all living tissues “If there are cells, there is DNA.”

Isolating 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 wallsSome 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.

To lyse - to break openOnce 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.

STEP 1 - Cell Lysis

Hydrophobic(Non-polar)

Hydrophilic

(Polar)

Outside of Cell

Inside of CellProteinsPolar

Lipid Bilayer

Cell Membrane

(Also called Sodium dodecyl sulfate (SDS))

The detergent dissolvesthe membrane byreplacing the lipid bilayerwith 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 toDenature 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.

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.)

We’ll use Ethanol – ice cold to decrease DNA’s solubility

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

Wheat Germ lysate

Alcohol/Aqueous interface

Ethanol

DNA