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

1. 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

2. 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.

3. 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

4. (Also called Sodium dodecyl sulfate (SDS))

5. The detergent dissolves the membrane by replacing the lipid bilayer with the hydrophobic portions of the detergent molecule. We’ll use SDS

6. 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.

7. 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): • A different type of enzyme called a protease, since enzymes are a type or protein (proteases don’t break themselves down.) • Heat (we cook food to denature the proteins.) • 3) Change the pH – use acid or base We’ll use HEAT

8. 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 pelletof 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.)

9. Review- 3 steps 1) cell lysis 2) denature DNAses 3) precipitate DNA (isolate it from the lysate)

10. We’ll use a rod to spool it up out of the solution DNA Ethanol Alcohol/Aqueous interface Wheat Germ lysate