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DNA Isolation

In this session, you will learn why we want to isolate DNA, the procedure for doing so, and you will perform the last steps of the process yourself.

Objectives
Activities
Discussion
Questions
Interesting web sites
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Objectives [top]

  1. Understand why and how DNA is extracted from cells
  2. Be able to explain the major steps involved in isolating DNA
  3. View DNA in a test tube

Activities [top]

  1. View computer animation
  2. Discussion of DNA isolation procedures
  3. Perform final steps of DNA isolation:
    1. Add 0.1 ml of protein precipitation solution to the cell lysate
    2. Vortex vigorously at high speed for 20 seconds to mix the solutions uniformly
    3. Centrifuge at 13-16,000 X g for 3 minutes. The precipitated proteins form a brown pellet
    4. Carefully pour off the supernatant containing the DNA into a clean 1.5 ml tube containing 0.3 ml of 100% isopropanol
    5. Mix the sample by inverting gently 50 times until the white threads of DNA form a clump

Discussion [top]

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To study DNA and learn how our genes function, scientists must be able to work with DNA itself, outside the protective walls of the nucleus and cell membrane. So researchers have learned to separate - to isolate - DNA from the proteins and other substances in the cell.

DNA is a hardy molecule, and one of the largest known. To work with it, we need to extract it from the surround cellular materials. Although DNA can be isolated from most types of cells, we'll be using the common approach of separating it from the cells contained in whole blood. This involves several steps that will be performed prior to the symposium because of time constraints. A very small amount of blood can provide a very large amount of DNA.

Whole blood includes a variety of types of cells, including the red cells that carry oxygen, the various types of white blood cells that are part of the immune system, and platelets, which play an important role in blood clotting. The first step in the DNA process, then, is to add whole blood to a tube containing a red blood cell (RBC) lysis solution - traditionally a detergent. Lysis means a process of disintegration, which is what happens to the red blood cells during this step. The RBCs explode into pieces while the other blood cells remain intact. Next, the tube is placed in a centrifuge for several seconds, which forces the white blood cells into a pellet at the end of the tube. The remnants of the red blood cells remain in the solution, which is removed using a micropipette. A small amount of liquid remains in the tube, which is shaken to allow the white blood cells to be suspended once again in the liquid.

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Next we want to separate the DNA from other proteins in the cells. After the white blood cells are re-mixed into the liquid, another lysis solution - an organic solvent - is added to break up the white blood cells and their nuclei, tearing down membrane walls and destroying the cytoplasm and enzymes. This leaves the DNA and RNA molecules floating in the liquid. Now a protein precipitation solution is added to the liquid, causing the "junk" created by the lysing process to clump together. After some additional shaking, the tube is again put into a centrifuge, causing a brown pellet to form in the bottom of the tube. The liquid above the pellet contains the DNA.

Now the liquid with the DNA - the supernatant - is poured into a tube containing isopropanol alcohol. The DNA molecules in the mixture are negatively charged, while the alcohol is not electrically charged. The result is an electrical imbalance that causes the DNA molecules to be no longer soluble. The tube is inverted gently about 50 times and the DNA molecules appear as white threads, forming a clump.

Questions: [top]

Why do we want to isolate DNA from the surrounding cells?

Why do we extract DNA from white blood cells rather than from red blood cells?

What other cellular "junk" would be in the brown pellet found after the lysing of the WBC?

Why do we add protein precipitation solution to the mixture?

Interesting web sites: [top]

Spooling the Stuff of Life (Scientific American)

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