Using Genetic Evidence
This lab is a modified version of a DNA extraction procedure used by scientists at the University of Arizona in Tucson. The lab has been adjusted to use equipment readily available within a secondary classroom and to fit within two 50-55 minute periods. Students like this lab because it is cool to see their own DNA and to get it from their "spit" (cheek cells). Some of my students carry it around for days!
DNA is present in all living things from bacteria to plants to animals. In animals, it is found in almost all cell types: muscles, reproductive cells, hair roots, and skin cells -- anything that has a nucleus. DNA is not found in red blood cells which lack nuclei. DNA can be obtained from white blood cells. The basic procedure for extracting DNA is the same, regardless of its source, although the specifics may vary:
· Collect cells.
· Split cells open and release contents (proteins, fats and carbohydrates).
· Destroy enzymes which break apart DNA.
· Separate DNA from other cell components (proteins, fats and carbohydrates).
· Precipitate DNA.
· Resuspend DNA in solution so it can be studied.
Extracting DNA and Why It Works
Studentscollect cheek cells by rinsing their mouths with a saline solution. The saline solution keeps the cells from lysing, or splitting open, too soon. The cheek cells are separated from the mouthwash by centrifugation. (spinning them in a centrifuge). The cells are heavier than the saline solution so they sink to the bottom and form a clump or pellet . The cell pellet remains stuck to the bottom of the tube and the saline solution can be poured off. Lysis buffer is added to the cell pellet in order to split the cells open (the DNA must be released from inside the nucleus). Lysis buffer contains soap (to break apart the fatty membranes), salts and ions (to increase the osmotic pressure outside the cell and help break apart the membranes) and buffers (to maintain the pH of the solution). The cells are incubated in a hot water bath in order to denature the cytoplasmic enzymes which break apart DNA. Students add a concentrated salt solution which changes the polarity of the solution; DNA dissolves in ionic solutions while fats, carbohydrates and many proteins will not. Centrifugation separates the DNA from the "junk" (proteins, carbohydrates and fats). The DNA is precipitated from the ionic solution by the addition of cold ethanol.
Students can use either 0.9% saline solution (9 g NaCl dissolved in 1 L distilled water) or Gatorade for their mouthwash. I prefer to use Gatorade because they won't complain about how "salty" it tastes. Choose a flavor which is light colored (such as Lemon Ice) to avoid staining the cells and DNA.
· Measure the Gatorade into sterile 15 mL conical tubes or paper cups. Don't let the students drink from or spit into them. They should be used for measuring purposes only and rinsed with water between uses. Have students pour the Gatorade into a separate cup, pour from the cup into their mouths, and spit back into the same cup. This will keep the measuring tubes clean and prevent students from spitting all over your room.
· Students need to swish hard and chew on their cheeks. The more cells they collect the better. No blood please! I have students do more than one mouthwash (2-3) so they gets lots of cells and DNA. Alternatively, each group member can contribute some of his/her cheek cells.
· After pouring their mouthwashes into 15 mL tubes, students should compare their samples and pair up with someone who has a similar volume. Their tubes should be placed opposite of each other in the centrifuge. Extra Gatorade can be added if needed to obtain similar volumes. This will keep the tubes balanced in the centrifuge.
· When removing the tubes from the centrifuge, check to see if a pellet (their cheek cells) has formed on the bottom of the tube. If the cell pellet is floating, spin the tube longer. The cell pellet should stay on the bottom while they pour the Gatorade out.
·For the second and subsequent mouthwashes, it doesn't matter if different students contribute the cheek cells. When they analyze their DNA they are only looking for the quantity of DNA they extracted and whether it is intact or broken in small pieces, not specific genetic markers. The contents of a second mouthwash should be added directly on top on the cell pellet once the liquid has been poured off. The students are trying to get a larger pellet from which to extract their DNA.
Don't worry too much about food, although the estraction procedure seems to work better if the students have not just finished eating. · Plan something for the students to do while their tubes are spinning in the centrifuge. It is a great time to demo the next part of the procedure, engage them in a discussion on DNA and its uses, or give them something to work on independently. This is particularly important if not all tubes can be spun at the same time.
· Keep the tubes with the cells and lysis buffer in the hot water bath at least 60 minutes, preferably overnight. Flick tubes to break up cell pellets. The tubes should be clear (no cells visible) before proceeding on.
· Research laboratories add 50-100 uL (microliters) of proteinase K (10 mg/mL) to each sample before incubating. Proteinase K helps to increase the yield of DNA, but it is very expensive (about $35 for 25 mg). If you choose to add proteinase K, decrease the incubation temperature to 55-60 C.
· The cell pellet should be mostly dissolved. After adding the NaCl, students should mix their tubes by flicking for about one minute before centrifuging. The volumes should be relatively the same. Don't worry as much about balancing them this time. Centrifuge the tube on HIGH power for 10 minutes.
· The DNA is now in the clear liquid. Using a pipette, transfer just the clear liquid into a clean tube. Avoid transferring the precipitate at the bottom of the tube; this is the proteins, carbohydrates and fat molecules which are not soluble in an aqueous solution. It is all right to leave some of the liquid behind.
· Add COLD alcohol. Rock the tube sideways to make the DNA precipitate out of solution. It should be visible as small pieces of white thread.
· If you want the students to look at their DNA on an agarose gel, they need to resuspend it in water or TE buffer. Otherwise, the DNA will stay crystallized and visible in the ethanol. You can make glass hooks by melting glass pipettes over a Bunsen burner and pulling the ends apart. You will be left with fine glass hooks on both ends.
The University of Arizona
Department of Biochemistry and Molecular Biophysics
General Biology Program for Secondary Teachers