Lab: DNA Extraction from Human Cheek Cells Introduction DNA…you hear about it all the time. DNA is used every day by scientists and lawyers to help in criminal investigation, paternity suits, and for cloning (to name just a few instances). Your DNA is your “genetic fingerprint”—this means that your DNA is like no one else’s in the world! The procedure that we will use to see your DNA includes the same basic processes that researchers use to isolate, analyze, and manipulate DNA in a laboratory setting (although the DNA isolated here is not nearly as “pure” as the research lab version). DNA is in the nucleus of almost every cell in your body. The length of DNA per cell is about 100,000 times as long as the cell itself. However, DNA only takes up about 10% of the cell’s volume. This is because DNA is specially packaged through a series of events to fit easily in the cell’s nucleus. The structure of DNA, the double helix, is wrapped around proteins, folded back onto itself, and coiled into a compact chromosome. Individual chromosomes can be studied using microscopes, but the double helix of a chromosome is so thin that it only be detected through innovative, high-tech procedures. Chromosomal DNA from a single cell is not visible to the naked eye. However, when chromosomal DNA is extracted from multiple cells, the amassed quantity can easily be seen and looks like strands of mucous-like, translucent cotton. Clumping the DNA... When DNA is released from a cell it typically breaks up into short strand fragments. The released DNA--highly soluble in water because the phosphate group of each nucleotide carries a negative charge--dissolves in the water. However, the positively charged sodium ions from the salt in the extraction solution are attracted to the negatively charged phosphate groups on the DNA backbone, effectively neutralizing the DNA's electric charge. This neutralization allows the DNA molecules to aggregate with one another. When the alcohol is added, the DNA clumps together and precipitates at the water/alcohol interface because the DNA is not soluble in alcohol. The colored layer of liquid above the cell solution will contain DNA and proteins. The DNA will then move up to a layer of alcohol carefully placed above the water. DNA does not stay dissolved in alcohol, but precipitates out and will appear as a white stringy mass. Spooling the DNA on a stick... Alcohol allows DNA fragments to stick together, or precipitate, producing a blob of DNA which you can examine. The DNA precipitate can be captured on a hook or spooled onto a wooden stick by placing the tool in the DNA and turning it. If you want to save your DNA, you can transfer it to a small container filled with alcohol. Introductory Questions: Before beginning the experiment, it is important that you realize the role each of the materials will be playing in the performance of this experiment. Answer the questions associated with starting this lab on a separate sheet of paper in complete sentences. A. Some basic, but cool, chemistry: DNA is the largest known molecule. A single unbroken strand of animal or plant DNA contains millions of atoms. The DNA from a single (diploid) human cell, if the 46 chromosomes in one cell were connected end-to-end and straightened, would have a length of approximately 2 meters and a width of approximately 2.4 nanometers. Releasing the DNA... In the DNA extraction process, the first step is to release the DNA from the cells of a living organism. Detergents and soaps are used to (1) dissolve the lipids in the cell membranes and nuclear envelope, releasing the DNA, and (2) break up proteins which may harm the DNA. (Enzymes may also be used to break up the proteins.) B. C. Detergent is being used to break open both the cell membrane and nuclear envelope. 1.) Which types of molecules compose the majority of your cell membrane? 2.) How does detergent interact with these molecules? 3.) If detergents can break open the nuclear envelope and cell membrane, which molecules can you infer are present in the nuclear envelope. DNA typically breaks apart when it is placed in water. 1.) Which substance will you use to avoid the fragmentation of DNA? 2.) Explain why your DNA will not break apart in water. How is the alcohol that is being added to your solution going to ensure your DNA sample is visible? Procedure Ingredients: Water Clear dish soap Food coloring Table salt Isopropyl alcohol And… you! 1. For the table, mix 200 ml drinking water with 2 tsp salt 2. Stir until salt is dissolved. Then transfer 5 tsp of salt water into a clear cup. 3. Gargle the salt water for 1 minute. 4. Spit the water back into the cup. Now your cheek cells are suspended in the salt water. 5. Gently stir the salt water with one drop of soap. (Avoid bubbles as much as possible). 6. In a separate cup, mix 10 ml isopropyl alcohol and 1drop of food coloring. 7. Tilt the salt water cup and gently pour the alcohol so that it forms a layer on top (about 2 cm thick). 8. Wait about 2.5 minutes. You should see white clumps and strings forming. That's your DNA! Discussion Questions- Please complete these questions on a separate sheet with your introduction questions. Your answers should still be in complete sentences. 1. Describe how long strands of double-helical DNA fit into the nucleus of a single cheek cell. 2. If DNA is so thin, how is it that we are able to see it during this simple lab exercise? 3. What does gargling with salt water do to your cheek cells? 4. If you had extracted DNA from plant cells instead of animal cells, what cell barrier would have been different? 5. Do you think that DNA from plant cells would look the same as DNA from animal cells (explain your reasoning)? 6. Why is DNA extraction important and what can it be used for? Give one example of something a scientist can do with extracted DNA.