GMO Investigator: Part 1

Transcription

1 Biology 211, North Seattle Community College Name: GMO Investigator: Part 1 OBJECTIVES To review the structure and function of DNA. Understand and perform the polymerase chain reaction (PCR) To gain experience using the micropipettes, thermocycler, and gel electrophoresis To explore the benefits and potential risks of genetically modified organisms (GMOs) INTRODUCTION A genetically modified organism is one whose DNA has been modified, usually by the introduction of a foreign gene. Many people are opposed to genetically modified crop plants, citing the risk of creating super-weeds (through cross-pollination with herbicide-resistant crops) or super-bugs that are no longer resistant to the toxins in the pest-resistant GM crops. Another commonly cited concern is the potential production of allergic reactions to the new proteins used to develop the crops. Advocates for the technology, however, argue that these crops can be better for the environment, requiring fewer toxic chemicals. In addition, they point to the ability to preserve farmland and improve the nutritional content of food. Regardless of your perspective, genetically modified food is now available, with corn, soy, and papaya being some of the most common. In the US, such foods do not have to be labeled for consumers, and foods with less than 5% genetically modified content can be labeled GMO-free. Europe and Asia, in contrast, have required genetically modified foods to be labeled if they contain more than 1% GM content. How do you make a GM plant? The first step in this process is to identify a gene of interest. For example, many GM crops include a gene from a soil bacterium, Bacillus thuringiensis (Bt). This gene produces a protein that is toxic to corn borers, a common agricultural pest. Once the gene has been identified, scientists clone the gene, or make identical copies of it. This cloned gene is then inserted into the target plant through one of several methods including the use of a naturally occurring bacterium that inserts its DNA into a host plant s genome. Plant cells may also be induced to take up foreign DNA using an electrical current (electroporation) or by physically shooting gold particles covered in DNA into the cells (biobalistics). Once the foreign DNA is inserted, the plants are carefully bred to ensure that the desired traits are correctly passed on. (For more information, please see Chapter 16 in your Life textbook.) How do we know if a food has been genetically modified? In the absence of effective tracking and labeling, GM foods can be identified experimentally by one of two methods. The first, an ELISA, uses antibodies to detect the presence of specific proteins. This method, however, can only test fresh produce and must be individualized according to the type of crop. The second available test utilizes the polymerase chain reaction (PCR) to test for sequences of DNA that have been inserted into a GM plant. As DNA is a much more stable molecule, fragments can be isolated from highly processed foods. We will use this technique to screen for GM foods in lab this week. Your group may choose to work either with a sample provided in lab, or with one you ve brought from home. If you choose to bring your own sample, consider selecting fresh corn, papaya, corn bread mix, corn meal, soy flour, soy burgers, or other similar products. 1

2 What is PCR? In 1983, Kary Mullis at Cetus Corporation developed the molecular biology technique known as the polymerase chain reaction (PCR). PCR revolutionized genetic research, allowing scientists to easily amplify short specific regions of DNA for a variety of purposes including gene mapping, cloning, DNA sequencing, and gene detection. The objective of any PCR is to produce a large amount of DNA in a test tube starting from only a trace amount. A researcher can take trace amounts of genomic DNA from a drop of blood, a single hair follicle, or a processed food sample and make enough to study. Prior to PCR, this would have been impossible! This dramatic amplification is possible because of the structure of DNA, and the way in which cells naturally copy their own DNA. DNA in our cells exists as a double-stranded molecule. These two strands, or sequences of bases, bind to one another in a very specific, predictable fashion. Specifically, A s will only pair with T s, and C s will only pair with G s. Thus if you know the sequence of one strand of DNA, you can accurately predict the sequence of the other. Both DNA replication and PCR take advantage of this predictability. In your cells, one strand of DNA is used as a template to copy the sequence of your DNA from every time a cell divides. PCR does essentially the same process, using one strand of your DNA as a template to produce copies of its sequence. PCR is conducted in three steps: 1) Denature the template DNA, 2) Allow the primers to anneal, and 3) Extend (copy) the template DNA. In the first step, the template DNA is heated up to break the hydrogen bonds holding the two strands together. This allows each strand to serve as a template for generating copies of the DNA. In the second step, the temperature is reduced to allow the primes to anneal, or bind, at their complimentary sequence on the template. (Primers are short, specific pieces of singlestranded DNA that provide a starting point for the enzyme that will do the copying.) In the third step, the temperature is raised again to allow the enzyme to bind at the primer and add bases to the growing DNA molecule. These three steps are repeated between 20 and 40 times in an instrument called a thermocycler. The power of this process is that it results in exponential growth. After the first round of copying, a single DNA molecule will have produced two identical copies. These two copies will generate four molecules in the next round. Those four molecules will create eight, and so on. Thus in 30 cycles we generate literally millions of copies of DNA from each template molecule! We will be able to visualize these millions of copies using a process called DNA electrophoresis, and thus determine whether or not our sample contained genetic modifications. (The process of electrophoresis is discussed in Part 2 of this lab handout.) This site is overly complicated in its explanation of PCR but has a great color photo that really brings the process home. PROCEDURES Part A: Extraction of DNA From Food and Control Samples 1. Find your screwcap tubes containing Instagene Matrix and label one control and one test. These are the tubes you will place the DNA from your food samples in. (The Instagene matrix will bind any ions released from your sample as you boil it that might otherwise interfere with your PCR.) 2. Weigh out grams of the GM- food sample provided and put it into a clean, uncontaminated mortar. 2

3 3 Add 5 volumes of high quality distilled water for every gram of food you weighed out. (Thus for 2 grams of sample, add 10 mls of water.) 4. Grind with the pestle for at least two minutes to form a slurry. 5. Add a second 5 volumes of water to your slurry and grind further until the mixture is smooth enough to pipette. 6. Use a disposable pipette to place 50 ul of ground slurry into the labeled screwcap tube labeled Control. 7. Clean out your mortar and pestle as indicated by your instructor. Repeat steps 2-7 using the food source you are testing. Place this sample in the screwcap tube you labeled test. 8. Shake or flick both tubes ( control and test ) to mix and place them in a 95 C waterbath for 5 minutes. 9. Place your tubes in a centrifuge in a balanced conformation (equal number of tubes on each side) and spin for 5 minutes at maximum speed. 10. When the centrifuge has stopped spinning, carefully remove your tubes and place them in a rack, taking care to avoid shaking. Your extracted DNA is ready! Part B: Setting up the PCR Reaction 1. Obtain 6 PCR tubes, and label them carefully with your group s letter and a number. Take care when handling these tubes as they are delicate and crush easily! The numbers on your tubes should correspond to the following tube contents: Tube Number Master Mix DNA Expected result 1 20 ul Plant MM (green) 2 20 ul GMO MM (red) 3 20 ul Plant MM (green) 4 20 ul GMO MM (red) 5 20 ul Plant MM (green) 6 20 ul GMO MM (red) 20 ul Non-GMO food 20 ul Non-GMO food 20 ul Test food DNA 20 ul Test food DNA 20 ul GMO positive 20 ul GMO positive +/-/? Actual result +/-/? 2. Obtain a Styrofoam cup with ice. Place each tube in an adaptor, and each adaptor into the ice to cool. 3. Referring to the table in Step 1, and using a fresh tip for each addition, add 20 ul of the indicated master mix to each PCR tube. Cap each tube to prevent contamination. 3

4 4. Again referring to the same table, and using a fresh tip for each tube, add 20 ul of the indicated DNA to each tube. Be sure to avoid the InstaGene pellet at the bottom of the sample tubes. As you add the DNA mixture to your reaction tube, pipette gently up and down to mix. 5. Be sure your tubes are tightly capped and place them in the thermocycler. Next Steps: The thermocycler will heat and cool your samples 40 times, making millions of copies of any regions of DNA that match the primers. This process will take several hours. When it has finished, your instructor will place the samples in the fridge for you to analyze during your next lab session. In the meantime, read Part 2 of this protocol carefully before coming to lab next time. LAB NOTEBOOK CHECK: Your lab notebook entry this week should contain a title and objective for this lab. As always, you should include detailed procedures. (You may simply tape them in from this handout if you like.) POSTLAB: No postlab this week! GMO Investigator, Part 2 BACKGROUND: Recall that in part 1 we ground up food samples, extracted their DNA, and used this DNA to do a Polymerase Chain Reaction (PCR). This PCR will copy specific sequences of DNA that are often used when a food crop is genetically modified. Thus if our sample contained modified food, we expect to see our reactions work; if the sample did not contain genetically modified food, no product should be formed. How can I tell if my PCR worked? Unfortunately, you cannot see individual DNA molecules with either your naked eye or our light microscopes. So we will need another technique to allow us to determine whether or not our PCR was successful. The technique we will use is known as DNA electrophoresis. How does DNA electrophoresis work? In DNA electrophoresis, a collection of DNA molecules is placed in wells at one end of a dense matrix called a gel. An electrical current is then conducted across the gel. As DNA is a negatively charged molecule, it will be drawn towards the positive pole. (Opposites attract!) Thus our DNA will move through the gel, and smaller fragments will travel faster, and therefore farther, than larger fragments. This allows us to sort the DNA fragments from each PCR based on its size. What will I see as my gel runs? We will mix our PCR products with a colored loading dye. This loading dye will make it easier to see and load our samples. In addition, the dye itself is also negatively charged; thus is will also migrate through our gel when the current is applied. You will see this dye moving. The DNA produced in the PCR reaction, however, is still not visible as it has no natural color. To see these DNA molecules, we will have to apply a DNA stain, FastBlast. This stain will bind to the DNA molecules produced by the PCR and allow us to see them.. 4

5 Part 2: DNA Electrophoresis Note: We will work today in the same group of 4-5 students, with each group running their own PCR products on their gel. Make sure everyone has a chance to practice with the micropipettes and load a sample or two! Part A: Preparing your gel. 1. Obtain a tray from the electrophoresis supply box. CAREFULLY tape both ends of the tray as demonstrated by your instructor. Note that these gels are prone to leaking! A little extra time now will be worth your while! Insert a comb into one of the sets of notches at the end of your tray. 2. Obtain a flask of melted agarose from the water bath. Carefully pour the melted agarose into your tray, up to the line on the side. Return the melted agarose to the water bath so other groups may use it! 3. Leave your gel in a level spot until the agarose has solidified (~10-15 minutes). The agarose will turn slightly opaque and will be solid to the touch. 4. Gently remove the comb and the tape from both ends of your tray. 5. Place your gel in an electrophoresis box. Recall that DNA is negatively charged, thus it will migrate towards the positive pole (red). This means that the wells in your gel should be at the negative (black) end of the box! 6. Carefully pour 1X TBE buffer over the surface of your gel until you have filled the chambers at either end of your gel. Part B: Preparing your Samples. 1. Carefully retrieve your PCR samples from last week. (Remember that these tubes have thin walls and should be handled gently. 2. Using a fresh tip each time, carefully add 5 ul of Orange G loading dye to each sample and mix well. On these pipettes, the dial should read if your pipette is set correctly. Remember to depress the plunger only to the first stopping point when drawing up liquids. Part C: Loading your Gel. When your group has reached this point, your instructor will demonstrate how to load a gel by loading a molecular size marker on your gel. This marker contains a set of DNA fragments of known size and can be used to determine the size of your PCR products. After your instructor has demonstrated the process, you may proceed. 1. Using a fresh tip each time, carefully remove 15 ul of the PCR/Loading dye mix. (Your pipette will read this time.) Place the pipette tip at the top of the well with the tip just below the surface and slowly dispense the solution, allowing it to sink into the bottom of the well. Note that one of the most common errors is to push the tip too far down and poke a hole in the bottom of the gel! 2. Record which well you placed the sample in, and continue as above to load the remaining five samples. Part D: Running the Gel. 1. When your gel is loaded, carefully place the cover on your electrophoresis chamber, taking care to align red with red and black with black. 2. Plug the ends of the wires into a power supply. We will run our gels at 100V for approximately 30 minutes. At the end of 30 minutes, your instructor will turn of the power supply. Part E: Staining your gel. 1. Carefully remove your gel from the electrophoresis box by lifting it out on its tray. Note that these gels are warm, slippery, and fragile at this point! 2. Slide your gel of the tray and into a large weigh boat. Label this weigh boat with your group s name. 3. Pour enough 100X Fast Blast solution into the weigh boat to completely cover the surface of your gel. Allow the solution to sit for 3-5 minutes. 5

6 4. Pour the FastBlast solution into the dedicated waste beaker provided. Begin washing the gel by filling your tray with water and shaking gently. As the water turns blue, pour it out and replace with fresh. You will need to repeat this washing procedure for 15 minutes or more. 5. When the bands are clearly visible, either sketch your gel, or obtain a picture (if the camera is available) to place in your lab notebook. 6. Clean-up: As you finish lab today, make sure your electrophoresis chambers and gel trays have been rinsed with tap water, dried, and returned to their storage boxes. Place these boxes on the carts in the classroom. Micropipettes and unused pipette tips should also be returned to their boxes. Take care to wipe down your workspace if any solutions have been spilled. The empty plastic tubes may be thrown in the trash. Analyzing Your Results: Indicate in your data table in your notebook the results obtained from running your gel POSTLAB Your post-lab assignment this week will help you gain some experience writing scientific papers. For this lab, we will practice writing just two of the critical sections included in scientific papers, the Results and Discussion. Your Results section should include your data, and just enough text to accurately describe it to your reader, noting any interesting trends or results. In this case, your results section should include a diagram or labeled picture of your gel. Like all figures in a scientific paper, this image should have both a descriptive title, and a 1-2 sentence caption explaining what s in the image. Clearly indicate what each lane in your gel contained, and how you interpret your results. Avoid discussing the implications of your results in this section! In other words just tell what you observed. Don t say what it means. For example There was a bright band in lane 2 Your Discussion section should consist of 2-3 paragraphs describing the implications of your work. Was the food source you tested genetically modified? How conclusive are your results? This is where you saw what it means. The bright band in lane 2 means this LAB NOTEBOOK CHECK: Your lab notebook entry this week should contain a title and objective for this lab. As always, you should include detailed procedures. (You may simply tape them in from this handout if you like.) You should also include your predictions and observations in the PCR table as well as a detailed sketch or photograph of your stained gel and the results and discussion as described above. 6