CH 112 Special Assignment #6 The Case of the Phony Fruit

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1 CH 112 Special Assignment #6 The Case of the Phony Fruit PRE-LAB ASSIGNMENT: Make sure that you read this handout and bring the essentials to lab with you. You may find it helpful to read parts of Chapter 13 in Adventures in Chemistry, specifically pp and Here are the pre-lab questions for this week. Make sure that you cite your sources and use your own words. 1. What are organic crops? 2. What are genetically modified crops? 3. Would you expect to find any differences in the DNA from organic crops and non-organic ones of the same variety? Would you expect to find any differences in the DNA from genetically modified crops and non-genetically modified ones of the same variety? Explain your answers. INTRODUCTION: CRaC s assignment this week comes from Colby s Dining Services division, which has recently been alerted to a scam involving phony fruits. Colby s produce supplier has been accused of selling several varieties of expensive organic fruits that are in fact genetically modified to be less susceptible to rotting. Apparently, the market has been flooded with illegal South American fruits produced by the Eureka Genetic Engineering Company. These fruits have not been approved by the FDA (Food and Drug Administration) for consumption in the United States. Dining Services wants to find out if their recent shipment of bananas and strawberries are of the certified organic quality that they paid for. That s where we come in. You will help to analyze the DNA of different fruit samples with relevance to this investigation to help determine whether Dining Services is getting what it paid for. CRaC is working cooperatively with scientists from Colby s DNA Sequencing Facility (CDSF)

2 on this project. DNA from suspect and control fruits will be analyzed for insertion of the gene that prevents rotting. A technique called the polymerase chain reaction (PCR) makes millions of copies of the region in question. If a new gene has been added, then the PCR product will be a different size than normal. The size of the PCR products can be analyzed through gel electrophoresis (as discussed on pg 574 of Adventures in Chemistry). The first step in the analysis of the fruit samples is to purify the DNA. Each investigator will choose one of the four available types of bananas (control organic, control regular, control genetically modified, and Dining Services banana). You will use salt and detergent to break open the cells, which results in release of the genetic material into solution. You will then add alcohol to the mixture, which causes DNA to precipitate out of solution, forming a cloudy solid that you can easily collect. This DNA will be sent downstairs for CDSF to perform PCR and subsequent analysis via electrophoresis. Because of the additional genes in the genetically modified specimens, the sizes of the products will differ from an ordinary control fruit of the same type. Because of the time required for PCR, each CRaC team will perform gel electrophoresis on strawberry samples previously isolated and PCR-amplified by CDSF. The PCR process used by CDSF amplifies two regions of DNA simultaneously. One region is for a strawberry-specific marker that should be identical in all types of strawberries, thereby leading to a band on the gel at the same position for all samples. The other region is the site at which the new gene has been inserted in the genetically modified samples, leading to a different-sized PCR product in the Eureka strawberries. A careful comparison of the bands produced by each fruit sample should allow you to make conclusions about the true identity of the produce purchased by Dining Services. LEARNING GOALS: Be able to: Be familiar with the technique of electrophoresis. Be familiar with the properties of DNA. Understand the steps in purification of DNA. PROCEDURE: Part 1. Electrophoresis of DNA from Strawberry Samples 1. Four investigators will share one agarose gel. The gel you are using is 0.8% agarose containing 6 µl of 0.5 mg/ml ethidium bromide per 30 ml gel. This gel is already prepared for you and was gently placed into the center of the electrophoresis chamber. You will load the gel with PCR samples that were 2

3 amplified from strawberry DNA. You must wear gloves for the following work. Note that each gel contains 8 lanes, but you will only load 4. Caution: Ethidium bromide is a mutagen that can be absorbed through the skin. It is also an irritant to the skin, eyes, mouth, and upper respiratory tract. Exercise caution when handling and dispose of pipette tips and any contaminated gloves or tips in the appropriate solid waste container. 2. Pour 250 ml of 1X TBE Running Buffer solution into the chamber to submerge the gel, if it is not already covered. 3. Acquire the strawberry samples within the PCR tubes as follows: green tube, ordinary strawberries from Hannaford supermarket; yellow tube, organic strawberries from Uncle Dean s supermarket; blue tube, suspect strawberries from Dining Services; orange tube, genetically modified Eureka strawberries. 4. Each investigator will pipette from one PCR tube into one well of the agarose gel. For best results the center wells should be used. Make sure that you note which sample was loaded into which well. 5. Pipette 15 µl of your specific strawberry sample, taking great care to avoid drawing up air bubbles in the pipette tip. Air bubbles mean that you have less than 15 µl, which can hamper your ability to visualize the DNA bands later on. 6. Slowly guide the pipette tip until it is sitting on the inside lip of a well, with the tip angled downwards into the well. Release the sample into the well, keeping your thumb down on the pipette s release button as you pull your pipette upwards. This will draw the tip back out of the agarose gel without accidentally pulling up the very DNA you just put into the well. Be careful not to puncture the bottom of the well, which is quite fragile. 7. When all samples are loaded in the agarose gel, record a sketch of the 8 wells noting the location and contents of each in your notebook. (Some will be empty.) 8. Place the cover on the chamber with the black lead (negative charge) nearest to the wells. This will enable the DNA to move towards the red lead (positive charge). 9. Plug the wire leads into the power source. The voltage will be set to 120 V. Once all samples are ready, the power source will be turned on. You want the front purple band of dye to have moved about half way across the agarose gel before stopping. This will take approximately one hour. Proceed to Part 2 while you are waiting. 10. When the gel is done, turn off the power, then unplug the black and red leads. 11. With freshly gloved hands, one investigator should carefully remove the cover from the chamber. 12. Using the handles, remove the gel tray from the chamber. Gently push the gel off its tray onto plastic wrap on the ultraviolet transilluminator (the plastic wrap protects the glass surface from chemical damage). 13. Once you have on UV glasses or a UV cover over the transilluminator, turn it on. Sketch the bands that appear in each lane, being careful to line up bands that line up on the gel. The position of each band reflects the size of the PCR product. 3

4 Caution: UV light can damage your eyes, so you either need to wear special glasses for viewing the gel, or you need to have the protective cover in place over the gel on the transilluminator. 14. Dispose of gel and any gloves that have touched ethidium bromide in the special disposal container. Wash your hands. Part 2. Isolation of DNA from Banana Cells 1. Pick one of the four types of bananas (ordinary banana from Hannaford supermarket; organic banana from Uncle Dean s supermarket; suspect banana from Dining Services; genetically modified Eureka banana). Grind up a piece of banana with a mortar and pestle. Make sure that your banana mush is liquid enough to pour easily by adding an equal amount of water to it. Using a funnel, pour about 25 ml of the mixture into a plastic 50 ml test tube. 2. Add 1 teaspoon each of salt and shampoo. Put the cap on tightly and carefully invert the tube five times to mix. The salt helps to break open the cell by osmosis. Because the cell contains a higher proportion of water than salt compared to the surroundings, water will exit the cell, causing it to burst open. This process is helped by the detergent in the shampoo, which dissolves the outside layer of the cell (the membrane), just like it washes away grease and dirt. 3. Put the tube into the beaker of water in the 60 C bath for 10 minutes. During this time, the cells completely break down, leaving a cell soup containing the freed DNA. 4. While the cell soup is heating, put a piece of cheesecloth inside a plastic funnel. Put the funnel on top of a second clean plastic 50-mL test tube. 5. After the 10 minutes are up, carefully pour the cell soup into the funnel. Let the liquid drip down into the tube below, leaving the solids to be trapped in the cheesecloth. 6. Carefully add an equal amount of ice-cold alcohol to your freshly collected liquid. Put the cap on the tube and turn it upside down 3 times to mix. Record your observations. 7. Pour your mixture into a plastic dish (a Petri plate). Use a toothpick to spool the DNA. Gently swirl the stick to wrap the DNA around it, much like cotton candy. Put the isolated banana DNA into a PCR tube of the appropriate color. PCR tubes as follows: green tube, ordinary banana from Hannaford supermarket; yellow tube, organic bananas from Uncle Dean s supermarket; blue tube, suspect banana from Dining Services; orange tube, genetically modified Eureka banana. These tubes will be passed along to CDSF for PCR amplification. References: Adapted from an experiment originally developed by Julie T. Millard and Lisa M. Miller of the Colby College Chemistry Department. Rowe, R. and Blaser, M. (2008) Adventures in Chemistry Laboratory Manual. 4

5 CraC Special Assignment #6 REPORT SHEET NAME PARTNER DATE Sketch your gel here. (Wells are depicted as lines at the far left of the gel.) DATA SUMMARY Describe your observations during the DNA purification. POST-LAB QUESTIONS 1. Ethanol was added to the aqueous solution to precipitate the DNA. Draw a Lewis dot structure for ethanol (CH 3 CH 2 OH). Why is DNA less soluble in ethanol than in water? 2. Why does DNA move towards the positive electrode during electrophoresis? 3. a) How do the sizes of the PCR products compare in the genetically modified and normal strawberries? Explain. b) What do the results of your agarose gel suggest about the fruit purchased by Dining Services? Explain. 5

6 4. Why was the PCR step necessary to detect differences in the samples? That is, why couldn t you have run the strawberry and banana DNA directly on the gel? 5. Assume that PCR was performed on banana samples as indicated to produce the results shown here. What can you conclude? Explain. Hannaford Organic Dining Services Eureka CONCLUSIONS Use the space below to report on your experiment to Dining Services, making sure that you address the original goals of this work. 6