Digging for DNA. Isolating Deoxyribonucleic Acid from Strawberries

Transcription

1 Biology Digging for DNA Isolating Deoxyribonucleic Acid from Strawberries MATERIALS AND RESOURCES EACH GROUP aprons bag, zipper-lock, quart balance beaker, 250 ml centrifuge tube, screw cap cheese cloth cup, 3-oz plastic dishwashing liquid ethanol, 95% goggles graduated cylinder, 10 ml ice and ice chest marker, Vis-à-vis meat tenderizer microcentrifuge tube paper clip, standard paper towels pipette, thin stem salt, non-iodized strawberry water, distilled weigh boat ABOUT THIS LESSON This lesson should be used following a discussion over the basic structure of the DNA molecule. The students will extract DNA from strawberries and from another substance of their choice. Included in this lesson is an explanation of the basic chemistry behind the extraction, which the students will use to construct a model that will serve as a visual representation of the sequential order for the reagents to proceed to have a successful extraction. The students then use this model to predict possible outcomes of errors made in a hypothetical extraction. OBJECTIVES Students will: Extract DNA from living cells Construct a model of the extraction process and use this model to predict outcomes of hypothetical extraction processes T E A C H E R P A G E S LEVEL Biology Copyright 2013 National Math + Science Initiative, Dallas, Texas. All rights reserved. Visit us online at i

2 NEXT GENERATION SCIENCE STANDARDS COMMON CORE STATE STANDARDS PLANNING/CARRYING OUT INVESTIGATIONS (LITERACY) RST Follow precisely a multistep procedure when carrying out experiments, taking measurements, or performing technical tasks, attending to special cases or exceptions defined in the text. STRUCTURE AND FUNCTION (LITERACY) W.3 Write narratives to develop real or imagined experiences or events using effective technique, wellchosen details, and well-structured event sequences. ASSESSMENTS LS1: STRUCTURES AND PROCESSES CONNECTIONS TO AP* AP BIOLOGY 3 A.1 DNA, and in some cases RNA, is the primary source of heritable information. The following types of formative assessments are embedded in this lesson: Visual assessment of correct procedure used within the lesson by the formation of lysate Both teacher and peer review of the constructed model The following assessments are located on our website: Middle Grades Life: Heredity Assessment DNA to Protein and Biotechnology Assessment ACKNOWLEDGEMENTS T E A C H E R P A G E S AP BIOLOGY 4 Vis-à-vis and EXPO are registered trademarks of Newell Rubbermaid, Inc. A.1 The subcomponents of biological molecules and their sequence determine the properties of that molecule. *Advanced Placement and AP are registered trademarks of the College Entrance Examination Board. The College Board was not involved in the production of this product. Copyright 2013 National Math + Science Initiative, Dallas, Texas. All rights reserved. Visit us online at ii

3 TEACHING SUGGESTIONS The strawberry DNA extraction lab serves as a simple and almost fail-proof way to extract DNA (and RNA) from a living cell. Other fruits will work as well, such as bananas and wheat germ. Citrus fruits such as oranges, lemons, and grapefruits do not work well for this lab. The results are stunning even if students make errors with the ingredients. The volumes and masses do not have to be exact for the procedure to still work. This lab not only serves as a good introduction to basic lab equipment but it can also be extended to an in-depth study of the chemistry of the DNA molecule. Students are typically very excited about this lab, and find isolating this important molecule highly rewarding. Before beginning this lab, review the basic structure of the DNA molecule with students. There are many paper or toothpick models of DNA available that can be created by students to provide a visual image for this molecule. Time also must be spent in class before the lab to discuss the basic chemistry behind this reaction. Depending on the level of your students, you can withhold as much of the information as you deem appropriate to make this lab more rigorous. The explanations are reiterated in the student procedure but should include: Macerating the strawberry breaks down the tissue, exposing the individual cells. The liquid soap binds to the lipid portion of the phospholipid bilayer comprising the cell membrane. This helps break the membrane down and keeps the phospholipids from re-associating. As a result, the cell membrane disintegrates. Adding non-iodized salt helps isolate and precipitate the DNA by shielding the phosphate groups along the sugar/phosphate backbone of the DNA molecule. Specifically, the Na + ions have an attraction to the negatively charged phosphates on the side chains, causing them to form a shield by occupying space around the phosphates and blocking out the polar water molecules. Chromatin is primarily made up of histone proteins and DNA. When chromatin condenses into a chromosome during the earliest stage of mitosis, it does so by wrapping and coiling itself around these histone proteins. Adding meat tenderizer releases papain, a protease enzyme that breaks down the histone proteins and frees the DNA. Papain also denatures or breaks down the DNase present. DNase is an enzyme that digests or breaks down and destroys the DNA. It is found naturally in all cells and will be released into your solution when maceration occurs. Adding cold ethanol will precipitate the DNA. DNA is water-soluble and in the presence of alcohol forms a precipitate and makes a viscous, white, stringy solid that can be spooled onto a pipette or a bent paper clip. Remember, the colder the ethanol, the less soluble the DNA and the greater the yield. T E A C H E R P A G E S Copyright 2013 National Math + Science Initiative, Dallas, Texas. All rights reserved. Visit us online at iii

4 TEACHING SUGGESTIONS (CONTINUED) Two critical ingredients for this lab are unseasoned meat tenderizer and 95% ethanol. With the exception of the 95% ethanol, all of the ingredients needed for this lab can be purchased at your local grocery store. The meat tenderizer should be purchased within a week of the lab and used immediately. The older the meat tenderizer, the less active its protease will be. The 95% ethanol can be ordered from a science supply company. As of this writing, the cost is approximately $27 for four liters, which should last your classroom a couple of years. Do not try to substitute isopropyl alcohol (rubbing alcohol), as it never seems to precipitate the DNA as well as ethanol. Any type of alcohol that is less than 95% pure will get less than anticipated results. The ethanol must be put in the freezer before class and kept there at all times when not being used. It is a good idea to place all glassware in the freezer as well. To keep the ethanol near-freezing during class, you can store the ethanol in a squeeze bottle and keep it on ice. The salt and the meat tenderizer can be kept at each lab table. It is important to keep these in re-sealable containers. One option is to use a 15 ml conical tube with a screw-on lid. The conical tubes should be labeled salt and meat tenderizer as they are both white granular powders. A very small amount (approximately 5 ml) of liquid dishwashing soap can be put in a small, disposable cup at each lab table. Always use distilled water, which is available at any grocery store. Tap water is filled with impurities, especially DNase, and will cause your DNA not to spool. Use the disposable transfer pipette to pull the DNA up from the alcohol layer. Alternately, straighten out a paper clip and bend it to make a hook (about 3 mm from the end) that can be used to fish out the DNA. One way to enhance student motivation is to provide inexpensive microcentrifuge tubes to each student to store the DNA after it is extracted. Each student can leave the lab with a microtube of DNA (topped off with ethanol) to show their friends and parents. Science can indeed be cool. For students who are struggling with the model, you might show them an example not one for the extraction process but rather another model that shows them an example of a model without giving away the answer. Some students might need a copy of a sample extraction model and then asked to complete the Conclusion Questions. This will depend on the level of the student. If you need to raise the rigor for students who can be pushed further than the parameters of this lab, you might try the following suggestion: Students can be asked to determine a quantitative lab protocol that produces the greatest yield of DNA. Each group can be given the same amount of strawberry (its mass measured on a balance) and are then allowed to experiment and design a procedure that maximizes the yield of DNA. The precipitated and isolated DNA can be dried on filter paper (or a small, pre-cut piece of paper towel) and weighed on an analytical balance after it has dried. Be sure and tell students to determine the mass of the filter paper before placing the DNA on it, and to write the mass of the paper near one edge in pencil. Some inks are water-soluble and will bleed, making it difficult to read the measurement. T E A C H E R P A G E S Copyright 2013 National Math + Science Initiative, Dallas, Texas. All rights reserved. Visit us online at iv

5 PROCEDURE The reagents available for your use are listed in Table 1. Discuss with your group how each might help isolate the DNA. Table 1. Reagents Reagent Meat tenderizer Function Chromatin is primarily composed of histone proteins and DNA. When chromatin condenses into a chromosome during the earliest stage of mitosis, it does so by wrapping and coiling itself around these histone proteins. Adding meat tenderizer releases papain, a protease enzyme that breaks down the histone proteins and frees the DNA. Papain is ph-specific, with an ideal range of 6.0 to 7.0. Papain also denatures or breaks down the DNase present. DNase is an enzyme that digests or breaks down and destroys DNA. DNase is found naturally in all cells and will be released into solution as maceration occurs. Salt Ethanol Adding non-iodized salt helps isolate and precipitate the DNA by shielding the phosphate groups along the sugar/phosphate backbone of the DNA molecule. Specifically, the Na + ions from the salt have an attraction to the negatively charged phosphates on the side chains, causing them to form a shield by occupying space around the phosphates and blocking out the polar water molecules. Adding cold ethanol will precipitate the DNA. DNA is water-soluble and in the presence of alcohol forms a precipitate and makes a viscous, white, stringy material that can be spooled onto a pipette or a bent paper clip. Remember, the colder the ethanol, the less soluble the DNA and the greater the yield. A N S W E R K E Y Detergent The liquid soap binds to the lipid portion of the phospholipid bilayer comprising the cell membrane. This helps break the membrane down and keeps the phospholipids from re-associating. As a result, the cell membrane disintegrates. v

6 EXTRACTION MODEL #1 - Maceration breaks down the tissue and exposes cells to the reagents. #4 - Salt s Na + ions shield DNA from attaching to polar water molecules and becoming less soluble in the aqueous solution. #2 - The detergent helps break open the phospholipid bilayers. #3 - Enzymes (protease) in meat tenderizer break down histones and DNases. #5 - The colder the ethanol, the less soluble the DNA, which will precipitate out. Figure A. Sample extraction model for DNA A N S W E R K E Y vi

7 CONCLUSION QUESTIONS 1. For each of the following scenarios, use your model to predict how far into the extraction process the experimenters would get and the effect on the amount of DNA extracted. For each independent scenario, justify your response. a. The detergent was not added to the mixture. If the detergent was not added, the strawberry tissue would be broken down into individual cells but the phospholipid bilayer would still be intact. Therefore, the rest of the reagents would have little to no effect getting the DNA to precipitate out. b. The salt was not added to the mixture. The entire process would still occur. However, most of the DNA would be attracted to the polar water molecule and would not precipitate out. The DNA yield would be greatly decreased. c. Instead of a strawberry, a lemon is used (ph 2.0). Papain is ph-specific, with the ideal ph range being 6.0 to 7.0. Enough hydrochloric acid was added to the bag to bring the ph down to 3.5. The process would proceed normally until the stage where the histone proteins bound to the DNA and the enzyme DNase were to be broken down. Because the protease in the meat tenderizer is ph-specific, the enzyme would denature at a ph of 3.5. This would prevent it from breaking down the histone proteins and the DNase, and would mean that the DNase would break the DNA down and the histone proteins would stay intact. This would cause the DNA to not spool when precipitated out. d. Ethanol at 50 C is poured into the test tube containing the strained lysate. The process would proceed normally until the ethanol stage. The colder the ethanol, the more DNA that will precipitate out. Therefore, very little to no DNA would show up in the ethanol layer. e. A strawberry is placed in a beaker of nearfreezing ethanol. Without maceration, the cells would not be isolated from the tissue, thus preventing the rest of the process from occurring. 2. Describe the contents of the material collected on the cheesecloth, i.e., what structures of the strawberry cells are contained on the cheesecloth? Be specific. There should be all of the various organelles, phospholipids (plasma membrane and nuclear membrane), cytosol, cellulose (cell wall), and so on basically, everything but the nucleic acids (DNA and RNA). 3. How would you modify this protocol for animal cells, such as from a cheek swab? The same protocol would work for animal cells. You will not have the cell wall to break down but everything else should be the same. A N S W E R K E Y vii

8 Biology Digging for Biology DNA Digging for DNA Isolating Deoxyribonucleic Acid from Strawberries MATERIALS aprons bag, zipper-lock, quart balance beaker, 250 ml centrifuge tube, screw cap cheese cloth cup, 3-oz plastic In the presence of near-freezing ethyl alcohol, deoxyribonucleic acid (DNA) will become an insoluble mucous-like, stringy material called a precipitate. However, simply putting substances such as strawberries in the alcohol will not cause the DNA to precipitate. Therefore, additional reagents must be used to extract the DNA. PURPOSE In this exercise, you will extract DNA from strawberries using common kitchen reagents. You will then use your extraction and reagent knowledge to derive a sequence of effective extraction steps and make a model (flowchart, concept map, or visual representation) showing this process. Your knowledge of basic chemistry, cell structure, DNA structure, and enzyme structure will help justify these steps. dishwashing liquid ethanol, 95% goggles graduated cylinder, 10 ml ice and ice chest marker, Vis-à-vis meat tenderizer SAFETY ALERT!» Safety goggles should be worn at all times.» Ethanol is flammable. Avoid close proximity to flames. microcentrifuge tube paper clip, standard paper towels pipette, thin stem salt, non-iodized strawberry water, distilled weigh boat 1

9 PROCEDURE Refer to the reagent descriptions in Table 1 and list, in order, the events necessary for an effective extraction. You and your group might want to consider the following: Where is the DNA in a strawberry cell? What structures will you have to go through to get to the DNA? What are the components of these structures? Which reagents and methods will allow you to accomplish this task? Why? Once you get to the DNA, will it precipitate? Why or why not? How is DNA stored in the cell? What is the difference between DNA, chromatin, and a chromosome? Which reagent might help get DNA by itself? Why? What is the DNA molecule made of? Why would this matter? What is the monomer of a nucleic acid? What are the three parts of this monomer? What is the charge of each of these parts? Based on this, how would DNA react with water? Which reagent might help prevent DNA from attaching to water so that it will precipitate? Why? 2

10 PROCEDURE (CONTINUED) The reagents available for your use are listed in Table 1. Discuss with your group how each might help isolate the DNA. Table 1. Reagents Reagent Function Meat tenderizer Salt Ethanol Detergent 3

11 PROCEDURE (CONTINUED) 1. Obtain a strawberry, a plastic bag, and 5 ml of water. Put the strawberry and the water into the plastic bag. 2. Place the following amount of reagents into the bag: 1 g salt 2 g meat tenderizer 1 ml detergent 3. Macerate, or crush up, the mixture until it is thoroughly mixed. However, make sure that you do not mix it so much that you form bubbles. Maceration of the strawberry tissue exposes the cells to the reagents dissolved in the water. 4. Once the crushed strawberry solution (lysate) is prepared, strain it through several layers of cheesecloth into a beaker. Transfer the strained lysate into a large test tube. 5. Carefully pour the cold ethyl alcohol down the inside of the test tube until you have about a one-inch layer on top of the strained lysate. Do not pour the alcohol directly into the lysate. Two layers should be visible: the pink fluid on bottom and the clear alcohol on top. 6. Using an unbent paper clip, swirl the alcohol layer. DNA (a white, stringy mass) should precipitate out of the pink fluid and into the alcohol layer. 4

12 ORDER OF EVENTS Refer to the reagent descriptions in Table 1 and list, in order, the events necessary for an effective extraction. Make sure that you include a justification for each step. EXTRACTION MODEL In this lab, many of the reagents are placed into the system (the plastic bag) at the same time. Each reagent takes its turn to isolate the DNA. Considering the reagent information provided in Table 1, construct a model (flowchart, concept map, or visual representation) depicting the order of extraction events as they occur at the cellular level. 5

13 CONCLUSION QUESTIONS 1. For each of the following scenarios, use your model to predict how far into the extraction process the experimenters would get and the effect on the amount of DNA extracted. For each independent scenario, justify your response. a. The detergent was not added to the mixture. b. The salt was not added to the mixture. c. Instead of a strawberry, a lemon is used (ph 2.0). d. Ethanol at 50 C is poured into the test tube containing the strained lysate. e. A strawberry is placed in a beaker of near-freezing ethanol. 6

14 CONCLUSION QUESTIONS (CONTINUED) 2. Describe the contents of the material collected on the cheesecloth, i.e., what structures of the strawberry cells are contained on the cheesecloth? Be specific. 3. How would you modify this protocol for animal cells, such as from a cheek swab? 7

15 EXTENSION Now it s your turn! 1. What steps could you take to get more DNA out of your strawberry? Devise a plan and, with your teacher s permission, try it out. Communicate the results of your experiment to the class. This could be in the form of a poster, graphic slides, a movie, and so on. 2. Within your group, decide on another substance from which you could extract DNA. Use the protocol from your strawberry DNA extraction to carry out this task. Communicate the results of your experiment to the class. This could be in the form of a poster, graphic slides, a movie, and so on. 8