http://www.biotech.iastate.edu/lab_protocols/DNA_Extraction_Kiwi.html http://www.exploratorium.edu/ti/human_body/dna.html DNA EXTRACTION FROM KIWI Prepared by the Office of Biotechnology, Iowa State University INTRODUCTION DNA is present in the cells of all living organisms. This procedure is designed to extract DNA from kiwi in sufficient quantity to be seen and spooled. It is based on the use of household equipment and supplies. MATERIALS For teacher preparation two 4-cup measuring cups (1000 ml) with ml markings one 1-cup measuring cup (250 ml) with ml markings measuring spoons knife for cutting kiwi large spoon for mixing and mashing kiwi thermometer that will measure 60ƒ C (140ƒ F), such as a candy thermometer strainer or funnel that will fit in a 4-cup measuring cup #6 coffee filter or cheese cloth hot tap water bath (60ƒ C) (a 3-quart saucepan works well to hold the water) ice water bath (a large mixing bowl works well) distilled water clear-colored shampoo, such as Suave Daily Clarifying Shampoo 2 to 3 kiwi table salt, either iodized or non-iodized Supplies provided to the class 1 test tube for each student, preferably with a cap, that contains the kiwi solution. (A narrow glass container or clear bud vase can substitute for the test tube.) pasteur pipettes or medicine droppers 95% ethanol (grain alcohol) laboratory instructions TEACHER PREPARATION 1. Set up hot water bath at 55-60 degrees C and an ice water bath. 2. For 2 to 3 kiwi, make a solution consisting of one tablespoon (10 ml) of liquid dishwashing detergent or shampoo and one level 1/4 teaspoon (1.5 g) of table salt. http://www.biotech.iastate.edu/lab_protocols/DNA_Extraction_Kiwi.html http://www.exploratorium.edu/ti/human_body/dna.html Put in a 1-cup measuring cup (250 ml beaker). Add distilled water to make a final volume of 100 ml. Dissolve the salt by stirring slowly to avoid foaming. 3. Peel the kiwi, cut them into about 12 pieces and put the pieces into a 4-cup measuring cup (1000 ml). 4. Cover the kiwi with the 100 ml of solution from step 2. The detergent dissolves the fatty molecules that hold the cell membranes together, which releases the DNA into the solution. The detergent, combined with the heat treatment used in step 5, causes lipids (fatty molecules) and proteins to precipitate out of the solution, leaving the DNA. The salt enables the DNA strands to come together. 5. Put the measuring cup in a hot water bath at 55-60 degrees C for 10-12 minutes. During this time, press the kiwi mixture against the side of the measuring cup with the back of the spoon. Do not keep the mixture in the hot water bath for more than 15 minutes because the DNA will begin to break down. 6. Cool the mixture in an ice water bath for 5 minutes. During this time, press the kiwi mixture against the side of the measuring cup with the back of the spoon. 7. Filter the mixture through a #6 coffee filter placed in a strainer over a 4-cup measuring cup. When pouring the mixture into the strainer, avoid letting foam get into the measuring cup. It can take more than an hour to recover most of the liquid. The filtering can be done in a refrigerator overnight. 8. Dispense the kiwi solution into test tubes, one for each student. The test tube should contain about 1 teaspoon of solution or be about 1/3 full. For most uniform results among test tubes, stir the solution frequently when dispensing it into the tubes. There is not an advantage to dispensing more than one teaspoon of solution into a test tube. The solution can be stored in a refrigerator for about a day before it is used for the laboratory exercise. When the solution is removed from the refrigerator, it should be gently mixed before the test tubes are filled. STUDENT INSTRUCTIONS The process of extracting DNA from a cell is the first step for many laboratory procedures in biotechnology. The scientist must be able to separate DNA from the unwanted substances of the cell gently enough so that the DNA is not broken up. Your teacher has already prepared a solution for you, made of kiwi treated with salt, distilled water and dishwashing detergent or shampoo. The salt allows the DNA to precipitate out of a cold alcohol solution. The detergent causes the cell membrane to break down by dissolving the lipids and proteins of the cell and disrupting the bonds that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution. http://www.biotech.iastate.edu/lab_protocols/DNA_Extraction_Kiwi.html http://www.exploratorium.edu/ti/human_body/dna.html PROCEDURE 1. Add cold alcohol to the test tube to create an alcohol layer on top of about 1 cm. For best results, the alcohol should be as cold as possible. The alcohol can be added to the solution in at least three ways. (a) Put about 1 cm of alcohol into the bottom of a test tube and add the kiwi solution. (b) Fill a pasteur pipette with alcohol, put it to bottom of the test tube, and release the alcohol. (c) Slowly pour the alcohol down the inside of the test tube with a pasteur pipette or medicine dropper. DNA is not soluble in alcohol. When alcohol is added to the mixture, the components of the mixture, except for DNA, stay in solution while the DNA precipitates out into the alcohol layer. 2. Let the solution sit for 2 to 3 minutes without disturbing it. It is important not to shake the test tube. You can watch the white DNA precipitate out into the alcohol layer. When good results are obtained, there will be enough DNA to spool on to a glass rod, a pasteur pipette that has been heated at the tip to form a hook, or similar device. DNA has the appearance of white mucus. After the Exploration Expected Results A slimy white material will precipitate at the interface of the ethanol and filtrate layers. This material consists of clumped-together DNA strands and some protein. What's Going On? The procedure used in this activity has the same essential elements as more advanced laboratory DNA extraction procedures: mechanical and thermal disruption of cells, liberation of the DNA, and precipitation of the DNA. In this procedure, the kiwi cell walls are broken down by the mechanical mashing and then the heating, and the detergent dissolves the lipids in the cell membranes and nuclear envelope (just like the detergent dissolves grease on your dishes). No longer confined inside nuclear membranes, the DNA--highly soluble in water because the phosphate group of each nucleotide carries a negative charge & goes into solution. 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 ethanol is added, the DNA clumps together and precipitates at the water/ethanol interface because the DNA is not soluble in ethanol. Each glob of material in the precipitate will contain millions of DNA strands clumped together, along with some of the protein that is normally associated with DNA. (Since the DNA was not highly purified, some protein precipitates out with the DNA.) http://www.biotech.iastate.edu/lab_protocols/DNA_Extraction_Kiwi.html http://www.exploratorium.edu/ti/human_body/dna.html It is possible to analyze the extracted DNA in a research laboratory to provide good evidence that it really is DNA. Each type of molecule, because of its unique structure, has a characteristic pattern of absorption of the electromagnetic spectrum. This pattern can be determined by an instrument called a spectrophotometer, which shines light of specific wavelengths through substances and records the degree of absorbance for each wavelength. DNA exhibits maximal absorbance at approximately 260 nm, while a typical protein shows peak absorbance at 280 nm. This difference can be used to distinguish the two types of molecules. Kiwi fruit DNA extracted by the procedure outlined here was removed from solution, dissolved in a buffer, and subjected to spectrophotometric analysis in order to obtain a crude idea of its constituents and purity. The material showed an absorbance peak at 264 nm, indicating that it likely contains DNA with some contaminating protein. Discussion Questions 1. Describe the structure of DNA that you can see and compare it to the chemical structure of DNA. 2. Explain the role of solubility and charge in the precipitation of DNA. 3. Calculate the concentration (M) of the salt solution used in moles per liter. 4. What characteristic does salt have that allows it to dissolve in water? 5. In terms of solubility, explain how the shampoo helped make the cell membrane permeable, allowing the extraction of the DNA. 6. Explain the role of temperature in DNA extraction. 7. Some people are concerned that we may be able to manipulate the DNA of people and that it will change them into something that they are not. Can you give some examples of the types of human genes that might be changed? Do you think that scientists should proceed with this type of research? Why or why not? Going Further: Ideas for Inquiry Try to extract DNA from other fruits or vegetables using this procedure. Research and try other types of DNA extraction procedures. Compare the yield of another procedure with the yield from the one described here. Calculate how many times to the moon and back a human's DNA would reach if it was removed from each cell and each strand laid end-to-end. Here is the information you need: Each cell nucleus in a human holds about 2 meters of DNA http://www.biotech.iastate.edu/lab_protocols/DNA_Extraction_Kiwi.html http://www.exploratorium.edu/ti/human_body/dna.html and a typical adult human is composed of 60 trillion cells. The distance from the earth to the moon is 380,000 kilometers. The Basics and Beyond Background Deoxyribonucleic acid (DNA) is the genetic material present in all organisms, from bacteria to humans. A single subunit of DNA is called a nucleotide and consists of a nitrogen-containing base, a sugar, and a phosphate group. Hundreds of thousands of nucleotides are hooked together to form a chain, and two chains are paired together and twisted into a double helix to form the finished DNA molecule (see Figure 1). In organisms with nucleated cells such as humans, DNA is coupled with protein in structures called chromosomes that are contained within a membrane-bound nucleus inside a cell. Very pure DNA can be easily extracted from cells in a research laboratory, and somewhat less-pure DNA can be extracted with some simple techniques easily performed at home or in the classroom. Tidbits There are about 3 x 109 nucleotide base pairs in the human genome (the complete set of genes in one cell). If you took all of the DNA from a single human cell and laid the strands end to end, it would be about 2 meters long! All of that DNA is folded and packed into the nucleus of a human cell. The diameter of the nucleus is about 0.005 mm or 1/500 the width of a dime. There are 6 billion bits of information coded by DNA in each of our nucleated cells (a bit is a measure of information). Each human cell contains twenty-one times the information that is found in the Encyclopædia Britannica, which is thought to have about 280 million letters. Acknowledgment Thanks to Dr. Robert Swezey of SRI International, Menlo Park, CA, for determining the absorption spectra of our DNA extracts.