Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Use scissors to cut the cheesecloth into 6-inch squares.
2. Prepare an 8.0% sodium chloride solution: Dissolve 200 g of sodium chloride in about 1.5 L of DI water. Dilute to a final volume of 2.5 L with deionized water. Place the solution in a labeled bottle.
3. Prepare the 2.5% ammonium molybdate in 3 M H₂SO₄ solution by pouring the entire contents of the 3 M sulfuric acid (30 mL) supplied with this kit into the ammonium molybdate bottle. Cap and mix well. Prepare just prior to the laboratory.
4. The 95% ethyl alcohol should be ice cold (about 0 °C) when used. Place it in an ice bath before class.
5. Label the three 600-mL beakers A, B and C and place into a chemical fume hood. These are the three shared disposal beakers.

Safety Precautions

Diphenylamine solution contains concentrated acetic acid and sulfuric acid. Ammonium molybdate solution also contains sulfuric acid. Tin(II) chloride solution contains hydrochloric acid. All three of these solutions are very corrosive to eyes, skin and other body tissues. They are toxic by ingestion. Avoid all body tissue contact. Acetic acid and hydrochloric acid are also toxic by inhalation. Ammonium hydroxide is extremely irritating to eyes and moderately toxic by inhalation and ingestion. Avoid breathing the vapors and dispense these chemicals in a fume hood. Silver nitrate solution is corrosive, irritating to skin and eyes and will stain skin and clothing. Ethyl alcohol is flammable and a dangerous fire risk—keep away from flames and other sources of ignition. Sodium dodecyl sulfate solutions may be irritating to skin. Any food-grade items that have been brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any food in the lab and do not remove any remaining food items. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The fruit solids may be disposed of in the regular trash according to Flinn Suggested Disposal Method #26a. The solution collected in Beaker A should remain in a chemical fume hood as the ethyl alcohol is allowed to evaporate according to Flinn Suggested Disposal Method #18a. After the ethyl alcohol has evaporated the remaining solution may be disposed of down the drain with plenty of excess water according to Flinn Suggested Disposal Method #26b. The solution collected in Beaker B and excess ammonium molybdate solution may be precipitated by calcium ions according to Flinn Suggested Disposal Method #6b. The solution collected in Beaker C and the excess diphenylamine solution and the solution tested with diphenylamine should be disposed of using a licensed hazardous waste company according to Flinn Suggested Disposal Method #26c. The excess ammonium hydroxide solution may be disposed of by neutralizing with acid then disposing of down the drain with plenty of excess water according to Flinn Suggested Disposal Method #10. The excess sulfuric acid and tin(II) chloride solution may be disposed of by neutralizing with base and then disposing of down the drain with plenty of excess water according to Flinn Suggested Disposal Method #24b. The excess EDTA, ethyl alcohol, sodium chloride, and SDS solution may be disposed of down the drain with plenty of water according to Flinn Suggested Disposal Method #26b. Leftover silver ions remaining in solution may be precipitated as silver chloride according to Flinn Suggested Disposal Method #11.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. Both parts of this laboratory activity can reasonably be completed in two 50-minute class periods. The prelaboratory assignment may be completed before coming to lab, and the Post-Lab Questions may be completed the day after the lab.
• If the lab must be broken into two class periods, cover the extracted banana DNA and store in a refrigerator.
• Sodium dodecyl sulfate is also known as dodecyl sulfate sodium salt and as sodium lauryl sulfate.
• Fresh banana, strawberries, kiwi, onions, raw wheat germ and liver will also provide ample yields of DNA.
• Many angiosperms are polyploids. For example, bananas have three copies of each chromosome (triploid), and strawberries have eight copies of each chromosome (octoploid).
• Spooled DNA may be analyzed using electrophoresis with further processing. The DNA should be cut using a restriction enzyme, otherwise it may be too large to run through the agarose gel. Results will likely appear as a smear or cloud as the restriction enzyme will cut the large macromolecule at numerous locations.

Answers to Prelab Questions

1. Explain why several tests instead of a single test are needed to determine the chemical composition of the substance isolated in Part I.

   DNA is composed of many different compounds. Just testing for phosphate or nitrogen would not rule out other biological compounds.

2. Review the Procedure and Safety Precautions. Why is personal protective equipment such as chemical-resistant gloves and chemical splash goggles recommended while performing this lab?

   Many of the chemicals in this laboratory are corrosive liquids or chemical irritants that may affect the skin, eyes, and mucous membranes. Personal protective equipment acts as a barrier to prevent the chemicals from coming into contact with the body.

Sample Data

Answers to Questions

1. In Part I, the rehydrated banana chips were broken into small pieces and the mixture was then filtered to remove the solids. Why not leave the banana whole and proceed with the remainder of the procedure?

   Surface area. In order to retrieve the maximum amount of DNA the sodium chloride must come in contact with as many cell membranes as possible.

2. In Part II, the nuclear material was tested for phosphate, purines and deoxyribose. Did the results of these chemical tests conclusively determine if the substance was DNA? Explain why or why not.

   Students may answer that these tests identified the substance as containing phosphate, purines and deoxyribose and therefore it must be DNA. Others may indicate that only three tests were performed and therefore further testing may reveal the presence of RNA, proteins or other compounds within a mixture of compounds removed from the interface.

3. What other components of DNA could be tested?

   Tests for nitrogen and pyrimidines could also be performed.

4. The phosphate backbone of DNA carries a negative charge causing the entire macromolecule to be negative. Histones interact with DNA to help coil and stabilize the DNA strand. What charge do the histones carry? How did you determine your answer?

   Histones have a positive charge, allowing them to form ionic bonds twith the DNA strand.

5. The procedure used in Part I is not specific to DNA. RNA may also be retrieved using this protocol. In order to test the nuclear material for RNA what component of RNA would need to be tested?

   A test for ribose should be performed to determine if RNA is also present.

Introduction

Nucleic acids, including RNA and DNA, are important macromolecules for their roles in the storage, transfer and expression of genetic information. A frequent misconception is that Watson and Crick discovered DNA in 1953. In fact, Watson and Crick simply provided a model for the structure of DNA. Watson and Crick made use of years of research conducted by numerous other scientists to build their double-helix model of DNA. Explore some of the same techniques used by these scientists to determine the chemical composition of nucleic acids.

Concepts

• DNA
• Nucleic acids
• Biochemical testing

Background

Nucleic acids were first extracted from cells in 1869 by Friedrich Miescher (1844–1895). Miescher determined that macromolecules isolated from the nucleus of leukocyte white blood cells contained carbon, oxygen, nitrogen, hydrogen and phosphorus but not sulfur. At the time of his discovery scientists knew that proteins contained sulfur but no phosphorus. Thus Miescher concluded that he had discovered a new class of macromolecule which he named “nuclein” since it came from the nucleus. Extracting only the nucleus from cells and then purifying nuclein was a new technique. Miescher’s nuclein settled to the bottom of the test tube after the nuclear material has been treated with alcohol to remove the lipids and with enzymes to digest away most of the proteins.

It was Albrecht Kossel (1853–1927) who determined the chemical structure of Miescher’s nuclein. Nuclein actually consists of nucleic acids and proteins. Kossel further determined the chemical structure of the nucleic acid portion of nuclein. Nucleic acids, it was found, could be broken into nitrogen-containing purines and pyrimidines plus a carbohydrate. Kossel devoted most of his experimentation to the protein portion of nuclein, later “discovering” histones. He was awarded the Nobel Prize in Medicine in 1910 for his work.

Before 1944 scientists thought that diverse, complex proteins were responsible for heredity. It wasn’t until 1944 when DNA’s role in the cell was determined. This finding led biochemist named Erwin Chargaff (1905–2002) to study DNA. Chargaff studied the composition of DNA from different species and found the ratios of adenine to thymine were always equal, as were the amounts of guanine and cytosine. Previously scientists had thought the amount of each of the four nitrogenous bases was equal (A=C=G=T).

DNA is found in the nucleus and mitochondria of organisms and in the chloroplasts of plants. DNA is a macromolecule composed of repeating subunits called nucleotides. A nucleotide is composed of three chemical parts: a phosphate group, a sugar called an aldopentose and a nitrogenous base (see Figure 1).

Phosphate groups alternate with the aldopentose sugar to form the backbone of DNA. A simple phosphate test is an easy way to determine if a cellular extract contains proteins or nucleic acids. This is possible because the amino acids that make up proteins do not contain phosphates. In the phosphate test, ammonium molybdate, tin(II) chloride and an acid are added to a sample of cellular extract. In acidic solutions, the phosphate ions found in DNA bond with ammonium molybdate to form phosphomolybdic acid (PMA). PMA is reduced by tin(II) to form a dark blue- or green-colored complex. The test only indicates the presence of phosphorus. It does not determine, on its own, that the extract is DNA.

Nitrogenous bases contain carbon, oxygen, hydrogen and nitrogen in an aromatic ring configuration. Two basic types of nitrogenous bases are found in DNA and RNA. The purines have two rings (see Figure 2a) while the pyrimidines have one ring (see Figure 2b). The two purines are adenine and guanine. Silver nitrate is used to test for purines. In the presence of a weak base, the purine and silver ion combine to form a white precipitate. The two pyrimidines in DNA are cytosine and thymine while RNA contains uracil instead of thymine. An aldopentose sugar is a monosaccharide with five carbon atoms and a parent aldehyde functional group. The aldopentose sugar in DNA is deoxyribose (see Figure 3a) while the aldopentose sugar in RNA is ribose (see Figure 3b). In deoxyribose, one of the OH groups has been replaced by a hydrogen atom. This change makes the DNA molecule more stable when it is bound in a chain with phosphate groups. The diphenylamine test is used to test for deoxyribose. When DNA is heated in the presence of concentrated sulfuric acid, the deoxyribose portion of DNA is converted to a molecule that binds with diphenylamine to form a dark blue–black complex. The intensity of the color is directly proportional to the amount of deoxyribose present. The process of extracting nucleic acids from cells for study is of primary importance in many fields of biotechnology. It is critical for genetic research, DNA fingerprinting and creating recombinant organisms which create beneficial products in the field of medicine. In this activity, the nucleic acid macromolecule of interest is DNA. The cell walls of a piece of fruit are lysed by mechanically smashing the fruit. Salt is added to the fruit before is it smashed so that the salt can coalesce (combine) the DNA strands that are freed from the nucleus. After the solids are filtered out, sodium dodecyl sulfate (SDS) is added to the remaining solution to break apart and emulsify the lipids and proteins that make up the cell and nuclear membranes. Next, the DNAdestroying enzyme DNAse is disabled by adding ethylenediaminetetracetic acid (EDTA). Finally, the DNA is precipitated from the solution using ethyl alcohol. DNA is soluble in water and insoluble in ethyl alcohol. Adding ethyl alcohol to the top of the chemically treated fruit mixture dehydrates and precipitates the DNA from the solution. The DNA precipitates at the water/alcohol interface and can be collected by “spooling” it onto a glass stirring rod or by evacuation into a pipet.

Experiment Overview

The purpose of this experiment is to extract the nuclear material from fruit and determine its composition using simple chemical tests. In Part I, the nuclear materials or nucleic acid portions will be isolated from a fruit. Many fruits are polyploids—they contain multiple copies of each chromosome within a single cell. In Part II, chemical tests will be completed on the isolated nucleic acid macromolecule to determine if it contains phosphate, deoxyribose and nucleotides.

Materials

Prelab Questions

1. Explain why several tests instead of a single test are needed to determine the chemical composition of the substance isolated in Part I.
2. Review the Procedure and Safety Precautions. Why is personal protective equipment such as chemical resistant gloves and chemical splash goggles recommended while performing this lab?

Safety Precautions

Ethyl alcohol is a flammable liquid and a dangerous fire risk—keep away from flames and other sources of ignition. Sodium dodecyl sulfate solution may be irritating to skin. Any food-grade items that have been brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any food in the lab and do not remove any remaining food items. Diphenylamine solution contains concentrated acetic acid and sulfuric acid. Ammonium molybdate solution also contains sulfuric acid. Tin(II) chloride solution contains hydrochloric acid. All three of these solutions are very corrosive to eyes, skin and other body tissues. They are also toxic by ingestion. Avoid all body tissue contact. Acetic acid and hydrochloric acid are also toxic by inhalation. Ammonium hydroxide is extremely irritating to eyes, and moderately toxic by inhalation and ingestion. Avoid breathing the vapors and dispense these chemicals in a fume hood. Silver nitrate solution is corrosive, irritating to skin and eyes and will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Part I. Isolating the Nuclear Material

1. Use the balance and a weighing dish or wax paper to mass 25 g of banana chips.
2. Place the banana chips and 150 mL of 8% sodium chloride solution into a resealable bag overnight. Note: Check the resealable bag for leaks.
3. The next day, gently knead the bag, breaking the soaked banana chips into pea-sized or smaller pieces.
4. Layer four layers of cheesecloth on top of one another and place into a funnel. Place the funnel into a clean 400-mL beaker.
5. Pour the banana mixture through four layers of cheesecloth so that the liquid drains into the beaker.
6. Gently squeeze the cheesecloth to remove most of the banana solution leaving the solids trapped in the cheesecloth. Typically, about 100 mL of banana solution is recovered from the original 150 mL of salt solution.
7. Use a clean graduated cylinder to add 10 mL of the 0.01 M EDTA to the beaker. Stir with a stirring rod.
8. Use a clean graduated cylinder to add 10 mL of the 10% SDS solution to the beaker. Stir with a stirring rod. Note: A cloud of precipitated proteins may form during this step.
9. Holding the beaker at a slight angle, use a clean graduated cylinder to carefully transfer 15 mL of ice-cold 95% ethyl alcohol down the side of the beaker so that the ethyl alcohol forms a layer on top of the banana solution in the beaker (see Figure 4).
   {11026_Procedure_Figure_4}
10. Carefully place the beaker back on the tabletop, making sure the two layers do not mix.
11. Allow the beaker to sit for one minute and observe the nuclear material precipitating out of the banana solution at the interface between the cold ethyl alcohol and the aqueous banana solution layers. Note: The nuclear material appears as a white cloud with numerous tiny bubbles attached to the precipitate.
12. Gently place the tip of a wide-stem pipet into the interface containing the nuclear material to collect the solid.
13. Transfer the solid nuclear material to the 50-mL beaker.
14. Repeat steps 12 and 13 until most of the solid substance has been collected.
15. In a chemical fume hood or well ventilated room, allow most of the ethyl alcohol to evaporate from the beaker.
16. Dispose of the banana solids in the regular trash, and pour the remaining solution into Beaker A located in a chemical fume hood.

Part II. Chemical Testing

1. Perform a deoxyribose test on the nuclear material.
   1. Use a graduated pipet to transfer 1 mL of the nuclear material to a clean test tube.
   2. Add 1 mL of DI water to a second test tube. This will be the negative control.
   3. Use a clean graduated pipet to transfer 2 mL of diphenylamine solution to each test tube.
   4. Place the two test tubes into a boiling water bath for 10 minutes.
   5. Record any observations in the data table on the Chemical Testing of DNA Worksheet.
2. Perform a phosphate test on the nuclear material.
   1. Use a clean graduated pipet to transfer 1 mL of the nuclear material to a clean test tube.
   2. Add 1 mL of DI water to a second test tube. This will be the negative control.
   3. Use a clean graduated pipet to transfer 1 mL of 2.5% ammonium molybdate solution to each test tube.
   4. Hold the two test tubes with one hand and gently tap near the bottom of the test tubes to mix.
   5. Place the test tubes in the rack and use a clean graduated pipet to add 1 drop of tin(II) chloride solution to each test tube.
   6. Mix the contents once again and record any observations in the data table on the Chemical Testing of DNA Worksheet.
3. Perform a test for purines on the nuclear material.
   1. Use a graduated pipet to transfer 1 mL of the nuclear material to a clean test tube.
   2. Add 1 mL of DI water to a second test tube. This will be the negative control.
   3. Use a clean graduated pipet to transfer 0.5 mL of silver nitrate solution to both test tubes.
   4. Use a clean graduated pipet to transfer 0.5 mL of 2 M ammonium hydroxide solution to each test tube.
   5. Record any observations in the data table on the Chemical Testing of DNA Worksheet.
4. Collect the solution from the phosphate test and the purine test into Beaker B located in a chemical fume hood. Collect the solution from the deoxyribose test into Beaker C located in a chemical fume hood.

Student Worksheet PDF

11026_Student1.pdf