Materials Included In Kit

Additional Materials Required

Prelab Preparation

Ammonium molybdate solution, 2.5% in 3 M H₂SO₄: Prepare 30 mL of 3 M sulfuric acid by appropriate dilution of a more concentrated solution. Add 0.75 g of ammonium molybdate and mix well. Prepare just prior to the laboratory.

Banana chips: Divide the banana chips into twelve 25-g portions in separate recloseable bags. Add 150 mL of saline (8% NaCl solution) and allow to soak overnight. Each student group uses one portion.

Ethyl alcohol, 95%: Must be chilled (ice-cold, about 0 °C) before use. Place in an ice bath prior to class.

Sodium chloride solution, 8%: Dissolve 200 g of sodium chloride (NaCl) in about 1.5 L of distilled or deionized water. Dilute to a final volume of 2.5 L with water. Place the solution in a labeled bottle.

Safety Precautions

Ethyl alcohol is a flammable liquid and a dangerous fire risk—keep away from flames and other sources of ignition. 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 severely corrosive to eyes, skin and other body tissues. They are also toxic by ingestion. 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. Sodium dodecyl sulfate solution may be irritating to skin. Avoid contact of all chemicals with eyes and skin. All food-grade items that are brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any materials in the chemistry laboratory and do not remove any remaining food items from the lab after use. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or 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 leftover water/alcohol mixture from Part A should have an alcohol content below 20% and may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b. Collect the leftover reaction mixtures from the phosphate test in a designated container. Excess molybdate in this solution may be precipitated with calcium according to Flinn Suggested Disposal Method #6b. Excess diphenylamine solution and the leftover reaction mixtures from diphenylamine testing should be collected for licensed hazardous waste disposal. These solutions are acidic and corrosive liquids. Excess tin(II) chloride solution may be neutralized with base according to Flinn Suggested Disposal Method #24b. Leftover EDTA, sodium chloride, and SDS solutions may be rinsed down the drain with water according to Flinn Suggested Disposal Method #26b. Leftover silver ions remaining in any test solutions may be precipitated as silver chloride according to Flinn Suggested Disposal Method #11.

Lab Hints

• Both parts of this laboratory activity can reasonably be completed in a typical 2-hr lab period. The Prelaboratory Assignment should be reviewed before class to reinforce the safety precautions.
• Sodium dodecyl sulfate is also known as sodium lauryl sulfate.
• Fresh banana, strawberries, kiwi, onions, raw wheat germ and liver will also provide ample yields of DNA using the procedure outlined in this activity.
• Many angiosperms are polyploids. For example, bananas have three copies of each chromosome (triploid) and strawberries have eight copies of each chromosome (octoploid).
• Cover the extracted banana DNA and store in a refrigerator, if desired, for further testing.
• Spooled DNA may be analyzed using electrophoresis after further processing. The DNA should be digested or cut using a restriction enzyme, otherwise it will be too large to run through a typical agarose gel. Results will likely appear as a smear or cloud as the restriction enzyme will cut the large macromolecule at numerous locations.
• The purity of the isolated DNA can be analyzed by UV spectroscopy. DNA has an absorption maximum at 260 nm, while proteins absorb at 280 nm. Pure DNA has an A₂₆₀/A₂₈₀ ratio = 1.9. Dissolve the precipitated DNA in pH 7 buffer (TRIS), so that A₂₆₀ is between 0.5 and 1.0. Measure A₂₆₀ and A₂₈₀ and calculate the ratio. Ratios < 1.9 indicate protein contamination.

Answers to Prelab Questions

1. Review the Procedure and Safety Precautions. Discuss the personal protective equipment required for performing this lab. What additional safety precautions are needed for glacial acetic acid and ammonium hydroxide solutions?

   Many of the chemicals in this laboratory are corrosive liquids or chemical irritants that may affect the skin, eyes and mucous membranes. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron as barriers to prevent the chemicals from coming into contact with eyes and skin. Glacial acetic acid and ammonium hydroxide solutions may be harmful if inhaled. Work with these reagents in an operating fume hood.

2. Explain why several tests instead of a single test are used to verify the chemical composition of the nuclear material.

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

3. The following bases represent a nucleotide sequence in a strand of DNA. What would be the base composition of its complementary strand?

   5′–AGCTTACGTCC
   3′–TCGAATGCAGG

4. Guanine and cytosine form three hydrogen bonds when they are aligned or paired in complementary strands of the DNA double helix. Draw dashed lines between the following stuctures to show the probable location of these hydrogen bonds.
   {14006_PreLabAnswers_Figure_5}

Answers to Questions

1. In Part A, 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?

   “Smashing” the banana chips increases their surface area for lysis of the cell walls and nuclear membrane. 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 B, 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.

   These tests identified the substance as containing phosphate, purines and deoxyribose, which is consistent with the structure of DNA. Further testing, however, might reveal the presence of RNA, proteins or other cell components within the 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 procedure used in Part A 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.

5. 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?

   Histones have a positive charge, allowing them to form ionic bonds with the DNA strands.

6. What was the purpose of each of the following reagents in the DNA extraction procedure: SDS and EDTA?

   SDS (sodium dodecyl sulfate) is a detergent that breaks apart cell and nuclear membranes, emulsifying and dissolving the lipid and protein components, respectively. EDTA complexes with Mg²⁺ ions needed for DNAase activity. It disables this enzyme, which otherwise would shear the chromosomal DNA.

Introduction

Nucleic acids, including RNA and DNA, are essential macromolecules for the storage, transfer and expression of genetic information. A frequent misconception is that Watson and Crick discovered DNA in 1953. Watson and Crick developed their doublehelix model for the structure of DNA based on research conducted by numerous other scientists over many years to study the chemical composition of DNA. Explore the procedures used in the chemical testing of nucleic acids.

Concepts

• DNA
• Nucleic acids
• Qualitative tests

Background

Nucleic acids were first extracted from cells in 1869 by Friedrich Miescher (1844–1895). Miescher determined that macromolecules isolated from the nucleus of 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 not phosphorus. Miescher concluded that he had discovered a new class of macromolecules which he named “nuclein” since it came from the nucleus. Miescher’s nuclein settled to the bottom of a test tube after the nuclear material had been treated with alcohol to remove the lipids and with enzymes to digest most of the proteins.

Building on Miescher’s work, Albrecht Kossel (1853– 1927) found that nuclein consisted of both nucleic acids and proteins. Kossel determined that the composition of the nucleic acid portion of nuclein could be broken down into nitrogen containing purines and pyrimidines plus a carbohydrate. Kossel devoted most of his subsequent research to the protein portion of nuclein, later “discovering” histones. He was awarded the Nobel Prize in Medicine in 1910 for his work.

Prior to 1944 scientists thought that complex, diverse proteins were responsible for heredity. In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty reported that the hereditary units, or genes, in bacteria were composed of DNA. This finding inspired Erwin Chargaff (1905–2002) to study the composition of DNA isolated from different species. Chargaff found that the number of adenine units was equal to the amount of thymine, and that the number of guanine units was equal to the amount of cytosine in the DNA from any cell in an organism.

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 deoxyribose, and a nitrogenous base (see Figure 1).

Phosphate groups alternate with deoxyribose 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 greencolored complex. The test only indicates the presence of phosphorus. It does not confirm, on its own, that the extract is DNA.

Nitrogenous bases contain carbon, oxygen, hydrogen and nitrogen in aromatic ring configurations. Two basic types of nitrogen 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. In DNA the pyrimidines are cytosine and thymine, whereas RNA contains uracil instead of thymine. An aldopentose sugar is a monosaccharide with five carbon atoms and a parent aldehyde functional group. The aldopentose in DNA is deoxyribose (see Figure 3a), while the aldopentose 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 (Part A) is of primary importance in many fields of biotechnology. It is critical for genetic research, DNA fingerprinting, and creating recombinant organisms that produce beneficial protein drugs for use in medicine. In this activity, the nucleic acid material of interest is DNA. The cell walls of fruit (bananas in this case) are lysed by mechanically smashing the fruit. Salt is added to soak 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 extract to break apart and emulsify the lipids and proteins that make up the cell and nuclear membranes. Next, the DNA-destroying enzyme DNAse is disabled by adding ethylenediaminetetraacetic acid (EDTA). Finally, 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 interface between the water and alcohol layers and can be collected by “spooling” it onto a glass stirring rod or by drawing it up 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 A, the nuclear material 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 B, chemical tests will be completed on the isolated nucleic acid macromolecule to verify the composition of phosphate, deoxyribose and nucleotide groups.

Materials

Prelab Questions

1. Review the Procedure and Safety Precautions. Discuss the personal protective equipment required for performing this lab. What additional safety precautions are needed for glacial acetic acid and ammonium hydroxide solutions?
2. Explain why several tests instead of a single test are used to verify the chemical composition of the nuclear material.
3. The following bases represent a nucleotide sequence in a strand of DNA. What would be the base composition of its complementary strand? 5′–AGCTTACGTCC
4. Guanine and cytosine form three hydrogen bonds when they are aligned or paired in complementary strands of the DNA double helix. Draw dashed lines between the following stuctures to show the probable location of these hydrogen bonds.
   {14006_PreLab_Figure_4}

Safety Precautions

Ethyl alcohol is a flammable liquid and a dangerous fire risk—keep away from flames and other sources of ignition. 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 severely corrosive to eyes, skin and other body tissues. They are also toxic by ingestion. 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. Sodium dodecyl sulfate solution may be irritating to skin. Avoid contact of all chemicals with eyes and skin. All 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 materials in the chemistry laboratory and do not remove any remaining food items from the lab after use. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part A. Isolating Nucleic Acids

1. Gently “knead” the banana chip mixture for several minutes, and then pour the mixture through four layers of cheesecloth. Collect the liquid in a 400-mL beaker.
2. 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.
3. Use a clean graduated cylinder to add 10 mL of the 0.01 M EDTA to the beaker. Stir with a stirring rod.
4. 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.
5. 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. Do not allow the layers to mix.
6. Carefully place the beaker back on the tabletop, making sure the two layers do not mix.
7. 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.
8. Gently place the tip of a wide-stem pipet into the interface containing the nuclear material to collect the solid.
9. Transfer the solid nuclear material to a 50-mL beaker.
10. Repeat steps 8 and 9 until most of the solid substance has been collected.
11. Dispose of the water/alcohol mixture as directed by the instructor.

Part B. Chemical Testing of Nuclein

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 distilled or deionized water to a second test tube. This will be the negative control (blank).
   3. Use a clean graduated pipet to transfer 2 mL of diphenylamine solution to each test tube.
   4. Place both test tubes into a boiling water bath for 10 minutes.
   5. Record observations in the data table.
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 distilled or deionized water to a second test tube. This will be the negative control (blank).
   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 observations in the data table.
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 distilled or deionized water to a second test tube. This will be the negative control (blank).
   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 observations in the data table.
4. Collect the solutions from the phosphate and purine tests in designated waste beakers located in the fume hood.
5. Pour the contents of the deoxyribose tests into an appropriately labeled waste container as directed by the instrucor.

Student Worksheet PDF

14006_Student1.pdf