Experiment #8: Nucleic Acids

I. Objectives

At the end of the experiment, students should be able to:

a. isolate RNA; and

b. test qualitatively for the presence of inorganic phosphate, purine bases, and reducing sugars.

II. Materials

Test tubes, beaker, graduated cylinder, cheesecloth, water bath, iron stand, Bunsen burner,
stirring rod, centrifuge, dropper, water,

III. Reagents

1% NaOH, Acetic Acid, 95% Ethyl Alcohol, conc. HCl, 10%H 2SO4, Orcinol, NH3, 6N HNO3,
Ammonium Molybdate, CuSO4, NaHSO3, Na2CO3, Benedict’s Reagent

IV. Procedures:

A. Separation of Ribonucleic acid (RNA) from yeast

1. Dilute 10mL of 1% NaOH with 50mL water.

2. Add 6 grams dried yeast and heat in water bath for 30 minutes with occasional
stirring. 3. Filter through cheesecloth and centrifuge for 10 minutes.
4. Decant into a beaker and cool the supernatant liquid.
5. Add glacial acetic acid dropwise until faintly acidic to litmus paper, if turbid, transfer in a test
tube and centrifuge for 5 minutes. Decant.
6. Evaporate supernatant liquid, filter if necessary.
7. Cool the filtrate below 40°C then pour in 40mL of 95% ethyl alcohol containing 2 drops of
conc. HCl.
8. Allow RNA to settle, decant and wash with 95% alcohol. Dry at room temperature.

B. Hydrolysis of RNA

1. Place a small portion of RNA from yeast in a test tube.

2, Add about 10mL of 10% sulfuric acid. Cover the test tube loosely and boil in water bath for 30
minutes.
 3. Use the solution in number 2 and the unhydrolyzed RNA (dissolved in water) for the following
tests and compare results:
∙ Orcinol test for ribose – Place 1mL dissolved RNA in a test tube and into another test tube
with 1mL of hydrolysate from number 2. Add 5 drops of orcinol reagent and heat in
water bath. Cool the tube immediately under the faucet and compare the results.
∙ Test for Inorganic Phosphates – Add excess NH 3 to 1mL of the test solution. Acidify with 6N
HNO3. Add 1mL of Ammonium Molybdate reagent and heat in water bath. ∙ Test for Purine
bases – Dissolve a pinch of the prepared RNA in 1mL of water, add to another test tube, 1mL
hydrolysate. Then add 1mL of 2N HCl. Mix and place in a boiling water bath for 20 minutes.
Add 1mL 2N NaOH and 2mL of Acetate buffer, mix and heat in water bath. Add 0.5mL of
10% CuSO4 and note formation of a bluish brown precipitate. Add 10 drops of NaHSO 3
solution and mix. After a few minutes of heating, a white or light tan flocculent precipitate
forms.
∙ Benedict’s test – Neutralize 2mL of the test solution with Na 2CO3. Decant. To 1mL of the
neutralized sample, add 1mL of Benedict’s reagent. Heat in boiling water bath and note
results.
Experiment #8

Analysis of Nucleic Acid

Name: Date:
Section/Group: Instructor:

I. Acid Hydrolysis of RNA

Test Hydrolyzed RNA Unhydrolyzed RNA

1. Orcinol test

2. Phosphate

3. Purine bases

4. Benedict’s test Free ribos, fructose, glucose RNA, DNA, ATP

II. Questions

1. Give the biological function of nucleic acid.

Large macromolecules called nucleic acids are necessary for all organisms and viruses to function. The
preservation and expression of genetic data is a key role of nucleic acids. The information cells require to
build proteins is encoded in deoxyribonucleic acid or DNA.

2. Why is yeast used in the isolation of RNA?

Preparations of ribonucleic acid are typically made from yeast. This is due to the highest RNA-DNA ratios
in these microorganisms. Technology for yeast fermentation and recovery has also been established.

3. What are the nitrogen bases found in nucleic acid?

In DNA There is Adenine (A), Thymine (T), Cytosine (C), Guanine (G). In RNA Thymine (T) will be replaced
by uracil (U)

4. What are the common sugars found in nucleic acid?

Ribose and Deoxyribose. The pentose sugar in DNA is called deoxyribose and the sugar in RNA is ribose.
The difference between sugars is the presence of a hydroxyl group at the 2’ carbon of ribose and the
absence of a hydroxyl group at the 2’ carbon of deoxyribose.

5. What do RNA and DNA stand for? How will you differentiate the two?

DNA stands for deoxyribonucleic acid, while RNA is ribonucleic acid. DNA replicates on its own, It is self
replicating. RNA does not replicate itself. The two forms of nucleic acid have similar structures, consisting
of two alternating chemical groups: phosphate and a sugar, ribose. Sugars in the backbone of DNA lack a
hydroxyl group (hydrogen plus oxygen), converting ribose to deoxyribose. This small difference has big
chemistry implications: with its more reactive ribose sugar, RNA is more likely to react with other
molecules and even with itself. The two nucleic acids also differ in structure. While RNA is typically a
single-stranded molecule, DNA forms the iconic double helix: double strands winding around each other
like a twisted ladder. The rungs of the ladder represent hydrogen bonds electrostatic attractions that
pair complementary letters on opposite strands: A with T, C with G. Such base pairing occurs throughout
DNA, Holding its strands together like teeth in two halves. Of a zipper unlike DNA, RNA does not
normally exist as a long double-stranded molecule, but as a single strand. And yet, complementary
letters from separate stretches of an RNA strand can pair up (“AAAA” matches “UUUU” for example),
creating double-stranded regions called stems. DNA is used to store information, a bit like a computer
hard drive. RNA is used to carry information or convert information into proteins. RNA has several forms.
Messenger RNA carries copies of genes to ribosomes. Transfer RNA: transports the building blocks of
protein called amino acids to the ribosomes. Ribosomal RNA: ribosomes are made up of RNA. DNA is
essentially the “master copy” of almost all cell instructions. It does not leave the cell nucleus.