Activity Extraction and Classification Tests for Nucleic Acids

No. 6 Title of the Activity

Instructions:

1. Search your answers from a reliable source.

2. All questions and answers should be typewritten. Font: Arial, size 12 and justified
paragraph.
3. Questions in black color and your answers in red color.
4. Submit as pdf file.
5. FILE NAME should be: Act6_MCPH30_SURNAME

I. Overview

Nucleic acids are mildly soluble in cold water, insoluble in alcohol, but mixes
instantly in weak alkali forming alkali metal salts. They are precipitated from alkaline
solution by the addition of acid.

In isolating RNA from yeast, heating with alkali is essential. This extracts the
nucleic acid is separated from associated protein and other interfering substances
by acid extraction at pH 4 to 5. The final step is treatment with alcohol and
concentrated HCl to precipitate the RNA, followed by repeated washing with alcohol
and other organic solvents to remove substances that may interfere with chemical
tests.

Complete hydrolysis of nucleotides yield a mixture of sugar (ribose), purine

(adenine and uracil) or pyrimidine (guanine and cytosine), and phosphate.

II. Procedures

A. Isolation of RNA from Yeast

Mix and grind 2g yeast with 2g white sand in a mortar. Then add freshly prepared
15 mL 0.2% NaOH to make a smooth creamy paste. Pour the mixture in a
beaker and dilute 0.2% NaOH solution to make 50 mL. Cover the beaker with
watch glass to avoud evaporation. Heat the beaker in water bath with a constant
temperature of 90 degC for 30 minutes. Filter the solution thrice using
cheesecloth and once through filter paper. Allow the filtrate to cool. Use filtrate
for the qualitative test for nucleic acids.

B. Qualitative Tests for Nucleic Acids

1. Test for Nucleoproteins:

 Place the filtrate in a test tube, and then add 10% NaOH solution and drops of
1% CuSO4 solution.

2. Mild acid hydrolysis:

Place 10% H2SO4 to the remaining filtrate in a beaker. Boil solution gently for
a few minutes. Use the solution for the following tests;

a. Test for inorganic phosphates

Add ammonia to the filtrate. Acidify it using 10% HNO 3 then add
ammonium molybdate. Boil the solution then allow it to stand for a few
minutes. Note the color of the precipitate.

b. Test for the presence of pentoses (ribose)

Test tube 1: 0.1% ribose solution

Test tube 2: 0.1% glucose solution
Test tube 3: Solution from acid hydrolysis

Add Bial orcinol reagent to each test tube. Place the test tubes in a
boiling water bath until the color changes. Note the color for each test
tube.

c. Test for the presence of purines

Add NH4OH to the filtrate in a test tube. Add drops of AgNO3 solution
to it. Note the color of the precipitate.

III. Results and Questions

Name of Test Visible Result Explanation

Biuret Test for

Nucleoproteins (filtrate
from yeast RNA)

a. Test for phosphates

(acid hydrolyzate)
b. Test for sugar

0.1% ribose

0.1% glucose

Acid hydrolyzate
(from mild hydrolysis)

c. Test for purine bases

(acid hydrolyzate)

Questions: Relate your answer in the activity

1. How do you account for the formation of precipitates in the test for purines?
2. Explain the principles of the qualitative tests for nuclei acids.