Nucleic-acid

EXPERIMENT NO. 8

NUCLEIC ACID BIOCHEMISTY GROUP 1

Our Agenda for Today

- Nucleic acid - Objectives - Materials - Isolation of RNA from Yeast - Qualitative test: Test for nucleoprotein Mild acid hydrolysis: Test for Inorganic phosphates Test for the presence of ribose (pentose) Test for the presence of purines

NUCLEIC ACID Nucleic acids, macromolecules made out of units called nucleotides, come in two naturally occurring varieties : deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the genetic material found in living organisms, all the way from single-celled bacteria to multicellular mammals like you and me. Some viruses use RNA, not DNA, as their genetic material, but aren’t technically considered to be alive (since they cannot reproduce without help from a host).

1.To isolate RNA from yeast

1.2. To test the properties of the isolated RNA

OBJECTIVES

Materials: - Mortar - 4 beakers (250 ml) - Graduated cylinder - Pipette - Aspirator - Watch glass - Tripod - Bunsen Burner - 12 test tubes - Cheese cloth - Filter paper

Reagents: - 5% ammonium molybdate - 0.1% ribose solution - 0.1 % glucose - Bial orcinal reagent - 10% 𝑁𝐻4 𝑂𝐻 - 5% 𝐴𝑔𝑁𝑂3 - Yeast - White sand - 0.2% NaOH - 10% NaOH - 1% 𝐶𝑢𝑆𝑂4 - 10% 𝐻2 𝑆𝑂4

Isolation of RNA from Yeast

Mix and grind 2 grams of yeast with 2 grams of white sand in a mortar. Then add 15 ml of freshly prepared 0.2% NaOH to make a smooth creamy paste. Pour the mixture in a 250 ml beaker and dilute with 0.2% NaOH solution to make 50 ml.

Isolation of RNA from Yeast

Cover the breaker with a watch glass to avoid evaporation. Heat the beaker in a water bath with a constant temperature of 90 degrees Celsius for 30 minutes. Filter the solution thrice using cheese cloth and once through filter paper.

Isolation of RNA from Yeast

Allow the filtrate to cool. Perform the following test on the filtrate.

QUALITATIVE TEST Test for nucleoprotein Mild acid hydrolysis: Test for Inorganic phosphates Test for the presence of ribose (pentose) Test for the presence of purines

QUALITATIVE TEST Test for nucleoprotein Mild acid hydrolysis: Test for Inorganic phosphates Test for the presence of ribose (pentose) Test for the presence of purines

PROCEDURE: Place 1ml of filtrate in a test tube, and then add 1ml of 10% solution and 5-10 drops of 1% CuSO4 solution. Notice the colour produced.

Tests Nucleoproteins (filtrate from RNA)

Observation

Explanation

When the filtrate and The proteins reacted yeast NaOH were added to the solution. together, the solution turned yellow. When 10 drops of CuSo4 was added the colour then changed to a light green-blue. Blue precipitate formed at bottom of the test tube

QUALITATIVE TEST Test for nucleoprotein Mild acid hydrolysis: Test for Inorganic phosphates Test for the presence of ribose (pentose) Test for the presence of purines

PROCEDURE: Place 20 ml of 10% H2SO4 to the remaining filtrate in a beaker. Boil the solution gently for a few minutes. Perform the following tests on the solution.

QUALITATIVE TEST Test for nucleoprotein Mild acid hydrolysis: Test for Inorganic phosphates Test for the presence of ribose (pentose) Test for the presence of purines

PROCEDURE: Add 1-2 ml ammonia to 1ml of the filtrate. Acidify it using 10% HNO3 then add 2 ml of ammonium molybdate. Boil the solution then allow it to stand for a few minutes. Notice the colour of the precipitate.

Hydrolysis of pyrophosphate yellow precipitate.

to

phosphate

forming

Tests

Phosphates (Acid Hydrolyzate)

Observation

Explanation

After mixing the After the mixture of 2mL solutions and boiling it ammonia to the 1mL of into the water bath there the filtrate, we acidified is a yellow precipation it using 10% HNO3 and formed and after some additional of 2mL of minutes the precipitation ammonium molybdate. formed dissolved. We have observed that there is a formation of yellow color in the liquid and after boiling it onto the water bath, solid yellow precipitation has shown and therefore it is a positive result for the test on inorganic phosphate.

QUALITATIVE TEST Test for nucleoprotein Mild acid hydrolysis: Test for Inorganic phosphates Test for the presence of ribose (pentose) Test for the presence of purines

PROCEDURE: Prepare 3 test tubes and place the following solutions as indicated below: Test tube Number 1: 1ml of 0.1% of ribose solution Test tube number 2: 1ml of 0.1% glucose solution Test tube Number 3: 1ml solution from the acid hydrolysis

QUALITATIVE TEST Test for nucleoprotein Mild acid hydrolysis: Test for Inorganic phosphates Test for the presence of ribose (pentose) Test for the presence of purines

PROCEDURE: Add 3 ml of bial orcinol reagent to each test tube. Place the test tubes in a boiling water bath until colour changes. Compare the colour formed with the one that contains the acid hydrolysis.

Bial’s test is useful in distinguishing pentoses sugar from hexoses sugars. Pentosses ( such as ribose sugar) form furfural in acidic medium which condense with orcinol in presence of ferric ion to give blue green colored complex which is soluble in butyl alcohol. Positive Bial’s test: formation of blue color ( eg. Ribose sugar)

Negative Bial’s test: formation of any other color indicates negative test. Hexose sugar ( glucose, fructose) generally gives green, red or brown color product.

TEST TUBE #1 Tests

0.1% Ribose

Observation

Explanation

The color of the solution A positive result in the (filtrate and 1ml of 0.1% experiment indicates ribose solution) changed change in color, the when we added 3 ml of specific color is blue and bial orcinol, it turned into formed a precipitate. The dark yellow. Then we result of the color, proves have observed after being the solution indicates heated, the color turned change in color but the into yellow orange or color is orange and not orange. blue, and it also did not form a precipitation in the solution. Therefore, the solution is negative.

TEST TUBE #2 Tests

0.1% Glucose

Observation

Explanation

The color of the solution A negative result in the (filtrate and 1ml of 0.1% experiment indicates glucose solution) changed change in color. The when we added 3 ml of result of the color, proves bial orcinol, it turned into the solution to be positive clear yellow. Then we since it did not turn into a have observed the bluish color nor solution after being precipitate. heated, the color turned into yellow.

TEST TUBE #3 Tests

Acid Hydrolyzate

Observation

Explanation

After adding Bial orcinol in the test tube containing 1ml of acid hydrolysis solution change in color appeared. The color of the solution turned into a dirty white color. Afterwards, we placed the test tube in the water bath, we check the solution for every two minutes if there’s a change in color happened. After boiling for about 6 minutes the solution turned into an unclear dirty white color.

QUALITATIVE TEST Test for nucleoprotein Mild acid hydrolysis: Test for Inorganic phosphates Test for the presence of ribose (pentose) Test for the presence of purines

PROCEDURE: Add 3ml 10% NH4OH to 2 ml of the filtrate in a test tube. Mix 2-3 drops of 5% AGNO3 solution to it. Notice the colour of the precipitate formed.

Hydrolysis of N-β-glucosidic bonds between purine bases and ribose or deoxyribose results in a release of purine bases(A and G) caused by NH4OH. Ag+ precipitate caused the formation of foamy gelatinous substance.

Tests Purine Bases (Acid Hydrolyzate)

Observation

Explanation

When added with 3 mL  Purine test: The result 10% NH4OH, white dots in this experiment was appeared within the negative becuase there solution. is no reaction from 10%  After mixing with 2-3 NHO4 after mixing with drops of 5% AgNO3, the the 5% AgNO3. white dots disappeared and the solution had an  Acid hydro: In this even color. experiment it was non acidified because of none color change simply means it is negative. The color turned into unclear dirty white color.

QUESTIONS: Why is the yeast used as a source of RNA? One of the many advantages to using yeast as a model system is that large quantities of biomacromolecules, including nucleic acids (DNA and RNA), can be purified from the cultured cells.

QUESTIONS: Name the purine bases found in nucleic acid: There are only two purine bases found in nucleic acid, which is the adenine and guanine.

QUESTIONS: Account for the formation of precipitates in the test of purines. The formation of precipitate in the test for purines is used because purines cannot be found in human DNA unless a test is performed to find their waste or byproducts. Purines cannot be tested for without boiling in alcohol to break them down.

FORMULA: YEAST: C19 H14 O 2

WHITE SAND: SiO 2

SOLUTIONS: - ammonium molybdate

- ribose solution C5H10O5 - glucose solution C6H12O6

FORMULA: SOLUTIONS: - Bial orcinol reagent - 𝑁𝐻4 𝑂𝐻

- 𝐴𝑔𝑁𝑂3

FORMULA: - NaOH - 1% 𝐶𝑢𝑆𝑂4

- 10% 𝐻2 𝑆𝑂4

SOLUTIONS:

Amines Aromatic Hydrocarbon Carbon Enzymes Ester Ether Monosaccharide Salt Substitution reaction

GROUP 1 Members: