EXPERIMENT #8 : Nucleic Acids

NUCLEIC ACIDS
◈ Polymers of nucleotides (building blocks)

◈ Components of nucleotides:

○ Sugar (Deoxyribose or ribose)

○ Nitrogen bases (Purine or Pyrimidine)

○ Phosphates

DNA
○ “Deoxyribonucleic acid”

○ Deoxyribose (sugar)

○ Double-stranded helix

○ Bases pairing:

■ Adenine – Thymine (A-T)

■ Cytosine – Guanine (C-G)

RNA
○ “Ribonucleic acid”

○ Ribose (sugar)

○ Single-stranded

○ Base pairing:

■ Adenine – Uracil (A-U)

■ Cytosine – Guanine (C-G)

EXPERIMENTAL RESULTS
A. Separation from yeast

NaOH (Sodium Hydroxide)

◈ Disrupts the cell membrane and lyse the cell extracting the nucleic acids.
 ◈ Increases the pH resulting in the denaturation of contaminant proteins.

◈ Inactivates nucleases that can degrade RNA.

HEAT
◈ Help loosen the cell membrane by increasing the kinetic energy of lipid molecules, releasing
more RNA.

CENTRIFUGATION
◈ Remove the denatured proteins, lysed lipid membrane and other contaminants.

◈ It was repeated to eliminate the precipitated proteins.

◈ Separates the RNA precipitate from the unneeded supernatant.

ACETIC ACID
◈ Lowers the pH to help denature more proteins.

◈ Prevents alkali RNA hydrolysis, ensuring that the desired RNA was not degraded.

ETHANOL (ETHYL ALCOHOL)

◈ Lowers the dielectric constant of the solution.

◈ Reduce the solubility of RNA causing it to precipitate.

◈ Washing with Ethanol

Removes any lipid residues and other nonpolar contaminants.

HCL (Hydrochloric Acid)

◈ Protonate the Phosphate groups in the nucleic acid backbones.

◈ Minimizes the charge repulsion between molecules and helped aggregate and precipitate.

B. Hydrolysis of RNA
ACID HYDROLYSIS
◈ Causes depurination

◈ Uses H2SO4 (Sulfuric acid)

○ Aids in hydrolysis

◈ Cleaves the N-glycosyl bonds and Phosphodiester bonds

QUALITATIVE TESTS
ORCINOL/BIAL’s TEST
◈ Test for pentoses

◈ Principle: Pentoses are dehydrated to furfural

◈ Positive result: Blue-green solution

ACID HYDROLYSATE UNHYDROLYZED (RNA)

BLUE-GREEN BLUE

INORGANIC PHOSPHATES
◈ Ammonium Molybdate test

◈ Principle: Presence of Phosphate ions/ Ammonium Phosphomolybdate

◈ Positive result: Yellow precipitate

ACID HYDROLYSATE UNHYDROLYZED (RNA)

YELLOW PRECIPITATE NO COLOR

CHANGE

PURINE BASES
◈ Detection of purine bases (Adenine/ Guanine)

◈ Principle: Hydrolysis of the bonds between sugars and bases results in release of purine bases.

◈ Positive result: White precipitate

ACID HYDROLYSATE UNHYDROLYZED (RNA)

WHITE NO PRECIPITATE
FORMED
PRECIPITATE
BENEDICT’s TEST
◈ Test for reducing sugars

◈ Principle: Copper (II) react with reducing sugars to precipitate into Cu2O.

◈ Green – small amount

◈ Orange – large amount

ACID HYDROLYSATE UNHYDROLYZED (RNA)

GREEN TO ORANGE BLUE

SOLUTION SOLUTION