EXPERIMENT 8:

NUCLEIC ACIDS
 NUCLEIC ACIDS
◈ Polymers of nucleotides (building blocks)
◈ Components of nucleotides:
○ Sugar (Deoxyribose or ribose)
○ Nitrogen bases (Purine or Pyrimidine)
○ Phosphates

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◈ DNA
○ “Deoxyribonucleic acid”
○ Deoxyribose (sugar)
○ Double-stranded helix
○ Bases pairing:
■ Adenine – Thymine (A-T)
■ Cytosine – Guanine (C-G)

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◈ RNA
○ “Ribonucleic acid”
○ Ribose (sugar)
○ Single-stranded
○ Base pairing:
■ Adenine – Uracil (A-U)
■ Cytosine – Guanine (C-G)

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EXPERIMENTAL
RESULTS:

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A. SEPARATION
OF
RNA 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.
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 HEAT

◈ Helploosen the cell membrane

by increasing the kinetic
energy of lipid molecules,
releasing more RNA.

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 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.

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 ACETIC ACID

◈ Lowers the pH to help denature

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

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 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.

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 HCl (HYDROCHLORIC ACID)

◈ Protonate the Phosphate groups in

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

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B. HYDROLYSIS
OF RNA
 ACID HYDROLYSIS

◈ Causes depurination
◈ Uses H2SO4 (Sulfuric acid)
○ Aids in hydrolysis
◈ Cleaves the N-glycosyl bonds
and Phosphodiester bonds

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QUALITATIVE
TESTS:
 A. 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

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 B. 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

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 C. 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

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 D. 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

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