Reyes, Sashley

Paringit, Jasmine Bijay

Goseingfiao, Jenifer

Leano, Florante

Albano, Kevin

EXPERIMENT X

NUCLEIC ACIDS

OBJECTIVES:

1) To isolate RNA from yeast.

2) To test the properties of the isolated RNA.

MATERIALS:

Reagents Instruments

METHODS:

I. Isolation of RNA from Yeast

II. Qualitative Tests for Nucleic Acids
A. Test for Nucleoproteins
B. Milc Acid Hydrolysiss
a. Test for Inorganic Phosphates
b. Test for the Presence of Pentoses
c. Test for the Presence of Purines

RESULT AND DISCUSSION:

Nucleic acids are made up of nucleotides. A component of each nucleotide is the purine base (adenine
or guanine) or pyrimidine base (cytosine, uracil or thymine), pentacarbon sugar: ribose or deoxyribose
and orthophosphoric acid residue. The base binds the sugar by the -N-glycosidic bond,
orthophosphoric acid residue binds the sugar component by an ester bond through the -OH group at
carbon 3' or 5' of ribose or deoxyribose. Individual nucleotides are bound with phosphodiester bonds
between carbons 3' and 5.
The acidic character of the nucleic acids is caused by orthophosphate residues, each of which contains
H+ capable of dissociation. Due to this, nucleic acids are polyanions - carriers of many negative charges,
and this makes them capable of interacting with polycations, particularly with alkaline proteins - which
are carriers of positive charges. They also bind with micromolecular compounds of an alkaline nature,
such as methylene blue. In the natural environment, DNA mainly binds with alkaline proteins - histones,
whereas RNA mainly binds with neutral proteins, which are part of the ribosome. Complexes of nucleic
acids with proteins are called nucleoproteins. In the student laboratory conditions, artificial
nucleoproteins can be obtained by mixing a solution of nucleic acid with blood serum. Nucleoproteins
dissociate into its constituents in concentrated salt solutions. In an alkaline environment, nucleic acids
form salts - nucleates, which are soluble in water. They can be precipitated from solution with ethanol.

Sugar constituents of nucleic acids: ribose and deoxyribose can be detected directly in the solutions of
these acids or their salts without prior hydrolysis. Ribose contained in the RNA, purine nucleosides and
nucleotides, heated with concentrated HCl dehydrates to furfural, which with orcin forms a complex of a
stable green colour. Deoxyribose, contained in the DNA, when heated with concentrated sulphuric acid
is converted into hydroxylevulinyl aldehyde. This compound forms a blue colour complex in reaction
with diphenylamine. Purine bases can only be detected in the hydrolysis products of nucleic acids. The
sample of nucleic acid, intended for the detection of purines, should be hydrolysed in sulphuric acid at
100C. Nucleic acids are hydrolysed, initially to mononucleotides. Purine mononucleotides are further
hydrolysed to bases, pentoses and orthophosphoric acid. The action of this acid leads to the hydrolytic
breakdown of the -N-glycosidic bonds between the purine and the ribose or deoxyribose. The following
purine bases are released: adenine and guanine. Purine bases precipitate easily as insoluble complexes
with the ions of copper or silver. In the same conditions, pyrimidine nucleotides are stable and do not
decompose.

Name of Test Observation Explanation

Biuret Test for Nucleoproteins ++ Purple with blue precipitates Formation of purple colored
product due to the reduction of
Cu2+ to Cu+
Test for Phosphates (Acid + Yellow solution with yellow Hydrolysis of pyrophosphate to
Hydrolysate) precipitates phosphate forming yellow
precipitate
Test for Sugar
0.1% Ribose +++ Olive green solution Pentose is dehydrated to furfural
and reacts with orcinol to form
green product
0.1% Glucose ++ Yellow clear solution No reaction with orcinol
Acid hydrolysate ++ Olive green solution Pentose is dehydrated to furfural
and reacts with orcinol to form
green product
Test for Purine Bases + White precipitates in foamy Hydrolysis of N-beta-glucosidic
 solution bonds between purine and
ribose result in a release of
purine bases (A and G) caused by
NH4OH
Ag+ precipitate cause the
formation of foamy gelatinous
substance

APPLICATION

Used in nucleic acid testing:

- Blood screening
- Detecting infectious disease
- Predicting cancer and guiding cancer treatment
- For personalized and precision medicine
- Screening for genetic disorders
- Diagnosis of mitochondrial diseases

QUESTIONS

1) What are the purine bases found in nucleic acids?

Adenine = 6-amino purine

Guanine = 2-amino-6-oxy purine
Hypoxanthine = 6-oxy purine
Xanthine = 2,6-dioxy purine

2) How do you account for the formation of precipitates in the test for purines?