EXPERIMENT 3: NUCLEIC ACIDS

Lloyd Patrick Gilig, Manuel Wilbert Guerrero, and Shaina Chelsey

Mainit
Locker no.71
Chemistry Department, Xavier University-Ateneo de Cagayan, Cagayan de
Oro City
Date Performed: January 4, 2017
Date Submitted: January 11, 2017

ABSTRACT
The nucleic acids are the building blocks of living organisms. DNA is just one
type of nucleic acid. Some other types are RNA, mRNA, and tRNA. All of these work
together to help cells replicate and build proteins. The objectives of this experiment
are to isolate yeast RNA from 5.0g dry yeast using 1% NaOH, to perform qualitative
tests on the hydrolysate and unhydrolyzed RNA, and to hydrolyze from yeast the
nucleic acid present in it.
INTRODUCTION
Nucleic acids, which are composed of nucleotides, are very large and
complex organic molecules that contain the genetic code for that organism. Two
closely related types are needed to transmit the genetic information from parent to
offspring: DNA and RNA. They also share structural similarities. Both DNA
(deoxyribonucleic acid) and RNA (ribonucleic acid) are polymers of individual
nucleotides. Each nucleotide has three components, namely, five-carbon-ring sugar
(deoxyribose or ribose), phosphate group, and nitrogen base. Both DNA and RNA
have four nitrogen bases available to construct nucleotides. Three of the nitrogen
bases are the same.
Nucleic acids allow organisms to transfer genetic information from one
generation to the next. When a cell divides, its DNA is copied and passed from one
cell generation to the next generation. DNA is organized into chromosomes and
found within the nucleus of our cells. It contains the "programmatic instructions" for
cellular activities. When organisms produce offspring, these instructions, in the form
of DNA, are passed down. RNA is essential to the synthesis of proteins. It is also a
component of cell organelles called ribosomes. Information contained within the
genetic code is typically passed from DNA to RNA to the resulting proteins.
In nucleic acid dehydration synthesis, nitrogenous bases are joined together
and a water molecule is lost in the process. Interestingly, some nucleotides perform
important cellular functions as "individual" molecules, the most common example
being ATP. DNA consists of the four nitrogenous bases: adenine (A), guanine (G),
cytosine (C), and thymine (T). In double stranded DNA, adenine pairs with thymine
(A-T) and guanine pairs with cytosine (G-C). Single stranded RNA contains the

nitrogenous bases adenine, guanine, cytosine and uracil (U). When DNA is
transcribed into an RNA transcript during DNA transcription, guanine pairs with
cytosine (G-C) and adenine pairs with uracil (A-U).

Results and Discussion

A. Isolation of yeast RNA

RNA isolation from yeast involves heating with NaOH, it serves to
disrupt the cell membrane and rupture the cell extracting the nucleic acids.
The NaOH also increases the pH of the solution resulting in the denaturation
of contaminant proteins, and inactivates nucleases which can degrade RNA.
the purpose of centrifuging the solution was to separate and get rid of the
denatured proteins, lysed lipid membranes and other contaminants. The
glacial acetate then was added to lower the pH to denature more
contaminant and unwanted proteins and prevent alkali RNA hydrolysis,
further purifying the isolation of RNA. The precipitated proteins was removed
by decantation. Ethanol helps lower the dielectric constant of the solution
and to reduce the solubility of RNA causing precipitation. The Hydrochloric
acid was then added to protonate the phosphate groups in nucleic acid
backbones. Also centrifugation separated the RNA from the unwanted
supernatant.

B. Qualitative Test
1. Benedicts
Test

SAMPLES
Hydrolysate

Unhyrdolized RNA

Colorized Soln

No observable
change

2. Orcinol Test

Gold Colored
Solution

Solution turned to
white

3. Test for Purine

Bases

Purple ppt formed

White ppt formed

4. Test for inorganic

phosphate

Lime colored solution Lime colored solution

A. RNA is a nucleotide containing a reducing sugar. If the test for

reducing sugars is done after RNA hydrolysis the RNA will react with
the Benedicts solution since hydrolysis releases the ribose sugar
present in the RNA. since Hydrolysis of RNA produces sugar base and
phosphate.
B. In the Orcinol test the concentrated Hydrochloric acid in the
orcinol reagent dehydrates the ribose, pentose sugar present in the
RNA molecule into furfural. The furfural then reacts wit the 3,5dihydrotoluene in the presence of ferric cholride present in the orcinol
reagent as a catalyst purine to give green color to the purine
nucleotide. The green coloration indicates that the reaction was
positive.
C. Hydrolisys of N-Beta-glucosidic bonds between purine bases and
ribose results in the release of purine bases that is due to the NH4OH
and Ag+ ppt present causes the formation of a foamy gelatinous
substance.
D. The test for inorganic phosphate or ammonium molybdate test
hydrolyzes the pyrophosphate to phosphate and reacts with the
reagent and produces the yellow precipitate.
In the experiment the hydrolyzed Rna is positive for orcinol test, and
test for purine bases. While Hydrolized RNA is positive only to test for
inorganic Phosphate.

Conclusion
RNA can be extracted from yeast by rupturing the yeast cells,
increasing the pH level to denature unwanted and contaminant proteins,
lower the pH level to denature the contaminant proteins and substances, and
centrifugation to separate the denatured substances and the unwanted
substances.
There are also different test to determine the content and composition
of nucleic acids in this case RNA. In the experiment the hydrolyzed Rna is
positive for orcinol test, and test for purine bases. While Hydrolyzed RNA is
positive only to test for inorganic Phosphate.

REFERENCES

1)

Nucleic Acids. (April 25, 2016.) About Education. Retrieved from

http://biology.about.com/od/molecularbiology/a/nucleicacids.htm.
2.) Malacinski, G. Essentials of Molecular Biology. Jones & Bartlett learning. 2005. Page
84
3.) Harisha,S. An Introduction to Practical Biotechnology. Firewall Media. 2005. Page 60
4.) Kuehner, J. Mechanism and Regulation of Yeast RNA Polymerase Transcription
Initiation and Termination. Proquest.2008. Page 144.