Experiment 7: Nucleic Acids
ABSTRACT
Nucleic acids are large biological molecules essential for all known forms of life. They
include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Together with proteins,
nucleic acids are the most important biological macromolecules. One DNA or RNA molecule
differs from another primarily in the sequence of nucleotides. Ribonucleic acid (RNA) functions
in converting genetic information from genes into the amino acid sequences of proteins. In this
experiment, RNA was isolated from yeast (Saccharomyces cerevisiae) by heating the active dry yeast with
alkaline NaOH. This method of RNA extraction involved the disruption of the cell membrane and subcellular
nucleus to break open and ischarge the nucleic acids. RNA was extracted from associated proteins with HCl
extraction and was treated with ethanol and ether to remove lipids.
INTRODUCTION
Nucleic acids are macrobiopolymers of high molecular weight with mononucleotide as
the repeating unit. The two structural kinds of nucleic acids are DNA and RNA which are
basically made up of nitrogenous bases, sugar, and phosphate group. The sequence of
nucleotides allows RNA to encode genetic information. For example, some viruses use RNA
instead of DNA as their genetic material. In isolating RNA, heating with alkali, acid extraction,
and treatment with alcohol are involved.
Yeast, Saccharomyces cerevisiae, is a unicellular fungus that contains 4% RNA by
weight; it has low d-ribonuclease and ribonuclease activity and can be readily obtained in
essentially pure form from this source. Many proteins in human biology were first discovered by
their homologs in yeast. S. cerevisiae was the first eukaryotic genome to be completely
sequenced. It was estimated that yeast shares 23% of its genome with humans.
MATERIALS AND METHODS
In a beaker, 5mL of 1% NaOH solution and 25mL of water were diluted and was added
with 5.0g of dry yeast. The mixture was set to heat in a water bath for 15 minutes with
occasional stirring. After heating, the mixture was strained with cheesecloth. The filtrate was
centrifuged and the supernate was transferred to a different test tube and was added glacial acetic
acid dropwise until faintly acidic. It was observed that the supernate was turbid; it was
centrifuged and decanted after. Twenty milliliters of 95% ethanol containing 0.2 mL of conc.
HCl was poured to the supernate while it was stirred vigorously.
All residues were decanted, centrifuged, and transferred in one big test tube and was
washed twice with 2mL of 95% ethanol. The decantation and centrifugation processes were
repeated every after washing. It was washed again twice with 2mL ether with the same process
as the previous; it was centrifuged and decanted every after washing. After the washing, the
residue was divided into two portions in two separate test tubes and cover. The test tubes were
refrigerated to be used in the following experiment in the next laboratory meeting.
RESULTS AND DISCUSSION
The isolation of RNA from yeast involves heating with NaOH which served to disrupt the
cell membrane and lyse the cell extracting the nucleic acids. The NaOH also increases the pH
level of the solution resulting in the denaturation of contaminant proteins, inactivates nucleases
which can degrade RNA. Heating helped loosen the cell membrane by increasing the kinetic
energy of the lipid molecules, making it release more RNA. The mixture was filtered and
centrifuged to get rid of the denatured proteins, lysed lipid membranes and other contaminants.
The purpose of the addition of glacial acetic acid was to lower the pH level to help denature
more proteins, prevented alkali RNA hydrolysis, ensuring that the desired RNA was not
degraded. The mixture was decanted and centrifuged repeatedly to eliminate the precipitated
proteins. The reason why the supernate was suggested to be 10mL or below was to increase the
RNA in the solution and for it to be easily isolated later on. Ethanol was added to lower the
dielectric constant of the solution and reduced the solubility of RNA causing it to precipitate
from solution. HCl was added to protonate the phosphate groups in nucleic acid backbones,
minimizing the charge repulsions between molecules and helped aggregate and precipitate.
Centrifugation also separated the RNA precipitate from the unneeded supernatant. Both
washings from ethanol and ether removed any lipid residues and other non-polar contaminants.
.
REFERENCES
1.)
Elson D (1965). "Metabolism of nucleic acids (macromolecular DNA and RNA)". Annu. Rev. Biochem. 34:
449–86
2.) Stryer, L.; Berg, J.; Tymoczko, J. (2007). Biochemistry. San Francisco: W.H. Freeman