Isolation and characterization of Nucleic acids from an onion (Allium cepa)

Christian D. Aceret, Harold James O. Alcantara, Ixzi Thia G. Alforque,

Jewel Hannah M. Angel, Rischthelle S. Aton, and Amiel Rovic T. Babaran
Group 1 2B Medical Technology, Biochemistry Laboratory

ABSTRACT
Nucleic acids, including DNA and RNA, are macromolecules that store genetic information and enable protein production
for metabolic functions and cell division. The experiment conducted activities on isolation of DNA from the vegetable
source onion (Allium cepa), Ultraviolet measurement of DNA, and chemical characterization of the DNA sample. The
DNA isolation was done following the procedure. The Ultraviolet determination of purity was carried out using UV-
Vis/Microplate Spectrophotometer, and then the absorbance ratio was calculated. Lastly, the DNA underwent Acid
hydrolysis with the use of Hydrochloric acid (HCl) for the preparation for the chemical characterization, after which the
procedure have utilized tests such as Deoxyribose test (Dische test), Phosphate test, Purine test (Murexide tes t), and
Pyrimidine test (Wheeler-Johnson test). Results in the Ultraviolet measurement has shown that for the absorbance of
the sample at 260nm, the Nucleic acid concentration is 7 μg/mL. For the absorbance at 280nm, the Protein concentration
is 0.8 μg/mL. Purity test showed that for the A260/A280 ratio for measurement protein contamination in the presence
of nucleic acids yielded 0.245. The A260/A230 ratio for secondary measure of nucleic acid purity yielded 1.14. In the
chemical characterization, results in the Deoxyribose test yielded blue solution, Phosphate test yielded yellow solution
with white fumes, Purine test yielded red residue, and lastly, Pyrimidine test yielded dark red precipitate.

INTRODUCTION DNA are adenine, guanine, cytosine and thymine

Nucleic Acids are one of the most important (exclusive to DNA). [2] These DNA are present in
biochemical molecules in every living organism. the chromosomes of the nuclei in eukaryotic cells.
They are most known for storing genetic
information which determines the expression of
our characteristics and traits. It functions to
encode, transmit and express genetic information
by means of protein synthesis. The two types of
nucleic acids are DNA (deoxyribonucleic acid) and
RNA (ribonucleic acid). The former was the focus
of the experiment.

Figure 1. The basic structure of a nucleic acid.
[1] Among the biochemical structures, DNA
molecules are one of the largest. The nucleic acid
molecules are 21 nucleotides long to large
chromosomes (1 chromosome in humans can
contain up to 247 million base pairs). They exist in
a double stranded, double helix form. In general,
nucleic acids are a chain of nucleotides. Each
nucleotide consists of a base (a purine or
pyrimidine nucleobase), a sugar and a phosphate
backbone.
The sugar in DNA is a 2-deoxy-D-ribose
and this sugar is bonded with the base through a
beta-N-glycosidic bond. The bases which exist in
Figure 2. The basic structure of a DNA double
helix.
[3] The nucleotides form polymers through
the bonding of the 3' sugar of one nucleotide and
the 5' phosphate group of another nucleotides
through phosphodiester linkages. This form of
bonding gives nucleic acids directionality and the
monomers of DNA are read from the 5'-end and
3'-end.
The objectives of the experiment are to
isolate the DNA in Allium cepa also known as
onion, determine the extracted DNA's purity,
perform acid hydrolysis on the extracted DNA and
after which characterize the acid hydrolysate.
EXPERIMENTAL ORGANIZATION amount of DNA [4]. Moreover, onion allows easier
visualization of DNA due to its low starch content
A. Reagents used for DNA Isolation from [5].
In order to recover the DNA, a homogenizing
an onion
solution is added causing the cell membrane to be
2 yellow onions
digested by dissolving the lipids and proteins of
Ice-cold 95% ethanol
the cell and disrupting the bonds stabilizing the cell
Commercial papain or meat tenderizer (6% in
membrane [5]. The DNA is then liberated when
water)
treated with Papain which hydrolyzes protein into
Homogenizing solution: 5% SDS
smaller peptides, exposing the nonpolar groups of
0.15 M NaCl
the amino acid, making it insoluble when mixed
0.15 M Na3C6H5O7
with ice-cold ethanol. Once precipitated out, the
0.001 M EDTA
DNA may be spooled. Onion DNA is characterized
B. Procedure by its white string appearance.
A 125 mL Erlenmeyer flask was placed in a
water bath with a 50 mL homogenizing solution
and the solution was heated until it reaches 60 Ultraviolet Measurement of Isolated DNA
degrees. Then immediately it was added and
stirred with a 25g of minced onion and let stood Upon isolation of DNA, it was subjected through
for about 5 minutes. After which, 1.5 g of papain spectrophotometric analysis to determine its
was added into the solution while keeping the absorbance as well as its degree of purity by
temperature of the water bath at 60 degrees for calculating the ratio of A230/A260 and
10 more minutes. A260/A280.
Next to water bath the flask was
immediately placed in an ice bath for about 5 Table 1. Absorbance of Isolated DNA at different
minutes and was swirled to impede the breakdown wavelengths
of DNA by deoxyribonucleases. After the ice bath Solutio Absorbance Absorbance Absorban
the contents of the flask was poured into a blender n at 230 nm at 260 nm ce at 280
and was homogenized for 45 seconds. The nm
homogenates were filtered through a 4 layered Blank 0 0 0
cheesecloth into a 250mL beaker and were cooled Isolated 0.021 0.024 0.098
off using ice. A 15-20mL of ice cold ethanol was DNA
immediately added using a pipette to slowly drip it
down the side of the tube so that it would form a The isolated DNA yielded varying
clear layer of ethanol on top of the filtrate. The absorbance with respect to the wavelength used.
DNA is precipitated after the process because all It is observed that as the wavelength increases,
the components of the mixture is soluble in the the absorbance also increases, exemplifying a
ice-cold solution except for DNA. direct relationship.
The tube was stood for 5 minutes without The nucleic acid and protein concentration
being disturbed and bubbles were formed as the may be assumed using the absorbance of the
DNA was precipitated out of the solution. The DNA absorbing species using the monograph. From the
became visible as white strings in the ethanol layer absorbance of the sample at 260nm, the nucleic
and these white strings was spooled out into a acid concentration may be assumed to be at 7
clean test tube to resuspend it with TE buffer or μg/mL. The protein concentration based on its
SSE solution. absorbance at 280 nm is assumed to be around
0.8 μg/mL.
RESULTS AND DISCUSSION
TABLE 2. Ultraviolet Measurement of Isolated
DNA
DNA isolation from an Onion
UV
DNA of an organism resides in its nucleus. In
Measurement A260/A230 A260/A280
isolating DNA, the preferred sample to be utilized
should have tissues containing cells with high 1.14 0.245
nuclear volume for better isolation of DNA since it
is concentrated in the nucleus. For this
experiment, DNA sample was liberated from an The A260/A280 ratio is A260/A280 is used
onion because their cells contain a relatively large to measure protein contamination in the presence
of nucleic acids [6]. The accepted range for this
ratio is around 1.8 - 2.0 for DNA [7]. From the
experiment, the ratio was 0.245, which is below
the suggested value. This may indicate that the
sample may have been contaminated by protein or
a reagent such as phenol or another possible cause
is insufficient amount of nucleic acid in the sample
[7].
The A260/A230 ratio is used as a secondary
measure of nucleic acid purity [6]. Compared to
the ratio of A260/A280, its value is usually higher
which may be observed in the experiment
performed. However, the accepted range for this
ratio of absorbance is around 2.0-2.2 [7]. The
A260/A230 ratio of the isolated DNA was
calculated to be at 1.14, which is below the
accepted range. Based on the ratio that the
experiment yielded, it may be inferred that there
might have been a contamination in the sample.
This contamination may be brought about by one
of the following: Carbohydrate carryover, a
common problem encountered with plants,
Residual phenol from nucleic acid extraction,
Residual guanidine and when is Glycogen used for
precipitation [7].
Chemical Characterization of DNA
Acid Hydrolysis
The isolated DNA was treated with 1M HCl and
heated to boiling point for 15 minutes. During this
process, the acid destroyed the different bonds
that stabilizes the DNA. This heating resulted in
the hydrolysis of the bonds shared by the bases,
Adenine, Guanine, Thymine and Cytidine, the
destruction of 3’-5’ phosphodiester bonds that
connects nucleotides, the bond between the
nucleoside and the phosphate group which is, the
Phosphoester bond, and lastly, the bond between
the pentose sugar and the base, the N-C glycosidic
bond.
TABLE 3. Results for chemical characterization of
DNA.
Chemical DNA DNA (Standard)
Test (onion)
Deoxyribose Blue Dark brown
solution precipitate, colorless
solution
Phosphate Yellow Colorless solution,
solution, white fumes
white fumes
Pyrimidine Dark red Yellow-orange
precipitate solution
Purine Red residue Yellow residue with
red precipitates
Table 3 demonstrates that the results in the
characterization of DNA are varying. In the
Deoxyribose test, we were able to obtain a blue
solution in the DNA of onions, while a dark brown
precipitate was found in a colorless solution for the
DNA standard. A yellow solution with white fumes
appeared for the DNA of onions, and colorless
solution with white fumes appeared in the DNA
standard. The test for pyrimidine produced a dark
red precipitate in the DNA of onions, and a yellow-
orange solution in the DNA standard. Lastly, the
test for purines yielded red residues in the DNA of
onion and yellow residues with red precipitates in
the DNA standard test.
These tests provided irregular results in
both the DNA of the onion and of the standard.
However, the DNA of the onion was more resistant
to the tests, thus creating different results.
Therefore, in the process of DNA characterization,
sample preparation highly affects the results and
the data. It is recommended that when measuring
solutions, accuracy and precision must be
observed. Vital laboratory safety guidelines must
always be followed as well. To be able to achieve
more accurate results, the procedures must be
done properly.
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Retrieved October 25, 2019, from
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