Group 8 2K-PH

Edmin Christian Tunod, Jant Nicole Joyce Ulep, Hannah Marie Villaroza, Christian Vizcarra

ISOLATION AND CHARACTERIZATION OF NUCLEIC ACIDS

Abstract

DNA is a type of nucleic acid that is double-stranded with a deoxyribose pentose. It is a

fundamental biochemical molecule of an organism that stores genetic information. The
experiment calls for the group to isolate DNA from the onion sample, then determine the
purity of the onion DNA in its isolated form. The method used was hydrolysis which is the
chemical breakdown of a compound with addition of a water molecule, colorimetric tests
then followed. Through disruption of its cell membranes, the DNA was isolated form the
onion sample. The result of the isolation, showed the DNA of the onion to have a white
thread-like appearance when extracted. UV spectroscopy was then used to identify its
purity. The sample was then subjected to Dische test (test for deoxyribose), Phosphate
test, Murexide test (test for purines), Wheeler-Johnson test (test for pyrimidines). During
the experiment, the test for deoxyribose resulted to a dark violet to brown hydrolysate
which is a positive result for deoxyribose. The test for phosphate resulted to a clear
colorless solution with a yellow precipitate indicating the presence of phospahate ions.
The test for purine resulted to a yellowish-brown precipitate which is a positive result for
purine. The test for pyrimidines resulted to a purple solution which is a positive result for
pyrimidine.

I. Introduction Studying DNA in the genomic level can

An organisms genetic material is found
in its genome. The genome provides the
necessary genetic information to encode
the base sequence of DNA. The
genome houses the genetic information
that is carried over from the parent to
the offspring.
For the case of haploid organisms like
bacteria, there is only one copy of the
genome per cell. As opposed to diploid
organisms like humans, there are two
copies of the genome per cell, one
passed down from each parent.
provide insight about not only the gene
itself but also on the structure and
physical relationship among the
individual genes.
Isolating the genomic DNA is the first
step in studying it and its applications.
The genomic DNA should be brought
into its purified and high molecular
weight state.
The DNA is a type of nucleic acid that
stores genetic information and unlike
RNA, it has deoxyribose as its pentose.
The DNA is most likely found in high
 Group 8 2K-PH
Edmin Christian Tunod, Jant Nicole Joyce Ulep, Hannah Marie Villaroza, Christian Vizcarra

cellular organs, in example the spleen,

liver, thymus, and other lymphoid
organs.
II. Methodology
DNA and RNA share similar
characteristics. They are sparingly DNA Isolation and UV Measurement
soluble in cold water, insoluble in
Ultraviolet Measurement of Isolated
alcohol, but readily soluble in weak
DNA
alkali with the formation of alkali metal
salts. [1] 1. 2.5 mL of the Tris-EDTA (TE)
buffer was added to the saved
The three primary steps in isolation of
portion of the isolated DNA for
nucleic acid is: disruption of the cell
the UV measurement.
membranes and the membrane of the
subcellular nucleus to release the 2. 50 L of the isolated DNA was
nucleic acid; dissociation from the pipetted and dispensed in the
nucleoproteins and denaturation of the microwell plate.
proteins; separation of the DNA from the 3. Absorbance of the DNA sample
other soluble cellular components. [2] at 260 and 280 nm was read.

UV spectroscopy/photometry measures Chemical Characterization of DNA

the purity and concentration of extracted
Dische Test (Test for deoxyribose)
DNA against the protein contaminants.
Specific contaminants in example Standard/s used: deoxyribose
protein absorb light at different
wavelengths. Proteins absorb UV light 1. In a test tube, 2 drops of the
strongly at 280 nm (A 280) and DNA at standard was added to the
260 nm (A260), providing the information isolated DNA.
2. 5 gtt of diphenylamine (DPA) was
needed to identify and measure the
added to the solution and it was
concentration. [3]
subjected under heat for 10
Objectives minutes.

Isolate DNA from microbial, plant, Phosphate Test

and animal sources;
Determine the purity if isolated Standard/s used: pH 12 buffer
DNA; and
1. In a test tube, 5 drops of the
Characterize DNA after acid
hydrolysis standard was added to the
isolated DNA.
2. 1 mL of concentrated H2SO4 was
added to the solution and it was
placed in a cold water bath.
 Group 8 2K-PH
Edmin Christian Tunod, Jant Nicole Joyce Ulep, Hannah Marie Villaroza, Christian Vizcarra

3. 1 mL of concentrated HNO3 was In homogenization, the onion was

added. After the addition, it was
placed again in a cold water bath.
4. 20 drops of 10% (NH4)2 was
added and was let stand for 5
minutes.
Murexide Test (Test for Purines)
Standard/s Used: Adenine & Guanine
1. In an evaporating dish, 5 drops of the
standard was placed with the isolated
DNA.
2. Three drops of concentrated HNO3
was added and heated to dry.
3. Five drops of 10% KOH was
subjected in heat with addition of water
and was heated to dry.
Wheeler-Johnson Test (Test for
Pyrimidines)
Standard/s used: Cytosine and Uracil
1. In a test tube, 5 drops of the standard
was added to the isolated DNA.
minced then heated. The heat treatment
was done to soften the cell and for the
breakdown of cell. Heating softens the
phospholipids in the cell membrane and
denatures the DNase, because if
present, this will cause the
fragmentation of DNA. After
homogenizing the sample and put in
cold ethanol, a thread-like structure was
formed in the ethanol layer. The layer
was the DNA isolate. Because the DNA
is not soluble in ethanol, the onion
filtrate contains the DNA and other
impurities. Ethanol was added to
precipitate out a pure DNA.
B. UV Spectroscopy
The table below shows the ultraviolet
measurement of the obtained sample.
From the date shown below, 0.70 was
obtained from the spectrophotometer. It
has been observed that the normal
range for a good quality DNA sample
should have A260 /A280 ratio of 1.8 to 2.0,
but the result the group gathered was
lower than the desired result, it can be
inferred that the DNA is protein-
contaminated.

2. Bromine water was added until the

solution turns yellow. It was heated until Table 1: Result of isolation of DNA and
the color fades. UV Measurement

3. Ba(OH)2 was then added to the DNA Plant DNA

solution. Physical white curdy
Description
UV Measurement 0.70
Protein Conc. 0.18
III. Results and Discussions (mg/mL)
Nucleic Acid 4.25
Isolation of DNA and UV
Measurement Conc. ( g /mL)

A. DNA Isolation from Onion

Acid hydrolysis
 Group 8 2K-PH
Edmin Christian Tunod, Jant Nicole Joyce Ulep, Hannah Marie Villaroza, Christian Vizcarra

DNA is generally quite stable. It will

resist attack in acid and alkali solutions.
However in mild actions at pH 4- the
beta-glycosidic bonds to the purine
bases are hydrolyzed. Protonation of
purine bases are good leaving groups
hence the hydrolysis. Once this
happens, the depurinated sugar can
easily isomerize into the open-chain
form and in this form the depurinated
DNA is susceptible to cleavage by
hydroxyl carbons
Table 2: Qualitative color reaction of
hydrolysate
Figure 1: Result of Dische test
Test Standard
Dische Test Purple solution
Phosphate Test Yellow precipitate Test for phosphate

Murexide Test This test is used to determine the

Formation of red residue
amount of phosphate ions present in the
Wheeler-Johnsons Test Purple solution substance. A small amount of nitric acid
and ammonium molybdate is added.
The presence of phosphate ions is
Test for deoxyribose (Dische indicated by the formation of a bright
test) yellow precipitate layer of ammonium
phosphomolybdate. This is also used to
This test is used to detect if there is detect arsenic, a yellow precipitate may
deoxyribose present in the sample in be formed. As seen from figure 2, the
which DNA contains. The group used group obtained a clear colorless solution
diphenylalanine and concentrated with a yellow precipitate indicating a
sulfuric acid to detect the presence of positive result which means it contains
DNA in substance. The reaction phosphate ions.
depends on the conversion of the
pentose to w-hydroxylaevulinic aldehyde
which then reacts with diphenylalanine
to give blue colored complex. The group
achieved a dark violet to brown (as seen
from figure 1.) solution which is positive
result and tell us that our sample
contains deoxyribose.
 Group 8 2K-PH
Edmin Christian Tunod, Jant Nicole Joyce Ulep, Hannah Marie Villaroza, Christian Vizcarra
Figure 2. Result of the test
phosphate
Test for Purines (Murexide test)
In the test for the presence of purines,
DNA is reacted with nitric acid since
purines are known to be readily soluble
in dilute acid. Nitric acid oxidized it
leaving a yellow precipitate upon
evaporation; however it turned red when
moistened with a base, a positive result
for the presence of purine bases. On the
table above the standard sample
achieved the expected result while the
DNA formed rather a yellowish brown
precipitate, which also a positive result
for the test.
Test for Pyrimidines (Wheeler-
Johnsons test)
The bromination of pyrimidine at C5
produced dibromohydroxyuracil which is
the observable yellow color in the
solution. After addition of barium
hydroxide Ba(OH)2 will give a result of
purple solution. The sample exhibited a
purple solution which indicates a
positive result as seen from figure 3.
for
Figure 3. Result of the test for
Pyrimidine
IV. Conclusion
These tests showed a significant
difference between the DNA of onions
and the standard DNA, the DNA of
onion may somewhat be more sensitive
or more resistive to the test, hence it
gave a different result. It can be inferred
that in the process of characterization of
DNA and the way of preparation of
solution can affect the resulting data. In
example the murexide test, the group
achieved the correct result in the
standard sample yet it had a different
outcome when the DNA sample was
tested, so therefore we recommend
utmost care in measuring the solutions
that will be used. It is also important to
properly and carefully practice the
laboratory safety guidelines for
chemicals that are being dealt with are
somewhat volatile and can easily be
harmful to the student. Proper
procedures should be done in order to
obtain accurate results.
 Group 8 2K-PH
Edmin Christian Tunod, Jant Nicole Joyce Ulep, Hannah Marie Villaroza, Christian Vizcarra

References
Articles Online
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Precipitation of DNA and RNA:
How it works. Retrieved from
http://bitesizebio.com/253/the-
basics-how-ethanol-precipitation-
of-dna-and-rna-works/
[2] Nucleic Acid Extraction and
Purification. (2017). Retrieved
March 29, 2017, from
http://www.bio-rad.com/en-
ph/applications-
technologies/nucleic-acid-
extraction-purification
Books
[3] Chomczynski P and Sacchi N (1987).
Single step method of RNA
isolation by acid guanidinium
thiocynate-phenol-chloroform
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156-9.