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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


provide insight about not only the gene
An organisms genetic material is found
itself but also on the structure and
in its genome. The genome provides the
physical relationship among the
necessary genetic information to encode
individual genes.
the base sequence of DNA. The
genome houses the genetic information Isolating the genomic DNA is the first
that is carried over from the parent to step in studying it and its applications.
the offspring. The genomic DNA should be brought
into its purified and high molecular
For the case of haploid organisms like
weight state.
bacteria, there is only one copy of the
genome per cell. As opposed to diploid The DNA is a type of nucleic acid that
organisms like humans, there are two stores genetic information and unlike
copies of the genome per cell, one RNA, it has deoxyribose as its pentose.
passed down from each parent. 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 minced then heated. The heat treatment
placed again in a cold water bath. was done to soften the cell and for the
4. 20 drops of 10% (NH4)2 was breakdown of cell. Heating softens the
phospholipids in the cell membrane and
added and was let stand for 5
denatures the DNase, because if
minutes. present, this will cause the
fragmentation of DNA. After
Murexide Test (Test for Purines)
homogenizing the sample and put in
Standard/s Used: Adenine & Guanine cold ethanol, a thread-like structure was
formed in the ethanol layer. The layer
1. In an evaporating dish, 5 drops of the was the DNA isolate. Because the DNA
standard was placed with the isolated is not soluble in ethanol, the onion
filtrate contains the DNA and other
DNA.
impurities. Ethanol was added to
2. Three drops of concentrated HNO3 precipitate out a pure DNA.
was added and heated to dry. B. UV Spectroscopy
3. Five drops of 10% KOH was The table below shows the ultraviolet
subjected in heat with addition of water measurement of the obtained sample.
and was heated to dry. From the date shown below, 0.70 was
obtained from the spectrophotometer. It
Wheeler-Johnson Test (Test for has been observed that the normal
Pyrimidines) range for a good quality DNA sample
should have A260 /A280 ratio of 1.8 to 2.0,
Standard/s used: Cytosine and Uracil but the result the group gathered was
lower than the desired result, it can be
1. In a test tube, 5 drops of the standard inferred that the DNA is protein-
was added to the isolated DNA. 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 for


phosphate Figure 3. Result of the test for
Pyrimidine

Test for Purines (Murexide test)


IV. Conclusion
In the test for the presence of purines,
DNA is reacted with nitric acid since These tests showed a significant
purines are known to be readily soluble difference between the DNA of onions
in dilute acid. Nitric acid oxidized it and the standard DNA, the DNA of
leaving a yellow precipitate upon onion may somewhat be more sensitive
evaporation; however it turned red when or more resistive to the test, hence it
moistened with a base, a positive result gave a different result. It can be inferred
for the presence of purine bases. On the that in the process of characterization of
table above the standard sample DNA and the way of preparation of
achieved the expected result while the solution can affect the resulting data. In
DNA formed rather a yellowish brown example the murexide test, the group
precipitate, which also a positive result achieved the correct result in the
for the test. standard sample yet it had a different
outcome when the DNA sample was
Test for Pyrimidines (Wheeler- tested, so therefore we recommend
Johnsons test) utmost care in measuring the solutions
that will be used. It is also important to
The bromination of pyrimidine at C5 properly and carefully practice the
produced dibromohydroxyuracil which is laboratory safety guidelines for
the observable yellow color in the chemicals that are being dealt with are
solution. After addition of barium somewhat volatile and can easily be
hydroxide Ba(OH)2 will give a result of harmful to the student. Proper
purple solution. The sample exhibited a procedures should be done in order to
purple solution which indicates a obtain accurate results.
positive result as seen from figure 3.
Group 8 2K-PH
Edmin Christian Tunod, Jant Nicole Joyce Ulep, Hannah Marie Villaroza, Christian Vizcarra

ph/applications-
technologies/nucleic-acid-
References extraction-purification
Articles Online Books
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Precipitation of DNA and RNA: Single step method of RNA
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http://bitesizebio.com/253/the- thiocynate-phenol-chloroform
basics-how-ethanol-precipitation- extraction. Anal Biochem 162,
of-dna-and-rna-works/ 156-9.
[2] Nucleic Acid Extraction and
Purification. (2017). Retrieved
March 29, 2017, from
http://www.bio-rad.com/en-