DNA ISOLATION FROM ONION, ULTRAVIOLET MEASUREMENT OF ISOLATED DNA AND CHEMICAL

CHARACTERIZATION OF DNA
Mendez, H.C.A., Miranda, M., Maravillias, M., Manlutac, D., Navarro, M.A.,
Panaligan, A, Pingol, E., Pique, V.M.,

Group 5-2B-Pharmacy

ABSTRACT
The Experiment was about the isolation of DNA from onion, Ultraviolet measurement of isolated DNA
and Chemical characterization of DNA. DNA was isolated form the plant source (onion). The Absorbance of
the DNA was then Determined with the use of a Spectrophotometer, the ratio of absorbance was then
computed. The DNA was then hydrolyzed with hydrochloric acid. The DNA was characterized with the use of
Tests ford Deoxyribose or The Dische reaction test, Test for Phosphate, Test for Purines a.k.a murexide Test
and Test for Pyrimidine namely as Wheeler-Johnson Test. The test for Deoxyribose produced a brownish
solution while the test for ribose produced a yellowish solution. The test for phosphate gave a colorless with
crystals solution while the test for purines formed a clear solution with yellow precipitate/crystals. Lastly, the
test for pyrimidine gave a purple solution.

INTRODUCTION DNA spends a lot of time in its chromosome

form. But during cell division, DNA unwinds so it can be
Deoxyribonucleic acid is a nucleic copied and the copies transferred to new cells. DNA
acid containing the genetic instructions used in the also unwinds so that its instructions can be used to
development and functioning of all known make proteins and for other biological processes.
living organisms. DNA is made of chemical building
blocks called nucleotides. These building blocks are Certain tests will be used in order to isolate and
made of three parts: a phosphate group, a sugar group classify the DNA ; Isolation of DNA from onion,
and one of four types of nitrogen bases. To form a Ultraviolet measurement of Isolated DNA, Acid
strand of DNA, nucleotides are linked into chains, with Hydrolysis, Dische Test, Test for Phosphates, Test for
the phosphate and sugar groups alternating. Purines, and Test for Pyrimidine.

The four types of nitrogen bases found in DNA ISOLATION FROM ONION
nucleotides are: adenine (A), , thymine (T), guanine (G)
and cytosine (C). The order, or sequence, of these bases Isolation of the DNA is the first step in the
determines what biological instructions are contained in experiment, The Raw Minced onion is then added with
a strand of DNA.
the Homogenizing solution, which involves breaking
down and removing cell walls and membrane.
DNA contains the instructions needed for an
organism to develop, survive and reproduce. To carry ULTRAVIOLET MEASUREMENT OF ISOLATED DNA
out these functions, DNA sequences must be converted
into messages that can be used to produce proteins, When DNA is isolated from organisms,
which are the complex molecules that do most of the frequently there remains protein present in the DNA
work in our bodies. solution; protein is tightly bound to DNA and complete
removal of protein is not possible. To determine the
DNA is found inside a special area of the cell concentration and purity of the protein it is therefore
called the nucleus. Because the cell is very small, and subjected for analysis in a spectrophotometer.
because organisms have many DNA molecules per cell,
each DNA molecule must be tightly packaged. This
packaged form of the DNA is called a chromosome.
ACID HYDROLYSIS Test for Pyrimidines

DNA is stable to bases, these is one of the Lastly the following were used in the test: 0.5ml
features that distinguish DNA from RNA. Strong acids at Nucleic Acid solution, Bromine Water, Barium
high temperatures are capable of breaking down DNA Hydroxide
molecules onto its basic components, at these
conditions both of the phosphate ester bond and the N-
DISCUSSION
glycosides bends between the Deoxyribose, the Purines Isolation of DNA from Onion.
and the pyrimidine bases.
A 50 ml Homogenizing solution is then prepared
in a 125 ml Erlenmeyer flask and was heated in a water
bath until the solution reached 60 deg C, 25 g minced
EXPERIMENTATION
onion was added to the preheated water bath. A 1.5g
Compounds Tested; Samples used and Devices papain was then added and was then kept for 10
used minutes. After heating place the Erlenmeyer to an ice
bath for about 5 minutes, after the cooling process,
DNA isolation from Onion place the solution to in a beaker to be homogenized;
after the homogenization process the homogenate was
The following were used for the isolation of
then filtered using a cheese cloth into a 250ml beaker
DNA; 2 White/yellow Onions, Homogenizing Solutions;
and cool it after wards. The cooled solution was then
5% SDS( Sodium Dodecyl Sulfate), 0.15 M NaCl, 0.15 M
added with 15-20ml of ice-cold ethanol, place it on the
Na3C6H5O7 and 0.001 M EDTA, Ice-Cold 95% ethanol.
sides and allow it to drip downwards, a clear layer of
Ultraviolet Measurement of DNA ethanol should form on top of the solution, DNA is not
soluble in ice-cold ethanol, when ethanol is added to
The Following were used in the Measurements: the mixture, all the components in the solution except
Quartz Cuvettes, UV-VIS Spectrophotometer. for the DNA stay in the solution, while the DNA
Acid Hydrolysis precipitates out. Let the mixture stand for 3-5 minutes
without disturbing, the DNA can be perceived by a
The Following were used in the Acid Hydrolysis: white precipitate/ strings. Spool the Formed DNA by
DNA Sample, 1M HCl using a pre-bent glass pipette, the obtained specimen
was then added with TE buffer solution.
Test for Deoxyribose
Ultraviolet Measurement of Isolated DNA
The following were used in the test: 3.5ml
Diphenylamine reagent, 1.5ml Hydrolyzed DNA The obtained DNA solution (0.5ml) is then
solution, 0.5 ml Standard DNA, Hot water Bath added to a 4.5 SSC or TE buffer solution. It is the
transferred to a quartz cuvette to determine the
Test for Phosphate
absorbance at the range of 260nm-280nm. The
The Following were used in the Test for obtained results are then calculated for the ratio to
phosphates: 1ml conc. H2SO4, 1ml Nucleic Acid Solution, determine the concentrations of protein and nucleic
1ml Standard Phosphate solution, 0.5ml conc.HNO3, acid.
1ml H2O, 1ml Molybdate Solution, 10ml water.
Acid Hydrolysis
Test for Purines
Mix the DNA sample with 1M HCl, then heat it
The Following were used in the Murexide Test: up to 100deg C for 60 minutes with occasional agitation,
5-10GTT Nucleic Acid solution, HNO3,10% KOH, water cover the tubes with marbles and placed under a
running water to neutralize the temperature and add
1M KOH. Adjust the PH to 4-6 using Acetic Acid. Use PH Next is the deproteinization, this process
paper to determine the acidity, Centrifuge and add to involves adding the protease enzyme papain. It is a
clean test tubes for chemical Characterization. common enzyme that removes or prevents proteins
from clinging to the DNA. DNA Precipitation is done by
Test for Deoxyribose means of adding ice-cold ethanol. This is done so the
Add 3.5 ml Diphenylamine reagent to 1.5 ml DNA will separate from the solution.
hydrolyzed DNA solution. The same procedure used The homogenizing solution is important in the
with the 0.5ml Standard Deoxyribose Solution. Place the experiment, it allows the release of the DNA from the
prepared mixture in a hot water bath for at least about clinging proteins, there are 3 chemicals that are used in
10 minutes. Cool immediately and observe the results. this process, it is Sodium Dodecyl Sulfate, it is a
Test for Purines detergent that helps facilitate in the breakdown of the
cell membrane. Ethylenediamine tetra acetic acid or
10 drops of nucleic acid solution into a small EDTA, it also weakens the cell by destabilizing the
evaporating dish, a few drops of conc. HNO3 is then cations. And lastly Sodium Chloride Facilitates the DNA
added. Carefully evaporate until try in water bath. to precipitate out of the alcohol solution.
Moisten the evaporated nucleic acid with some 10%
KOH and heat further, note the color changes upon Ultraviolet measurement of DNA
adding the KOH and upon heating. Add a few drops of Table 1: Isolation and UV measurement of DNA
water then observe the color, then lastly evaporate and
note the color changes. DNA UV PROTEIN NUCLEIC
measurement Mg/mL ACID
Test for Pyrimidine µg/mL
PLANT 1.o85(260nm)/ 1/0.7=1.43 24/18=1.33
Treat 0.5 ml nucleic acid solution with an excess DNA 0.801(280nm)= mg/ml mg/ml
of bromine water until the solution turns yellow, 1.35 ratio
remove the excess by boiling the solution until it turns
yellow, note the changes, add the excess Ba(OH)2
The chart above shows the ultraviolet
solution, then test with litmus paper. Take note of the
measurements of the obtained samples. As shown from
appearance of the solution.
above, 1.085 at 260nm and 0.801 at 280nm is obtained
RESULTS AND DISCUSSION from the spectrophotometer. At normal condition it has
been observed that the normal range for a good quality
Isolation of DNA and Ultraviolet Measurement DNA sample should have an A260/A280 ratio of 1.7-2.0,
of DNA but we obtained a ratio of 1.35, it can be inferred that
our DNA sample is contaminated or sensitive for it not
Isolation of DNA can be done in three steps
to be at the normal range. The amount of protein
which are Homogenization of deproteinization and
present in the DNA is about 1.43mg/mL, we obtained
precipitation.
this by dividing 1 with 0.7 we obtained both data from
In homogenization the onion is minced, then the given chart of the amount of proteins present.
heated. The heat treatment is done in order to soften Lastly the amount of Nucleic acid is 1.33mg/ml, we
the cell wall and for the breakdown of the cell. Heating obtained both of the given data from the given chart.
softens the phospholipids in the cell membrane and
Chemical Characterizations
denatures the ribonuclease enzymes, which if presents
will cause the fragmentation of the DNA and thus Acid Hydrolysis
prevent it from being spooled.
Strong acids at high temperatures are capable
of breaking down DNA molecules onto its basic
components, at these conditions both of the phosphate REFERENCES
ester bond and the N-glycosidic bends between the
Deoxyribose, the Purines and the pyrimidine bases

Table 2: Chemical Characterization BOOKS

Chemical Test DNA(onion) DNA(standard) Biochemistry (3rd Edition) by Christopher K.

Mathews.

Deoxyribose Yellow Solution Brown Solution Lehninger Principles of Biochemistry, Third

Ed..by David L. Nelson
Phosphate No result Clear Solution
w/yellowcrystals Crisostomo., Laboratory Manual in General
Pyrimidine Purple Solution Purple Solution Chemistry., 2010.

WEBSITES
Purines Reddish brown Yellow residue
w/ red Dots 1. http://www.uniregensburg.de/Fakultaeten/n
at_Fak_IV/Organische_Chemie/Didaktik/Keusch/p31_d
_rib-e.htm

As shown from the chart, there are various

results for the characterization of DNA. In the 2. http://www.medicalhealthtests.com/blog/bl
Deoxyribose Test we obtained a Yellow solution in the ood-tests/blood-phosphate-level.html
DNA Of onions while we obtained Brown solution in the
standard DNA. The test for phosphates didn’t show any
results with the DNA in onions while The Standard DNA 3. http://www.biochemia.amb.edu.pl/pdf/class
resulted in a Clear solution w/ yellow crystals, test for 9.pdf
Pyrimidine resulted in a Purple Solution in the DNA of
the onion, same as The Standard DNA. Lastly the Test
for purines Resulted in a Reddish Brown solution in the
DNA of the onion, while the standard DNA gave a yellow
residue w/ red dots. These tests showed a significant
difference between the DNA in onions compared to the
Standard DNA, the DNA of the onion may somewhat be
a more sensitive or may be more resistive to the test,
thus it gave a different result. It can be inferred that in
the process of characterization of DNA, the way of
preparation of the solution can affect the resulting data,
for example, our DNA string were not successfully
obtained partially due to a process that was not
properly done, we recommend the utmost care in
measuring the solutions that will be used, it is also
important to properly practice laboratory safety
guidelines, for the chemicals that are being dealt with
are somewhat volatile and can easily harm the
examiner. Proper procedures should also be done in
order to achieve accurate data of the results.