Isolation, Acid Hydrolysis and Qualitative Color Reaction of DNA from

Onion
HAG Gutierrez, KL Hagiwara, CBDL Javier, AMF Labajo, AP Lansangan
Group 5, 2CMT, Faculty of Pharmacy, UST
Abstract
The experiment aims to isolate DNA from microbial, plant and animal sources, it
also aims to determine the purity of isolated DNA and characterize DNA following
acid hydrolysis. DNA was isolated from the plant source, onion. Absorbance was
measure under the wavelengths 260nm and 280nm which was followed by
hydrolysis using hydrochloric acid after which was subjected to different qualitative
color reaction: Dishe Test, a test for deoxyribose, Test for Phosphates, Murexide test,
a test for Purines and Wheeler-Johnson Test, a test for Pyrimidines. The computed
absorbance ratio is 0.56. Dische Test produced a blue solution, Test for Phosphates
produced a yellow precipitate, Murexide test yields a yellow to red residue while
Wheeler-Johnson test yields a pale yellow turbid solution.
Introduction
Nucleic acids are the biological
molecule essential for life. They make
up
the
most
important
macromolecules.
They
are
high
molecular weight biopolymers of
mononucleotides. The backbone of a
nucleic acid is
made
of
alternating sugar and
phosphate
molecules bonded together in a long
chain. Each of the sugar groups in the
backbone is attached to a third type
of molecule called a nucleotide base.
Though
only
four
different nucleotide bases can occur in
a nucleic
acid,
each
nucleic acid contains millions of bases
bonded to it. The order in which these
nucleotide bases appear in the nucleic
acid is the coding for the information
carried in the molecule.
Two structural classes occur in
cell: DNA and RNA. These are
macromolecular structures composed
of regular repeating polymers formed
from nucleotides. These are the basic
building blocks of nucleic acids and
are derived from nucleoside which has
two components: a five-membered
pentose
carbon
sugar
and
a
nitrogenous base.

DNA or deoxyribonucleic acid is the

hereditary material in humans and
almost all other organism. Most DNA is
located in the cell nucleus but a small
amount can also be found in the
mitochondria. DNA is a two stranded
structure
consisting
of
two
polynucleotide chain twisted about
each other in a double helix. Both
chains are right handed however,
since each strand has both free 5
hydroxyl group at one end and a free
3 hydroxyl on the other end, each
strand has a polarity or directionality.
The polarity of the two strands of the
molecule is in opposite directions and
thus, DNA is described as anti-parallel
structure.

Figure1. The double helical structure of

DNA

The nucleotide bases of

the
DNA molecule form complementary
pairs:
The
nucleotides hydrogen
bond to another nucleotide base in a
strand of DNA opposite to the original.
The Purines bases adenine (A) and
Guanine (G) are found in both RNA
and DNA, as is the Pyrimidines
Cytosine (C). The other Pyrimidines
are each restricted to one type of
nucleic
acid:
Uracil
(U)
occurs
exclusively in RNA, whilst, Thymine (T)
is limited to DNA. This bonding is
specific, and adenine always bonds to
thymine (and vice versa) and guanine
always bonds to cytosine (and vice
versa). This bonding occurs across the
molecule, leading to a doublestranded system

DNA
is
also
capable
of
replicating. Each strand of DNA in the
double helix can serve as a pattern for
duplicating the sequence of bases.
This is critical when cells divide
because each new cell needs to have
an exact DNA copy present in old cell.
The Nucleic Acid isolation and
procedures involve three steps. 1)
Disruption of cell membrane and
membranes of sub cellular nucleus to
release nucleic acids. 2) Disassociation
from nucleoproteins and denaturation
of proteins and 3) separation of DNA
from other soluble cellular component.
Materials and Method
A. Isolation of DNA from Onion

Figure2. GC base pairing with 3

hydrogen bonds (below) and AT base
pairing with 2 hydrogen bonds
(upper).

To isolate DNA from its source, onion,

50 ml of homogenizing solution was
placed in a 125 ml Erlenmeyer flask
and was heated in a water bath until
the solution reached 60oC. 25 g of
minced onion was weighed out and
was
added
to
the
pre-heated
homogenizing solution which was left
seated for 5 minutes with eventual
stirring in the water bath. 1.5 g of
papain (or 4ml of meat tenderizer
solution) was added and was kept in
the 60oC water bath for another 10
minutes after which was immediately
placed in an ice bath for 5 minutes.
The solution was swirled gently to
allow even cooling. After, the contents
of the flask were poured in a blender
and were homogenized for 45
seconds. The homogenate was filtered
through 4 layers of cheesecloth into a
250 ml beaker. The solution was
cooled on ice. Using a pipette, 20 ml
of ice cold ethanol was immediately
added, and was allowed to slowly drip
down the sides of tube. The tube was
left to stand for 5 minutes without
disturbing it. The DNA was spooled by

snagging it with a Pasteur pipette with

a hook bent on the tip. The DNA was
transferred into a clean test tube and
was resuspended in TE buffer.
B. Ultraviolet
Measurement
Isolated DNA

of

A 0.5 ml aliquot of the DNA solution

was dissolved in 4.5 ml of TE Buffer.
The solution was transferred to a
quartz cuvette and the absorbance
was determined at 260nm and at 280
nm. The buffer solution was used as
the blank reagent. The A260/A280 ratio
was calculated.
C. Acid Hydrolysis
For Acid Hydrolysis, DNA sample was
mixed with 1M HCl. The mixture was
heated at 100oC for 60 minutes with
occasional agitation. The tubes were
covered with marbles. After, the test
tube was carefully cooled under
running water and was neutralized
with 1M KOH. The pH was adjusted to
4-6 with glacial acetic acid using pH
paper. The mixture was centrifuged
and was decanted into clean test
tubes for chemical characterization
D. Qualitative Color Reaction
1. Test for Deoxyribose ( Dische
test)
3.5 ml diphenylamine reagent was
added to 1.5 ml of hydrolyzed DNA
solution. The same was done to the
0.5
ml
standard
deoxyribose
solution. Both tubes were placed in
a boiling water bath for 10 minutes
and were immediately cooled.
2. Test for Phosphates
A volume of 1ml conc. H2SO4 was
added to 1 ml nucleic acid solution
and standard phosphate solution.

Both tubes were heated over a

small flame and were shaken
frequently until the contents of the
tube turned brown. The mixture
was cooled after which, 0.5 ml
conc. HNO3 was introduced. The
solution was heated until white
fumes appeared and the solution
was colorless. 1 ml of water was
added to the colorless liquid and
was heated for 5 minutes in a
boiling water bath. The mixture
was cooled after. Introduced to the
mixture was 1 ml 10% (NH4)2MoO4
solution. The solution was mixed
well and was diluted to 10 ml with
water. It was left to stand for 5
minutes.
3. Test for
Test)

Purines

(Murexide

Into a small evaporating dish, 5- 10

drops of nucleic acid solution was
placed. A few drops of conc. HNO3
were introduced. The same was
done to the standards adenine or
guanine solution. The mixture was
carefully evaporated in a water
bath until dry. The residues formed
were moistened with 10% KOH and
was further heated. The changes in
color were noted upon addition of
KOH. A few drops of water were
introduced to the mixture and was
warmed.
The
residue
was
evaporated and the color was
noted.
4. Test for Pyrimidines (WheelerJohnson Test)
A 0.5 ml of nucleic acid solution
was treated with an excess of
bromine water until the solution
turned yellow. The same was done
to the standards cytosine or uracil.
Excess were removed by boiling
the solution until it turned light
yellow or colorless. Excess Ba(OH)2
solution
was
introduced.
The

solution was tested with litmus

paper
Results and Discussion
A. Isolation of DNA from Onion
In isolation of DNA from source, onion
a first step is homogenization.
Homogenization media includes: 1)
SDS (Sodium Dodecyl Sulfate) A
biological detergent which causes the
cell membrane to break down further
and emulsifies the lipids and proteins
of the cell by disrupting the polar
interactions
that
hold
the
cell
membrane together. The detergent
forms complexes with these lipids and
proteins causing them to precipitate
out of the solution. SDS is the major
ingredient in laundry detergent. 2)
EDTA (Ethylenediamine
tetracetic
acid) weakens the cell by binding the
divalent cations (Mg++ and Ca++) which
are needed for membrane stability.
This further aids in breaking open the
cells of the onion and lastly, 3) NaCl
(Sodium chloride) enables nucleic
acids to precipitate out of an alcohol
solution because it shields the
negative phosphate end of DNA
causing the strands to come closer
together and coalesce.
Homogenization involves heating
and blending the onion tissue in order
to break down the cells. The heat
treatment softens the phospholipid in
the cell membrane and denatures the
DNAse enzymes which if present,
would cut the DNA into small
fragments so that it would not spool.
The onion tissue is mixed in a blender
with homogenization media, which
breaks down the cell wall, cell

membrane and nuclear membrane

allowing the release of DNA.
The next step is followed by
deproteinization.
Deproteinization
involves adding a protease enzyme
Papain, a common enzyme used to
clean soft contact lenses. This will
denature the proteins clinging to the
DNA making the molecule flexible and
easy to spool. Precipitation of DNA
involves adding ethanol alcohol which
causes every component in the filtrate
to stay in solution except DNA.
Table1. DNA isolation
DNA
Physical
Descriptio
n
Plant DNA
Light Yellow
turbid
Solution
B. Ultraviolet
Measurement
Isolated DNA

of

The
measurement
of
the
absorbances allows measurement
of the DNA concentration and
provides information about the
contaminant levels. DNA absorbs
light most strongly at 260nm so the
absorbance
value
at
this
wavelength (called A260) can be
used
to
estimate
the
DNA
concentration while the absorbance
at 280nm is used as an indicator of
protein contamination. A good
quality DNA sample should have an
A260/A280 ratio of 1.7-2.0

Ratioof Absorbance=

C. Acid Hydrolysis

A 260 0.55
=
=0.56
A 280 0.98

DNA is generally quite stable. It will

resist attack in acid and alkali
solutions. However, in mild acid
solutions - at pH 4 - the betaglycosidic bonds to the purine
bases are hydrolyzed. Protonation
of purine bases occurs at this pH.
Protonated
purines
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
(or apurinic) DNA is susceptible to
cleavage by hydroxyl ions.

D. Qualitative Color
Hydrolyzate

reaction

of

then
with

reacts

diphenylamine to give a a blue

colored complex (test tube 1 and
2). The intensity of the blue color is
proportional to the concentration of
DNA.
Figure3. Dische Test Reaction
2. Test for Phosphates
In the test for presence of
phosphates for DNA, a yellow
precipitate was obtained. The
ammonium
molybdate
solution
reacted with the sample which
yields
yellow
crystals,
phosphoammonium
molybdate
which is a positive result.

Table2. Results of Qualitative Color

Reaction of Acid hydrolyzed DNA
TEST
Standard
Hydrolyz
ate
Dische
Light Blue
Blue
Test
solution
solution
Test for
Formation
Formation
Phosphate
of Yellow
of yellow
s
precipitate
precipitate
Murexide
Formation
Formation
Test
of red
of yellow
precipitate
to red
precipitate
WheelerFormation
Pale yellow
johnson
of Purple
turbid
Test
precipitate
solution

3. Test for Pyrimidines (WheelerJohnson Test)

Bromine water reacted with the
sample
to
form
5-bromo6hydroxyhydroxo derivative which
produces a green coloration. Upon
addition of Ba(OH)2 will give a
result of purple precipitate.

1. Test for Deoxyribose (Dische

test)

4. Test for
Test)

DNA can be identified chemically

with the Dische diphenylamine
test. The reaction between the
Dische
reagent
and
2deoxypentose
results
in
the
development of a blue color. The
reaction depends on the conversion
of
the
pentose
to whydroxylaevulinic aldehyde which

Purines

(Murexide

In the test for 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 presence of purine bases.

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