Biochemistry Laboratory Formal Report

Isolation and characterization of

deoxyribonucleic acid (DNA)
from Allium cepa

CHEMISTRY

600L
EXPT 08
PAGE 13 - 18

Marj Hipolito, Von Gabriel S. Tan, Reina Justina P. Tolete, Clarisse

Anne D. Yung, Angelo D. Villaroman*
Department of Chemistry, College of Science
Date submitted: 23 November 2015
*Corresponding author; e-mail: angelo.villaroman@yahoo.com

Abstract
The purpose of this experiment is to isolate and characterize
deoxyribonucleic acid (DNA) from onion (Allium cepa) cells. In the
experiment, isolation of onion cells DNA was undergone through
homogenization process using sodium dodecyl sulfate (SDS), sodium
citrate (NaC6H5O7) and sodium chloride (NaCl). Afterwhich, the isolate is
tested for its concentration and purity (via spectrophotometric analysis)
and characterized using chemical tests (Murexide test, Dische reaction,
Wheeler-Johnson test, and phosphate test). The results obtained were: no
DNA were extracted based form absorbance reading and a positive result
for Wheeler-Johnson Test only.

Keywords: nucleic acids, deoxyribonucleic acid (DNA), spectrophotometry

Introduction
Life will never be possible without reproduction. The propagation and multiplication of
life is ultimately dependent on the so-called genes. The main component of these genes
are nucleic acids which are of two types: deoxyribonucleic acids or, DNA, (the focus of
this experiment) and ribonucleic acids or, RNA. Nucleic acids are essential
biomolecules that are responsible for the transfer and storage of genetic information

Biochemistry Laboratory Formal Report

from generation to generation. It is composed of monomers called nucleotides which
consist of three (3) parts: a ribose sugar, a phosphate group, and a nitrogenous base.
Figure 1 shows the general structure of a nucleotide. The
sugar component is an aldopentose, a -D-ribose (in RNA)
Figure 1. General
structure of a nucleotide

or a -D-deoxyribose (in DNA) (Boyer, 2012). In DNA, the

hydroxyl group (-OH) in C-2 position of the sugar is

replaced by a hydrogen, thus, deoxygenated. The phosphate group, on the other hand,
makes the nucleotide negatively charged and enables it to bond to other nucleotides
(polymerization). Nucleotides are held together by a 3,5-phosphodiester bonds and
experience directionality -- one end of the chain has a 3-hydroxyl (or phosphate) group
and the other end has a 5-hydroxyl (or phosphate) group. Both the sugar and the
phosphate groups constitute the common, invariant region of the nucleotide (referred to
as the backbone)(Boyer, 2012). On the other hand, the nitrogenous base is the variable
region of the compound. There are 2 types of nitrogenous bases: pyrimidines and
purines. Both are named such for they resemble either a
pyramidine or a purine structure. Pyramidine bases are singlering aromatic compound which includes cytosine, thymine, and
uracil. Cytosine is present in both DNA or RNA. Thymine, on
the other hand, is substituted for uracil in DNA. Uracil occurs
only in RNA. Purine bases, however, are double-ring aromatic

Figure 2. Purines
and pyrimidines

compounds. It includes adenine and guanine, both of which is found in DNA and in RNA
(Campbell & Farell, 2012). Figure 2 shows the five (5) heterocyclic bases.

Biochemistry Laboratory Formal Report

Native DNA exists as two, complementary, antiparallel strands arranged in a double
helix held by noncovalent bonding. The two strands of DNA wound around each other
with the bases inside and the sugar-phosphate backbone on the outside. The most
important and significant feature of the double helix is its complementary base pairing.
In each pair, a purine and a pyrimidine pairs together, thus, adenine (A) always pairs
with thymine (T), and guanine (G) always pairs with cytosine (C). The forces that holds
the double helix are: the hydrogen bonds of the base pairs, and the hydrophobic
interactions and van der Waals forces between stacked bases (Boyer, 2012).
Today, there are a lot of isolation and characterization methods of nucleic acids. The
isolation techniques used in this experiment are through mechanical, enzymatic, and
chemical means. Mechanical method employs physical treatment like grinding, stirring,
chopping, and other disruptive procedures. On one hand, enzymatic involves using
enzymes to hydrolyze certain bonds e.g. proteases. Lastly, using chemicals like
detergents disrupt the lipid components of the cell. Moreover, characterization of nucleic
acid in this experiment involves spectrophotometric analysis, which runs under the
principle of light absorbance (chromophores; in DNA conjugated bases (A,T,C,G)) and
chemical tests (qualitative) like Dische, Murexide, Wheeler-Johnson, and phosphate
test. Dische test detects the presence of sugars in a solution. A blue solution indicates a
positive result to this test. Murexide test identifies presence of caffeine and purine
derivatives. A pink residue is positive for this test. Wheeler-Johnson test detects uracil
or cytosine in a solution. A purple or violet-blue color is positive in this test (Wheeler,
H.L., & Johnson, T.B, 1907). Lastly phosphate test detects presence of phosphate in a
solution, if phosphate ions are present, a bright yellow precipitate is observed.
The objective of this experiment is to isolate and characterize DNA from onion cells.
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Biochemistry Laboratory Formal Report

Results and discussion
The experiment is divisible into three parts as already said previously -- isolation, test
for concentration and purity, and chemical characterization.
I. Isolation of DNA
Three (3) methods are involved in extracting the DNA namely, mechanical, enzymatic,
and chemical. The initial treatment to the onion, cutting, is part of the mechanical
degradation which helps in the preliminary disruption of the onion cell. However, too
much mechanical stress can induce the activity of DNase, which if present, would cut
the DNA into smaller fragments; this would not allow the DNA to be spooled. Also, the
heat treatment softens the phospholipid in the cell membrane (easier to degrade) and
denatures DNases, thus removing its function.
Chemical

part

is

observed

in

the

homogenizing

solution

which

contains

ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), sodium chloride

(NaCl), and sodium citrate (NaC6H5O7). This solution will allow the isolation of DNA by
degradating other cell components while protecting the DNA itself. In this case, the
EDTA serves two (2) purpose. First, it binds to the divalent metal ions (Ca+2, Mg+2, Mn+2)
which could form salts with the anionic phosphate groups of the DNA. Second, in
relation to the first, it also inhibits DNases because it chelates metal ions, specifically
Mg+2 or Mn+2, which are necessary cofactors of nucleases (Boyer,2012). SDS also
performs two (2) functions, first, it solubilizes the cell membranes (emulsifying lipids and
proteins) and, second, being an anionic detergent, it disrupts the ionic interactions
between the positively charged histones and the negatively charged backbone of the

Biochemistry Laboratory Formal Report

DNA (Boyer,2012). The detergent then forms complexes with these lipids and proteins
causing them to precipitate out of the solution (Extraction of DNA, n.d.).Sodium chloride
(NaCl) provides a shielding effect to nucleic acids negative phosphate ends from
cations (diminishing ionic interactions) causing the strands to come together and
coalesce thus, enabling the DNA to precipitate when alcohol is added.
The enzymatic part is observed in the addition of papain. Papain is a cysteine protease
that denatures proteins which are clinging to strands of DNA. This allows the DNA to
furtherly uncoil making it easier to spool, and remove impurities brought about by
proteins (Extraction of DNA, n.d.). The results of the experiment are shown below.
Table 1. Results of the experiment
Absorbance

Description

Weight
(mg)

A260

A280

White, thread-like material

33.8

-0.339

-0.296

Ratio

Protein
(mg/mL)

---

---

Nucleic
acid
(g/mL)
---

II. Test for Concentration and Purity Using UV-Vis

From the given data above, negative absorption readings were recorded in which
theoretically are not possible. UV-Vis spectroscopy uses the principle that conjugated pi
bonds absorbs light. From this, the degree of absorption is calculated by noting the
difference after the light had passed through a solution. The readings from the sample is
compared to a reagent blank which only contains the solvent used to dissolve the DNA.
This cancels any unwanted readings that may be scanned in the process, thus,
producing only the DNAs absorbance. This is the reason why negative absorbance
values are not possible. Possible explanations to these bad readings could be: wrong or
different solvent was placed in the reagent blank (a solvent which has even more

Biochemistry Laboratory Formal Report

content than the DNA-SSC solution), or wrong side of the cuvette is exposed (matted
side). Using a nomograph, the purity and the concentration can be quantify. Pureness of
DNA isolate is based on A260/A280 ratio. Pure DNA has a ratio of ~1.8.
Before reading, DNA is dissolved in SSC solution. The reason behind this is that DNA is
more stable in high saline solution. In addition, SSC is a buffer, thus, preventing any
change in pH that might denature DNA and affecting the reading. Single stranded DNA
(denatured) absorbs more light than those in double helix form (Sharma, A.K., 2011).
III. Chemical Characterization
Table 2. Chemical characterization results
Chemical Test
Standard Sample
Dische Test
Dark-purple solution
Test for Phosphate
Clear solution w/ yellow ppt*
Murexide Test
Orange substance (crust-like)
Clear, colorless solution w/
Wheeler-Johnson Test
white ppt; purple litmus

DNA Hydrolyzate
Clear, colorless solution
Clear colorless solution
Yellow substance (crust-like)
Clear, colorless solution w/
white ppt; purple litmus

Before characterizing, DNA isolate was first hydrolyzed using an acid. Strong acids at a
high temperature are capable of breaking DNA molecule into its components. The
phosphate ester bonds and N-glycosidic bond between the sugar and the nitrogenous
bases are broken by hydrolysis at this extreme condition. This, in turn, releases a
mixture of 4 nitrogenous bases, deoxyribose, and phosphoric acid.
First for the Dische reaction, this test can
identify DNA by its sugar component, the

Scheme 1. Dische reaction

deoxyribose.

Basically,

between

Dische

the

the
reagent

reaction
or

the

diphenylamine reagent and 2-deoxyribose constitute to the development of a blue color.

The reaction is dependent to the conversion of the pentose sugar to -hydroxylaevulinic
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Biochemistry Laboratory Formal Report

aldehyde which reacts to diphenylamine to produce a blue colored compound (Scheme
1). The concentration of the DNA is directly proportional to the intensity of the color
(Dische reaction, n.d.). The result of the DNA hydrolyzate in this test is colorless
because the amount DNA used is extremely low, therefore, there is no noticeable
results.
For the phosphate test, this test is basically based on the reaction of phosphate ions
with

ammonium

molybdate

which

yields

ammonium

phosphomolybdate

((NH4)3PMo12O40), hence, the yellow crystals (Damodaran, 2011). Again, the

hydrolyzate does not form any yellow precipitate indicating a negative result.
For Murexide test, this test is really for uric acid, however, uric acid is the end product of
purine catabolism. In this reaction, purines when reacted to concentrated nitric acid
(HNO3) are oxidized to dialuric acid and alloxan which then condense to form alloxantin.
This is then reacted to potassium hydroxide (KOH) to form ammonium purpurate or
murexide which is a red (pink) residue. But, purplish violet in color may also be seen
due to the potassium salt (Damodaran, 2011). Once more, due to the lack of DNA for
this part of the experiment, a yellow tint
was just seen and not a red color.
Scheme 2. Murexide formation from a purine
Lastly, for the Wheeler-Johnson test, pyrimidines undergo bromination upon reaction
with bromine water to produce dibromohydroxyuracil which is a yellow solution. The
addition of Ba(OH)2 forms dialuric acid and excess of it produces the purple barium salt
of dialuric acid. The DNA hydrolyzate is positive for pyrimidines in this case, eventhough
the observed color is white. This might be due to poor color recognition.

Biochemistry Laboratory Formal Report

Experimental methodology
Three (3) parts were involve in this experiment, namely, isolation of DNA, concentration
and purity determination, and chemical characterization. First, for the isolation part, 50
mL of homogenizing solution was placed in a 250mL Erlenmeyer Flask and pre-heated
at 60C in a water bath. 25g of peeled and minced onion was made up and mixed with
the homogenizing solution for 5 minutes still under 60C and was intermittently stirred
every 2 minutes. After that, 3 pieces of papain tablets were pulvurized and added to the
solution for another 10 minutes. After which, the flask was placed immediately to an ice
bath and swirled occasionally. Then, the mixture was placed in a blender and blended
for 45 seconds. The resulting homogenate was then filtered using 4 layers of
cheesecloth. The filtrate was collected with a 100mL graduated cylinder and the volume
was noted. The residue, on the other hand, was discarded. The filtrate was then
transferred to a 250mL beaker. After which, the beaker was placed in an ice bath in a
position tilted to 45. Cold ethanol, with a volume twice that of the homogenate, was
poured slowly onto the side of the beaker to prevent disrupting the upper layer where
DNA will precipitate. It was let stood for 8 minutes, and after which, the DNA was
spooled using an improvised spooler made during the course of the preliminary part of
the experiment. The spooled DNA strings was placed in a pre-weighed watch glass and
weighed in an analytical balance. The resulting weight was noted.
In determination of DNAs concentration and purity, 2.0mg of the DNA isolate was
weighed and placed in a large test tube. After that, 6.6mL saline-sodium citrate (SSC)
solution was added to the test tube (1.0mg DNA:3.3mL SSC) and mixed using a vortex.
Then, using a UV-Vis, the solution was subjected to two (2) wavelengths of light (260nm

Biochemistry Laboratory Formal Report

& 280nm) at the same time and the absorbance reading was noted. The readings were
plotted using a nomograph.
Lastly, for the chemical characterization part, four different tests were included in this
experiment: Dische (Test for deoxyribose), Murexide (Test for purines), WheelerJohnson (Test for pyrimidines), and phosphate test. Before these tests, the DNA isolate
was first hydrolyzed with an acid. The excess DNA isolate was transferred first in a
large test tube and 1mL of 1M HCl (hydrochloric acid) was added to it. Then, the test
tube was covered with a marble and was heated in a boiling water bath for 1 hour with
occasional shaking. After that, it was cooled to room temperature and, then, added with
2.5mL distilled water. It was then neutralized with 1M NaOH (sodium hydroxide) and
filtered. The resulting filtrate was diluted with a distilled water to make 4mL of a solution.
Then, the DNA hydrolyzate was ready for chemical characterization.
First, for Dische reaction, 1.5mL of diphenylamine reagent was added to 1.0mL of the
DNA hydrolyzate. The solution was heated in a boiling water bath for exactly 10 minutes.
Then, it was cooled immediately and the results were observed.
For the test for phosphate, 1mL of concentrated H2SO4 (hydrochloric acid) was added to
1mL of DNA hydrolyzate. Then, 0.5mL of concentrated HNO3 (nitric acid) and 1mL of
distilled water were added. After that, it was heated in a boiling water bath for 5 minutes.
The solution was allowed to cool and, after cooling, 1mL of ammonium molybdenate
solution was added to it. The solution was mixed well and diluted with 10mL of water. It
was let stood for 10 minutes. The solution was then observed (if there are precipitate
formed and color change).

Biochemistry Laboratory Formal Report

For the Murexide test, a small amount of the DNA hydrolyzate (about 3-5 drops) was
placed in a small evaporating dish. Then, a few drops of concentrated nitric acid (HNO3)
was added to it. It was then evaporated to dryness in a water bath. The resulting
residue was moistened with 10% KOH (potassium hydroxide). The change in color upon
the addition of KOH was noted. Then, a few drops of water were added and was heated
(warmed) again. Then, the solutions color was observed. After which, the solution is
evaporated again and the resulting residue was observed for its color.
Lastly, for the Wheeler-Johnson test, 0.5mL of DNA hydrolyzate was treated with
excess bromine water (Br2H2O) until the solution turned yellow. To remove the excess
bromine water, the solution was boiled using a water bath until it became colorless or
light yellow. After which, barium hydroxide (Ba(OH)2) was added in excess. Then, the
color was noted.
The same treatment was done to the each standard affiliated to each test. The results
from the standard was also noted and compared to that of the sample.

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Biochemistry Laboratory Formal Report

References
For journal resources
Wheeler, H.L., & Johnson, T.B. (1907). Researches on pyrimidines: On color test for
uracil and cytosin. J. Biol. Chem. , 3, 183-189.
For book references
Boyer, R.F. (2012). Biochemistry laboratory : modern theory and techniques (2nd Ed.).
Upper Saddle River, N.J.: Pearson Prentice Hall.
Campbell, M.K., & Farell, S.O. (2012). Biochemistry (7th Ed.). USA: Brooks/Cole,
Cengage Learning
Damodaran, G.K. (2011). Practical biochemistry. New Delhi: Jaypee Brothers Medical
Publishers (P) Ltd.
Sharma, A.K. (2011). Biochemistry of nucleic acid. New Delhi: Random Publications.
For web resources
Dische reaction. (n.d.) Retrieved from:
http://product.lookchem.com/item/251/diphenylamine-test.html
Extraction of DNA from onion cells. (n.d.) Retrieved from:
http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/cpmcnet.columbia.edu/d
ept/physio/tchrplan/oniondna.html

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