CHEMICAL CHARACTERIZATION OF DEOXYRIBONUCLEIC ACID

Redentor D.L. Laureta III, Bea Czarina T. Loque, Lara Mae S. Lorenzo,
Jeanette T. Lusung and Tobias Manuel Y. Maningat
Group 5 2H Medical Technology Biochemistry Laboratory

ABSTRACT
In order to characterize DNA, several procedures must be performed, namely, Wheeler-Johnson's test for Pyrimidines,
Murexide test for Purines, the Phosphate test, and the Dishe, or deoxyribose test. Each of these tests were performed
to note if the DNA sample is truly DNA. The Dische test reacts with doexyribose to create a blue solution, while the
Phosphate test reacts with the phosphate found in the backbone of the nucleic acid to form a yellow precipitate. The
Wheeler Johnson's test and the Murexide tests all react with their respective substances to form a red residue and a
violet ring.

INTRODUCTION Nucleic acid solution, evaporating dish, conc.

DNA isolated from plants are said to be nitric acid, beaker, water, 10% Potassium
condensed and often duplicated. In order to hydroxide
further study these nucleotides, one needs to • Test for Pyrimidines (Wheeler-Johnson
determine its reactivity through different test):
chemical tests after hydrolysis. Procedures like
Dische test, which is also known a deoxyribose 0.5mL Nucleic acid solution, Bromine water,
test that gives a blue color when performed in a beaker, water, 20 drops Barium hydroxide, litmus
solution with deoxyribose. Another technique to paper
use in order to classify if the solution has purine
or pyrimidine is by making it undergo into B. Procedure
Murexide Test, a test in which treatment of a
substance, usually urine, with nitric acid and
1. Test for Deoxyribose (Dische Test)
ammonia indicates the presence of uric acid by
formation of murexide; and the Wheeler-Johnson
test. Lastly, to prove that a nucleic acid has its
Two test tubes were prepared for the set-up.
phosphate group, it must yield a yellow
The diphenylamine reagent with an amount of
precipitate in the test for Phosphate can give
3.5 mL was added to 1.5 mL of hydrolyzed DNA
data about the composition a plant DNA that is
solution. The same procedure was done to 0.5
likely to be the almost the same as human DNA.
mL of the standard deoxyribose solution. Both of
These chemical tests will also help students to
the test tubes were placed in water bath for 10
classify if a solution has RNA or DNA but still, the
minutes and were cooled immediately for
main objective of this experiment is to give the
observation.
students an idea of what is the DNA made of.
2. Test for Phosphate

Two test tubes were used in the test. In the

EXPERIMENTAL
A. Materials and Compounds Used
• Test for Deoxyribose (Dische Test):
2 test tubes, 3.5mL diphenylamine reagent,
1.5mL hydrolyzed DNA solution, beaker with
water, hot plate
• Test for Phosphate:
2 test tubes, 1mL conc. Sulfuric acid,1mL nucleic
acid solution, standard phosphate solution, 0.5
mL conc. Nitric acid, 1mL and 10mL water,
beaker, 1mL 10% Ammonium molybdate solution
• Test for Purines (Murexide Test):
first test tube, 1 mL of concentrated sulfuric acid
was added to 1 mL nucleic acid solution. The
same procedure was done in the second test tube
using the standard phosphate solution. Both test
tubes were heated over a small flame, shaking
frequently until the contents of the tube turn
brown. The mixture was then cooled and was
further added with 0.5 mL of concentrated nitric
acid. The solution was heated until white fumes
appear. The resulting colorless liquid was added
with 1 mL water and was heated for 5 minutes
over water bath. The solution was cooled and
added with 1 mL 10% ammonium molybdate
solution and was mixed evenly followed by
dilution with 10 mL water. The solution was left
for 5 minutes for the observation of the color of test for DNA was indicated by a blue color
the resulting precipitate. change. This procedure involves chemical
3. Test for Purines (Murexide Test) hydrolysis of DNA: when heated in acid, the
reaction requires a deoxyribose sugar and
therefore is specific for DNA. Under these
In a small evaporating dish, 5-10 drops of conditions, the 2-deoxyribose was converted to
nucleic acid solution was placed and added with a ω -hydroxylaevulinic aldehyde. This resulted to a
few drops of concentrated nitric acid. The blue solution indicating the presence of protein in
solution was carefully evaporated until it was dry DNA.
through water bath. The residue was moistened
with 10% potassium hydroxide and was further
heated. The change in color was noted upon the
addition of potassium hydroxide and further
heating. Few drops of water was then added and
the solution was warmed. Again, the solution’s
color was noted. The solution was evaporated
and the color of the residue was observed.
4. Test for Pyrimidines (Wheeler-
Johnson Test)

Nucleic acid solution with an amount of 0.5 mL

was treated with an excess of bromine water Figure 1. Result for Dische test
until the solution turns yellow. The excess was
removed by boiling the solution until it turns light
yellow or colorless. An excess of barium
hydroxide was then added followed by the testing
with litmus paper. Finally, the solution’s color was
noted.

RESULTS AND DISCUSSION

Table 1. Results for the chemical characterization

of DNA

CHEMICAL DESCRIPTION HYDROLYZATE

TEST (STD.)
Test for Light blue Blue solution
Deoxyribose solution
Test for Formation of Formation of
Phosphate yellow yellow Figure 2. Dische Test Reaction
precipitation precipitation
Test for Formation of red Formation of 2. Test for Phosphate
Purines precipitation yellow to red
Phosphate Test gave out a yellow result for the
precipitation
precipitation which simply means that a
Test for Red litmus paper Red litmus paper
Pyrimidines turned into blue; turned into blue;
phosphodiester bond exists between DNA and
Formation of Pale yellow RNA between the 3’ Carbon atom and the 5’
purple turbid solution Carbon of the ribose sugar. This was made
precipitation possible because of the reaction between the
ammonium molybdate solution and the sample
which yields yellow crystals, phosphoammonium
1. Test for Deoxyribose (Dische Test)
molybdate which indicates a positive result.
Dische Test was used to indicate that DNA can
3. Test for Purines (Murexide Test)
be identified chemically with the Dische
In the test for the presence of purines, DNA
diphenylamine test. DNA and RNA are aldehydes,
reacted with Nitric acid since purines are known
which reacts with the compound, diphenylamine,
to be readily soluble in acids. Nitric acid oxidized
to produce blue colored compound. A positive
it leaving a yellow precipitation upon
evaporation; however it turned red when
moistened with a base (10% potassium
hydroxide), a positive result for presence of
purine bases.
Soodak, M. (n.d.) A Colorimetric Method for the
Estimation of Uracil and Cytosine. Retrieved from
http://www.jbc.org/content/181/2/713.full.pdf
2/16/2011
Test for Phosphate. (2011). Retrieved from
http://www.tpub.com/content/construction/1427
9/css/14279_41.htm 2/16/2011

Figure 3. Result for Murexide Test

4. Test for Pyrimidines (Wheeler-Johnson
Test)
In the Wheeler Johnson Test, a qualitative test
for the pyrimidine bases cytosine and uracil,
yellow coloration was produced when the
samples were treated with excess bromine water.
A purple precipitate was achieved and formed
upon addition of barium hydroxide. Cytosine was
used in the test which is a Pyrimidine derivative
heterocylic in nature.

REFERENCES
From Books
Boyer, R. F. (2006). Concepts in Biochemistry
(3rd ed.). Hoboken, NJ: Wiley.
Campbell, M.K., and Farrell, S.O. (2006).
Biochemistry, Fifth Edition. California, USA:
Thomson Brooks/Cole.
Champe, P.C., and Harvey, R.A. (1994).
Biochemistry, 2nd Edition. Philadelhphia,
Pennsylvania: J.B. Lippincott Company.
Delvin, T.M., (2002). Textbook of Biochemistry
with Clinical Correlations. New York, USA: Wiley-
Liss.
Wilson, K. (2010). Principles and technique in
Biochmeistry and molecular Biology 7th Ed.
Cambridge University Press: New York

From the Internet

DNA. (n.d.) Retrieved from
http://en.wikipedia.org/wiki/DNA#Supercoiling
2/12/2011
Malik, S. (2006). Physical chemical
characterization of DNA–SWNT suspensions and
associated composites. Retrieved from
http://www.sciencedirect.com/science 2/17/2011
Murexide test - Acid, Colour, and Uric. (n.d.)
Retreived from http://science.jrank.org/pages/
36674/murexide-test.html 2/16/2011
Projection Experiment: Test for Deoxyribose in
DNA. (2003). Retrieved from
http://www.demochem.de/p31_d_rib-e.htm.
2/16/2011