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BIOORGANIC
CHEMISTRY
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BioOrganic Chemistry Laboratory CH205 (2015-2016) Experiment 12

Characterization of Nucleic Acids

Mary Bernardine G. Bagalay, Julie Ann Kim F. Berdonado,
Jeanne Isabelle B. Bilasano, and Assumpta Minette C. Burgos*
Department of Speech Language Pathology, College of Rehabilitation Sciences
University of Santo Tomas, Espana Street, Manila 1008
Date Submitted: May 12, 2016
Abstract:
Nucleic acids are made up of nucleotides. A chain of nucleotides make up a polymer that is RNA or DNA. RNA nucleotide is
comprises of a heterocyclic base (adenine, guanine, cytosine and thymine), a pentose sugar (2-deoxy-d-ribofuranose), and a
phosphate group. Deoxyribonucleic acid or DNA is a double helix formed by nitrogenous base pairs (adenine with thymine,
and guanine with cytosine) attached to a sugar-phosphate (deoxyribose) backbone. Conducting of Dische Test, Murexide
Test, Wheeler-Johnson Test, and Test for Phosphate yielded to results that characterizes the structural features of nucleic acid.
Nucleic acid tested positive in Dische Test; guanine and nucleic acid tested positive for Murexide Test; DNA and cytosine
tested positive for Wheeler-Johnson Test; and both phosphoric acid and nucleic acid tested negative for the Test for
Phosphate.
Keywords: nucleic acids, DNA, RNA,Dische Test, Murexide Test, Wheeler-Johnson Test, and Test for Phosphate.

Introduction
Nucleic acids are made up of nucleotides. Each nucleotide made up of purine base (adenine or
guanine) or pyrimidine base (cytosine, uracil or thymine), pentacarbon sugar: ribose or deoxyribose and
orthophosphoric acid residue. The base binds the sugar by the -N-glycosidic bond, orthophosphoric
acid residue binds the sugar component by an ester bond through the -OH group at carbon 3' or 5' of
ribose or deoxyribose. Individual nucleotides are bound with phosphodiester bonds between carbons 3'
and 5. A representation of nucleotide can be seen in Figure 1.

Figure 1. Nucleotide
*Assumpta Minette C. Burgos

A chain of nucleotides make up a polymer: RNA or DNA. Figure 2 shows the structure of RNA
and DNA. Ribonucleic acid or RNA has a hydroxyl group attached to the 2-position of the sugar. RNA
nucleotide is comprises of a heterocyclic base, a pentose sugar (2-deoxy-D-ribofuranose), and a
phosphate group. The heterocyclic bases of RNA are adenine (A), guanine (G), cytosine (C) and
thymine (T). Adenine is pairs to uracil; guanine is pairs to cytosine; cytosine is pairs to guanine; and,
thymine is pairs to adenine.
Moreover, RNA aids in the conversion of genetic information from genes into amino acid
sequences that forms proteins. There are three types of RNA: transfer RNA (tRNA), messenger RNA
(mRNA), and ribosomal RNA (rRNA). The messenger RNA transcribes the genetic sequences.
Ribosomal RNA translates the sequence catalyzing in into a peptiode bond formation. And transfer RNA
carries the molecule to be used in and decodes the mRNA (Marieb, 2014).

Figure 2. Structure of RNA and DNA

Deoxyribonucleic acid or DNA is a double helix formed by nitrogenous base pairs attached to a
sugar-phosphate (deoxyribose) backbone. DNA can be located either in the cell nucleus (nuclear DNA)
or in a mitochondria (mitochondrial DNA or mtDNA). The nitrogenous bases of DNA include adenine
(A), guanine (G), cytosine (C), and thymine (T). DNA bases pair up with each other, A with T, T with A,
G with C and C with G, to form units called base pairs (What is DNA?, n.d).
DNA serves as the
blueprint of protein synthesis carried by the gene (Marieb, 2014).
The groups objective was to identify different structural features that make up the complex
structure of nucleic acids. Moreover, the group is to give specific chemical test to characterize structural
features of a nucleic acid. Thereafter, they are to provide the principles that involved in each chemical
test.
Methodology
Time constraints and test exclusivity limited the number of compounds to be tested by each
group. Groups 1 and 2 conducted the Dische Test for ribose (RNA), deoxyribose (DNA), and nucleic
acid. Ribose was unavailable and was disregarded. Groups 3, 4, and 9 did the Murexide Test for
guanine, adenine, and nucleic acid. However, adenine was unavailable for that time and was neglected.
Groups 5, 6, 10, and 11 did the Wheeler-Johnson Test for thymine, cytosine, uracil, and nucleic acid.
*Assumpta Minette C. Burgos

Again, thymine and uracil were unavailable for that time and was neglected. Lastly, groups 7 and 8
conducted the Test for Phosphate for phosphoric acid (H3PO4) and nucleic acid.
As aformentioned, group 1 and 2 performed the Dische Test for deoxyribose (DNA) and nucleic
acid. Group 1 did the test for nucleic acid whereas, group 2 for DNA. The apparatus and materials used
were 13x100 test tubes, diphenylamine reagent, DNA solution, deoxyribose, nucleic acid and a boiling
water bath. Prior to obtaining the deoxyribose, the group prepared a beaker quarterly filled with water
and set-upped the Bunsen burner, tripod, and wire gauze for boiling. Thereafter, 10 drops of deoxyribose
and 10 drops of nucleic acid were collected in two seprate test tubes then, 20 drops of diphenylamine
reagent was added. After 6 drops of diphenylanine, a change of color of the solution from clear colorless
solution to foggy white solution was observed. Upon boiling of water bath, the prepared test tube was
heated for 10 minutes. A change of color from white foggy to peach color was observed after 3 minutes
of boiling. After 10 minutes, the test tube was then taken out of the boiling water bath and cooled.
Lastly, color of the solution was observed and noted.
Next, the Murexide Test entailed the use of concentrated HNO 3, 10% KOH, DNA solution,
purine (guanine and/or adenine), evaporating dish, and boiling water bath. 10 drops of guanine and 10
drops of nucleic acid were collected in two separate test tubes was collected in two separate test tubes.
Then, 5 drops of concentrated HNO3 was collected in an evaporating dish. The dryness was then
evaporated over a water bath and moistened with 10% KOH. Afterwards, it was warmed over a water
bath. The color was then observed and noted. 10 drops of warm water was added and the color produced
was again noted.
The Wheeler-Johnson Test required 13x100 test tubes, bromine water, 10%Ba(OH)2, DNA
solution, thymine, pyrimidine (cytosine and/or uracil), and water bath. Since, thymine and uracil were
unavailable, only cytosine was used. 10 drops of cytosine was collected in a test tube, then the group
gradually added bromine water until the solution turned yellow (approximately 10mL). It was then
placed in a boiling water bath until it became colorless (to expel excess bromine). Ba(OH) 2 was added in
excess until the red litmus paper turns blue. Then, the color of the solution was then observed and noted.
Lastly, in Test for Phosphate needed apparatus and materials such as potassium nitrate (KNO 3),
sodium carbonate (Na2CO3), 3M nitric acid (HNO3), 2.5% NM4M0O4 solution, DNA solution, H3PO4,
nucleic acid, and a warm water bath. 20 drops of H 3PO4 was collected in a porcelain tube. 0.2 g KNo 3
and 0.1g Na2CO3 were added then mixed. Afterwards, the porcelain crucible was directly heated on a
Bunsen burner until the mixture turned white or gray. The porcelain crucible was cooled and 5 mL warm
distilled water was added to the mixture. Next, the mixture was filtered into a test tube and acidified
filtrate with 3M HNO3. 3mL 2.5% NM4M0O4 solution was added and warmed in a water bath. The test
tube was left to stand for 10 minutes. The formation and color of the precipitate was then observed and
noted. These steps were repeated but with the nucleic acid.

*Assumpta Minette C. Burgos

Results and Discussion

Table 1:
Results of the Experiment
Dische Test
Ribose (RNA)
Deoxyribose
(DNA)
Guanine

Murexide Test

peach solution

Wheeler-Johnson
Test

for

red litmus paper

turned blue
red
orange
solution to
yellow
clear
solution

Adenine
Thymine
Cytosine

purple
solution
with
violet
precipitate

Uracil
H3PO4
(phosphoric acid)
Nucleic acid

Test
Phosphate

blue solution

yellow solution

clear
solution

clear
colorless
solution with no
precipitate
cloudy cloudy
solution
with white floating
precipitate

Dische test is used for the detection of deoxyribose (DNA). As aforementioned, there are two
types of nucleic acids: DNA and RNA. Their sugar component makes them distinct from one another.
RNA has ribose whereas, DNA has deoxyribose. To chemically identify DNA, diphenylamine can be
used as an indicator; it also aids in the production of stabilizers in explosives and fuel. Diphenylamine,
with a molecular formula of C12H11N, is an organic compound classified as an aromatic amine. The
molecular structure of diphenylamine can be observed in Figure 3.

Figure 3. Molecular structure of diphenylamine.

A reaction between Dische reagent and 2-deoxypentose yields a blue color. This reaction is due
to the conversion of pentose to w-hydroxylaevulinic aldehyde which then reacts with diphenylamine to
give a blue colored complex. The intensity of the blue colored complex is proportional to the
*Assumpta Minette C. Burgos

concentration of DNA used. The greater the concentration of DNA, the stronger the hue (Damodaran,
2011).
In the experiment, RNA, DNA, and nucleic acid were the compounds to be tested. As RNA was
unavailable, DNA and nucleic acid were the only ones tested. DNA displayed a peach solution whereas;
nucleic acid displayed a blue solution. The DNA was expected to express a dark deep purple or blue
color, an error in handling or contamination can be some possible reasons for the wrong result. Nucleic
acid confirmed the presence of DNA as it expressed a blue color.
The second test, Murexide Test, is a test for uric acids. Uric acid is the end product of purine
catabolism. Purines are heterocyclic aromatic organic compound that consists a pyrimidine ring fused to
an imidazole ring. Further catabolism of purine yields uric acid. Some examples of which are adenine
(6-amino purine), guanine (2-amino-6-oxy purine), hypoxanthine (6-oxy purine), and xanthine (2,6dioxy purine). Adenine and guanine are the two purines present in a nucleic acid. Their molecular
structure can be seen in Figure 4.

Figure 4. Molecular structure of purines: adenine and guanine.

Reaction of purines with concentrated nitric acid (HNO3) yields to the oxidation of dialuric acid
and alloxan which then condense to form alloxantin. Then, this is reacted to potassium hydroxide (KOH)
to form ammonium purpurate or murexide which is a red (pink) residue(dry state). In solution, its color
ranges from yellow in strong acidic pH through reddish-purple in weakly acidic solutions to blue-purple
in alkaline solutions. Figure 5 shows the formation of a murexide from a purine.

Figure 5. Formation of a murexide from a purine.

In this specific test, guanine and nucleic acid were used. Guanine exhibited a yellow clear
solution from a red orange solution. On the other hand, nucleic acid exhibited yellow solution. Both
results points verifies to the presence of a purine in structure.
Wheeler-Johnson Test is test exclusive for pyrimidine bases: cytosine, uracil and thymine.
Pyrimidines have only a six-membered nitrogen-containing ring. Some examples of which pyrimidine
are uracil (2,4-dioxy pyrimidine), thymine (2,4-dioxy-5-methyl), cytosine (2-oxy-4-amino pyrimidine),
*Assumpta Minette C. Burgos

and orotic acid (2,4-dioxy-6-carboxy pyrimidine). Among of which, uracil, thymine, and cytosine are
found in nucleic acids. Their molecular structure can be seen in Figure 6.

Figure 6. Molecular structure of pyrimidines: cytosine, thymine and uracil.

Pyrimidine goes through bromination as it reacts to bromine water and yield a dialuric acid
which gives it green coloration. Addition of the barium hydroxide turned the mixture into purple. A
display of purple or violet solution with violet precipitate indicates a presence of pyrimidine in a
compound.
In the test done by the students, out of the three pyrimidines mentioned only cytosine was
available and tested. Besides, cytosine, DNA and nucleic acid were also tested. Cytosine exhibited a
purple solution with violet precipitate- which indicates a positive result. DNA showed a positive result.
Nucleic acid showed a clear cloudy solution which indicates a negative result. Although, nucleic acid
was also expected to contain pyrimidine, it did not transpire.
Lastly, the Test for Phosphate is to test the presence of phosphate in a DNA. The phosphate
group can be found at the 5- or 3-position of the sugar depending on the method used to break down
DNA to produce the nucleotides. In this test, the phosphate ions react with ammonium molybdate and
form yellow sediment of ammonium phosphomolybdate (NH 4)3P(Mo3O10)4, which is reduced by metol
to molybdenic blue (mixture of molybdenium oxides at lower oxidation state). Hence, formation of
bright canary-yellow crystals/precipitate could be observed (Damodaran, 2011).
The phosphoric acid in yielded a clear colorless solution with no precipitate while, the nucleic
acid yielded cloudy solution with white floating precipitate. The phosphoric acid is expected to show a
precipitate. Moreover, the precipitate of the nucleic acid should have been yellow in color and not white.
This may be brought about by poor color recognition of the group.
To sum it all up, table 2 shows the expected result and the actual result yielded.
Table 2.
Comparison of the expected result and actual result
Tests
Dische

Expected
Blue solution

*Assumpta Minette C. Burgos

Actual
DNA: peach solution
Nucleic acid: blue solution

Positive (+) or
Negative (--)
-+

Murexide

yellow in strong acidic pH

reddish-purple
weakly
acidic solutions
blue-purple in alkaline
solutions
Wheeler-Johnson purple or violet solution
with violet precipitate

Phosphate

Bright canary-yellow
precipitate

Guanine:
red
orange
solution to yellow clear
solution
Nucleic
acid:
yellow
solution
DNA: red litmus paper
turned blue
Cytosine: purple solution
with violet precipitate
Nucleic acid: clear cloudy
solution
Phosphoric acid: clear
colorless solution with no
precipitate
Nucleic
acid:
cloudy
solution with white floating
precipitate

+
+

+
+
--

--

--

Conclusion

Nucleic acids are made up of nucleotides. Each nucleotide made up of purine base (adenine or
guanine) or pyrimidine base (cytosine, uracil or thymine), pentacarbon sugar: ribose or deoxyribose and
orthophosphoric acid residue. Dische test is used for the detection of deoxyribose (DNA). The greater
the concentration of DNA, the stronger the hue. Murexide Test is used for the detection of purines. In
solution, murexide color ranges from yellow in strong acidic pH through reddish-purple in weakly acidic
solutions to blue-purple in alkaline solutions Wheeler-Johnson Test is test exclusive for pyrimidine base.
Test for Phosphate is to test the presence of phosphate in a DNA.

*Assumpta Minette C. Burgos

References
Damodaran, G.K. (2011). Practical biochemistry. New Delhi: Jaypee Brothers Medical
Marieb, E. (2014). Essentials of Human Anatomy and Physiology (10th ed.) Pearson.
Publishers (P) Ltd.
What is DNA?. (n.d). retrieved on May 11, 2016 at https://ghr.nlm.nih.gov/primer/basics/dna
Figures:
Molecular structure of pyrimidine. Retrieved on May 11, 2016 at chemwiki.ucdavis.edu
Molecular structure of purine. Retrieved on May 11, 2016 http://www.scienceprofonline.com/chemistry/
Molecular structure of diphenylamine. Retrieved on May 11, 2016 at
https://www.mpbio.com/product.php?pid=05217746&country=168
Nucleotide. Retrieved on May 11, 2016 at http://eternagame.wikia.com/wiki/Nucleotides
Structure of RNA and DNA. . Retrieved on May 11, 2016 at http://www.scienceprofonline.com/
chemistry/

*Assumpta Minette C. Burgos