I.

Name:
(GROUP 05) BSN - 1H
ORTONIO, Eunice Jireh F. (Leader)
LLANILLO, Piolo Angelo E.
MOPAL, Gerhard D.
NARVAZA, Donna Daniela A.
PABLICO, Jessica Lorenz M.
PAGUIA, Norhanna T.
PIZARRAS, Hannah Gale G.

II. Title of experiment and no.:

Analysis of Nucleic Acids, Experiment 4B

III. Objectives:
‣ To qualitative test the DNA sample gathered from Experiment 4A.
‣ Differentiate unhydrolyzed DNA to acid hydrolyzed DNA.

IV. Theoretical Background:

DNA is a biological molecule that contains genetic information. It is a nucleic acid that is organized into
chromosomes. The genetic code found in DNA provides instructions for the production of proteins and all components
necessary for the reproduction of life. Extracting DNA from a banana may sound like a difficult task, but it is not very
difficult at all. The process involves a few general steps, including mashing, filtration, precipitation, and extraction.Mashing
the banana exposes a greater surface area from which to extract the DNA. The liquid soap is added to help break down cell
membranes to release the DNA. The filtration step (pouring the mixture through the strainer) allows for the collection of
the DNA and other cellular substances. The precipitation step (pouring the cold alcohol down the side of the glass) allows
the DNA to separate from other cellular substances. Finally, the DNA is removed from the solution by extraction.

ACID HYDROLYSIS OF DNA

For DNA (also RNA) acid hydrolysis cleaves the predisposed purine N-glycosyl bonds. If the nucleic acids are
placed in a dilute acid solution,like the sulfuric acid, coupled with heating, the adenine and guanine residues are liberated.
What remains are apurinic sites. Mild heating doesn’t release pyrimidines. Further heating in sealed test tube or autoclave
is required to cleave pyrimide N-glycosyl bonds. It can be deduced that the depurination occurs is promoted by the
protonation of the purine base, thus, weakening the N-glycosyl bond. The N glycosyl bond is then irreversibly broken by
the neighboring oxygen atom giving a free purine base and an apurinic nucleic acid. Acid-catalyzed depyrimidization also
proceeds in a similar mechanism as depurination. Depyrimidization releases cytosine and uracil, depending on the nucleic
acid being considered. However, in acidic conditions, cytosine can be easily deaminated to uracil. The products of hydrolysis
of DNA are purine and pyramidine bases, oligonucleotide, nucleosides, deoxyribose, and phosphate.

Benedict's Test is used to test for simple carbohydrates. It identifies reducing sugars (monosaccharide's and some
disaccharides), which have free ketone or aldehyde functional groups. Benedict's reagent is a chemical reagent and complex
mixture of sodium carbonate, sodium citrate and copper(II) sulfate pentahydrate. The presence of other reducing substances
also gives a positive reaction. Such tests that use this reagent are called the Benedict's tests. A positive test with Benedict's
reagent is shown by a color change from clear blue to a brick-orange precipitate. Benedict's reagent starts out aqua-blue. As
it is heated in the presence of reducing sugars, it turns yellow to orange. The "hotter" the final color of the reagent, the
higher the concentration of reducing sugar. In general, blue to blue-green or yellow-green, traces found, yellowish to bright
yellow is a moderate positive, and bright orange is a very strong positive.
(http://www.bio.brandeis.edu/classes/bio18/sample1.html)

1 | 4B - Analysis of Nucleic Acids

 Purine is a heterocyclic aromatic organic compound that consists of a pyrimidine ring fused to an imidazole ring. It
is water-soluble. Purine also gives its name to the wider class of molecules, purines, which include substituted purines and
their tautomers. Test for purines base was used to detect two-carbon nitrogen ring bases which are the adenine and guanine
and the positive indicator of this test is a gelatinous white precipitate if guanine is found and grayish blue gelatinous
precipitate if adenine is present. The role of silver nitrate/basic condition is to precipitate purines via its interaction with the
purines nitrogens. Test for purines: Add excess 2M ammonia solution and a few drops of 0.1M silver nitrate to 1 mL of
DNA extract. A white precipitate indicates the presence of purines. The precipitation mechanism is by a reaction of Ag+
with the nitrogens of purines . Depurination, the release of purine bases from nucleic acids by the hydrolysis of N-glycosidic
bonds, has aroused considerable interest for a long time because of its close relationship with mutation and repair of nucleic
acids. At apurinic sites caused by depurination, the covalent structure of DNA becomes more susceptible to damage, which
induces spontaneous mutagenesis, carcinogenesis and cellular aging. The silver nitrate/basic condition is to precipitate
purines via its interaction with the purines nitrogens. https://www.ncbi.nlm.nih.gov/pmc/articles

Figure 1.1 shows the positive and negative indicator of Benedict’s test

Bial's test is a chemical test for the presence of pentoses. The components include orcinol, hydrochloric acid, and
ferric chloride. A pentose, if present, will be dehydrated to form furfural which then reacts with the orcinol to generate a
colored substance. The solution will turn bluish and a precipitate may form. The reagents are the chemical compounds
which are necessary to perform some chemical test. Just like any other test, there are few reagents that are used in this test.
The compounds being used in this test acid are Ferric acid, hydrochloric acid, and orcinol. The reagent is prepared by
combining these compounds in a balanced manner. The reagent for Bial’s test is easily prepared by adding these compounds

in a balanced proportion. The amount of orcinol in the reagent is 0.4g is added to the 200 ml of hydrochloric acid and 0.5
ml of ferric chloride solution is also added to complete the making of reagent. The reaction works in a way that pentose is
dehydrated by the reagent and a furfural form is formed. Orcinol then reacts with this furfural which then reacts with iron
to give a bluish colored product and the presence of pentose is detected. A positive result in indicated when a bluish or
greenish color appears in the solution. Remember that only bluish or green color indicates a positive test. If some other color
appears then the result is negative. http://allmedtests.com/bials-test/

The phosphate forms the structural framework of nucleic acids, including DNA. This phosphate is composed of
alternating sugar and phosphate groups, and defines directionality of the molecule. A simple qualitative method to
determine the presence of phosphate ions in a sample is as follows. A small amount of the sample is acidified with

2 | 4B - Analysis of Nucleic Acids

concentrated nitric acid, to which a little ammonium molybdate is added. The Ammonium molybdate will react to the
acidified solution creating a yellow solution and precipitate . The Yellow solution indicates the presence of phosphate ions
in the test solution. The yellow precipitate at the bottom is the presence of ammonium-phosphomolybdate. The appearance
of the precipitate can be facilitated by gentle heating. This test is also used to detect arsenic, a yellow precipitate being
formed.

V. Data & Results

Table 1.1 Bendict’s Test Table 1.2 Test for Purine

Test Tube A Test Tube B Test Tube A Test Tube B
Positive (+) Positive (+) Negative (-) Positive (+)

The solution turned from

blue-colored solution to The basic solution slightly
The solution turned from No precipitation occurred. precipitate after 3 drops of
green. After undergoing
blue-colored solution to Thus, no formation of 1% Silver Nitrate solution.
boiling water bath. It
red-colored solution gelatin-like substance. The color of precipitate is
developed yellow-orange
precipitate at the bottom. moss green.

VI. Data Analysis

In this experiment we analyzed the DNA sample using qualitative test. We divided the DNA sample into two; the
acid hydrolyzed DNA and the unhydrolyzed DNA. After, we determined the individual components of nucleotide using

3 | 4B - Analysis of Nucleic Acids

 Table 1.3 Bial’s Test Table 1.4 Test for Phosphate
Test Tube A Test Tube B Test Tube A Test Tube B
Negative (-) Negative (-) Postive (+) Negative (-)

After boiling, the sample

There is precipitation solution did not developed
There is coagulation, where After boiling, the color of
where the supernatant’s any yellow precipitate but
it turned to pale yellow- the solution turned to light
color turned to light blue. only heavy white
orange. yellow.
precipitation.

Benedict’s Test (used to detect glucose), Test for Purine Bases (used to detect purine), Bial’s Test (detect presence of
pentose) and Test for Phosphate (detect presence of phosphate).

Benedict’s Test

Benedict’s test was used to detect the presence of reducing sugars(Brandeis University, n.d.) with a positive
indicator of green colored solution (trace of reducing sugar) yellowish or yellow solution (moderate)bright orange solution
or brick red solution (large amount of reducing sugar). In test tube A with hydrolyzed DNA, the sample turned from blue
colored solution to red-colored solution which means that presence of reducing sugars is found (positive). In test tube B
with unhydrolyzed DNA, the blue colored solution turned to green colored solution which indicates a trace of reducing
sugar (positive). Therefore, both acid hydrolyzed and unhydrolyzed DNA resulted to positive result which means that
reducing sugar is found. However, unhydrolyzed DNA has lesser reducing sugar than acid hydrolyzed. Since DNA contains
deoxyribose sugar, a reducing sugar, it is theoretically correct that both samples exhibited a positive result, the factor of
color differences lies in the hydrolysis of DNA in which it has reduced in its sugar form which is the deoxyribose sugar
which explains the large amount of reducing sugar found by yielding a red-colored solution while the unhydrolyzed one
yielded only traces of the reducing sugar.

Test for Purine Bases

Test for purines base was used to detect two-carbon nitrogen ring bases which are the adenine and guanine and the
positive indicator of this test is a gelatinous white precipitate if guanine is found and grayish blue gelatinous precipitate if
adenine is present. In the test, the one with the hydrolyzed DNA should exhibit a positive result it is because base hydrolysis
acts on the cleavage of the phosphodiester bond and not on the purine N-glycosyl bonds. The experiment exhibited an
erroneous result. In test tube with hydrolyzed DNA, it resulted to no precipitation which indicates a negative result. This
may have been caused by a contaminated solution or factors like inadequate DNA sample in the solution. In test tube B, a
slight precipitation occurred but is moss green colored . Therefore, it is still considered as a negative result since we are

4 | 4B - Analysis of Nucleic Acids

looking for a white or grayish blue gelatinous precipitate. This result is true because the sample was unhydrolyzed meaning
that there is no cleavage of the phosphodiester bond that would expose or liberate the purines nitrogen that reacts with the
silver nitrate.

Bial’s Test

Bial’s test is a test used to detect the presence of pentose. A positive test for pentose is indicated by a blue or green
condensation product (Rao, 1992). All samples are expected to give a negative result. Though DNA has a ribose as a sugar
component, it is a deoxyribose. Without a free hydroxyl group, it cannot further dehydrate to a furfural derivative that would
give a positive result to the test. Both samples exhibited a negative result which coincides with the theoretical basis. In test
tube A with hydrolyzed DNA, the color of the solution turned to pale yellow-orange which means that the presence of
pentose is not found (negative). In test tube B with unhydrolyzed DNA, the color of the solution turned light yellow
indicating that there is no pentose found (negative). Thus, both the acid hydrolyzed and unhydrolyzed DNA resulted to a
negative result which means that pentose is not found.

Test for Phosphate

The phosphate sample hydrolyzes phosphate and reacts with the reagent, producing the yellow precipitate. The
cleavage of the phosphodiester bonds in the Hydrolyzed DNA enables phosphate ions to be liberated thus its reaction with
ammonium molybdate produced a light yellow precipitation which indicates a positive result. In test tube b with an
unhydrolyzed DNA, the sample solution did not develop a yellow precipitation because there is no phosphate ions identified
since the DNA is still intact, without a free phosphate ion to interact with the reagent, but it produced a white heavy
precipitation caused by the reaction between the acidified solution and ammonium molybdate, which indicates a negative
result. Therefore, in acid unhydrolyzed DNA no presence of phosphate was found while in the hydrolyzed DNA phosphate
was found.

VII. Conclusion

The experiment obtained a solution made from experiment 4a which is the extraction of DNA. This DNA is
separated into two: hydrolyzed and unhydrolyzed DNA. Acid Hydrolyzed DNA cleaves the predisposed purine N-glycosyl
bonds. If the nucleic acids are placed in a dilute acid solution, like the sulfuric acid, coupled with heating, the adenine and
guanine residues are liberated, while the unhydrolyzed DNA is intact and no cleavage on its phosphodiester bonds. In this
experiment there are 4 qualitative tests done on both solutions these tests are: Benedict’s test, Test for Purine, Bial’s Test
and Test for phosphate.

Benedict’s test is a test used to test for simple carbohydrates. It identifies reducing sugars (monosaccharide's and
some disaccharides), which have free ketone or aldehyde functional groups. The hydrolyzed DNA turned to red-colored
solution which indicates a large amount of reducing sugars present in the solution while the unhydrolyzed DNA developed
a green colored solution which indicates traces of reducing sugar in the solution.

5 | 4B - Analysis of Nucleic Acids

 Test for Purine identifies purine nitrogens in solution using the silver nitrate/basic condition is to precipitate purines
via its interaction with the purines nitrogens. The hydrolyzed solution, theoretically, should have exhibited white gelatinous
precipitates but the sample did not exhibit any precipitation thus a negative result. The unhydrolyzed DNA on the other
hand, developed slight moss-colored precipitates which is still negative since we are looking for white gelatinous
precipitates.

Bial’s test is a chemical test for the presence of pentoses. Both the hydrolyzed and unhydrolyzed DNA exhibited a
negative result with their solution turned to yellowish one. Even though DNA has a ribose as a sugar component, it is a
deoxyribose. Without a free hydroxyl group, it cannot further dehydrate to a furfural derivative that would give a positive
result to the test.

In the Test for phosphate in the experiment, the hydrolyzed DNA exhibited a Positive result with its precipitation
color turned to light yellow which is a positive indicator meaning that there are phosphate ions and ammonium-
phosphomolybdate present in the sample while the unhydrolyzed DNA tested negative even though it developed white
precipitates.

REFERENCES

Brandeis University, (1997), Biology 18 B Laboratory Manual. Brandeis University. Waltham, MA. Dr. Judith Tsipis Ed,

Fall 1997. Lab 2-3. Retrieved from: http://www.bio.brandeis.edu/classes/bio18/sample1.html

Course Hero. (n.d.) Bial’s Test is a Test for the Presence of Pentose it. Retrieved from:

https://www.coursehero.com/file/p8v62j/Bials-test-is-a-test-for-the-presence-of-pentose-It-can-be-used-as-a-

test-for/

6 | 4B - Analysis of Nucleic Acids

Gilig, L.P., (n.d). Experiment 3: Nucleic Acids Retrieved from:

https://www.scribd.com/document/338035486/Experiment-3-Nucleic-Acids

Sayres, M.W.,. (n.d). Rennin Enzyme. Retrieved from: https://www.britannica.com/science/rennin

Science Direct. (n.d). Diphenylamine. Retrieved from: https://www.sciencedirect.com/topics/medicine-and-

dentistry/diphenylamine

The American Phytopathological Society. (2019). Activity 1 - DNA Extraction. Retrieved from:

https://openlab.citytech.cuny.edu/bio-oer/chemistry/biologically-important-macromolecules/nucleic-

acids/nucleic-acids-dna-extraction-and-disches-diphenylamine-test-activity/

The Open Lab. (n.d.). Nucleic Acids: DNA extraction and Dische’s Diphenylamine test (Activity). Retrieved from:

https://openlab.citytech.cuny.edu/bio-oer/chemistry/biologically-important-macromolecules/nucleic-

acids/nucleic-acids-dna-extraction-and-disches-diphenylamine-test-activity/

7 | 4B - Analysis of Nucleic Acids