EXPERIMENT 4B

ANALYSIS OF NUCLEIC ACIDS

I. INTRODUCTION

Nucleic acids are composed of monomer units called nucleotides that are connected by
phosphodiester bonds. There are four types of bases present in the nucleic acids DNA and RNA, namely:
Adenine, Guanine, Cytosine, Thymine (present only in DNA), and Uracil (present only in RNA). Adenine
and Guanine are called Purine bases, containing two-carbon nitrogen ring. Cytosine, Thymine, and Uracil
are called Pyrimidine bases, containing one-carbon nitrogen ring.

The main aim of this experiment was to analyze the individual components of nucleotide through
testing the DNA sample gathered from experiment 4A qualitatively by means of conducting four different
tests, namely: Benedict’s test for reducing sugars, Test for Purine to detect the presence of Purine bases,
Bial’s test to detect the presence of pentoses, and Test for Phosphate for the presence of
phosphates/phosphate groups in the DNA sample. This qualitative analysis is dependent upon the
hydrolysis of the sample DNA’s chain to nucleotides and subsequently to phosphorus, deoxyribose, and
purine/pyrimidine bases. Thus, two sets of samples for each test were prepared, one unhydrolyzed set
and one hydrolyzed set.

Knowledge of basic Nucleic acid analysis procedures is essential for medically-inclined students as
this can help better familiarize them with the valuable clinical information that both DNA and RNA can
provide such as in the process of genotype analysis and infectious disease testing.

II. OBJECTIVES

This experiment aimed to:

1. Test the DNA sample gathered from experiment 4A qualitatively.

2. Differentiate unhydrolyzed DNA to acid hydrolyzed DNA.
III. METHODOLOGY

Materials:

Graduated cylinder Test tube brush

Test tube rack Blender

Beaker Hot plate

Test tube holder Test tubes

Stirring rod Water bath

Reagents:

Distilled water Benedict’s reagent

1% silver nitrate 6N nitric acid

10% sulfuric acid 1% ammonium hydroxide

Bial’s reagent Ammonium molybdate

A. ACID HYDROLYSIS B. ANALYSIS OF DATA

Gathered DNA 1. BENEDICT’S TEST

Test tube 1A Test tube 1B

Test tube A Test tube B
(Hydrolyzed) (Unhydrolyzed) Neutralized with
Added Solid of Na2CO3

10ml of 10% 10mL of distilled water Tested with

sulfuric acid Divided in Litmus Paper
Covered
Test Test Test Test
Marble Set aside for
tube tube tube tube
two minutes
Heated 1B 2B 3B 4B
One-hour 60°C
Water bath Decanted
Cooled at room temperature
Centrifuged at 2rpm Set aside for the 1mL solution 1ml solution
for 10 minutes following experiment from from
extracted DNA decanted 1B
Discarded precipitate and placed in
Added
Test Test Test Test 0.5mL Benedict’s
tube tube tube tube reagent
1A 2A 3A 4A
Five-minute boiling
water bath

Observed results

2. TEST FOR PURINE BASES

Test tube 2A Treated until basic with

Tested Added
1% ammonium
Litmus Paper 1% silver nitrate solution
hydroxide
Test tube 2B Set aside
Looked for precipitate
3. BIAL’S TEST 4. TEST FOR PHOSPHATE

Test tube 3A Test tube 3B Test tube 4A Test tube 4B

Added Added until basic

0.5mL Bial’s reagent 1% ammonium hydroxide
Placed in
Tested with
Five to ten-minute Litmus Paper
boiling water bath
Added until acidic
6N nitric acid
Observed results Tested with
Litmus Paper
Added
Heated
1mL of ammonium
Observed results Water bath
molybdate

IV. RESULTS AND DISCUSSION

Table 1. Analysis of DNA

TESTS OBSERVATIONS RESULT

GREEN POSITIVE
BENEDICTS
ORANGE PRECIPITATES POSITIVE

WHITE PRECIPITATES/ POSITIVE

TEST FOR PURINE WHITE CLOUDY SOLUTION

BROWNISH COLORATION AND NEGATIVE

PRECIPITATES SANK AT THE BOTTOM

BRIGHT YELLOW SOLUTION NEGATIVE

BIAL’S TEST
ORANGE SOLUTION NEGATIVE

WHITE PRECIPITATES NEGATIVE

TEST FOR PHOSPHATE
OFF WHITE SOLUTION W/
BROWN AND DARK YELLOW PRECIPITATES POSITIVE
 According to the Lincoln High School (n.d), the presence of smaller carbohydrates like glucose can
be determined by using Benedict's solution. Benedict's solution has copper ions (Cu2+) that have a light
blue color. When this solution is heated in the presence of sugars like glucose the copper ions change and
turn anywhere from a light green to rusty orange-brown color. In this experiment, it showed a green
coloration of sample which means it possessed reducing sugars albeit with varying concentrations with
sample A, having only traces of reducing sugars and sample B, having a higher amount. The hydrolyzed
sample (see Figure 1- left) was greenish orange and the unhydrolyzed sample (Figure 1- right) showed an
orange coloration which indicates that they are both positive in reducing sugars but only differed in
concentrations.

Figure 1. Benedict’s Test Results

The most important biological substituted purines are adenine and guanine, which are the major
purine bases found in RNA and DNA and this test indicates the presence of these purines. As shown on
the table above, it is positive since the silver nitrite (AgNO3) reacts with the purine nitrogen bases which
resulted in the white formation and white precipitations. According to Lloyd Gilig (2017), this experiment
involves a hydrolysis of N-Beta-glycosidic bonds between purine bases and ribose results in the release of
purine bases that is due to the ammonium oxide (NH4OH) and Ag+ ppt present causes the formation of a
foamy gelatinous substance.
 Figure 2. Test for Purine

The Bial's test indicates the presence of pentose. Both tests resulted negative since the
unhydrolyzed sample became yellowish in color (see Figure 3). Positive results in Bial’s test is the
formation of a bluish product. All other colors indicate a negative result for pentoses. Hexoses generally
react to form green, red, or brown products. This means that no pentose was found in the group's
samples. Bial’s reagent consists of reagents which promotes the dehydration of ribose to furfural.
According to Harper College (n.d), the Bial’s test on DNA, all samples are expected to give a negative result.
Though DNA has a ribose as a sugar component, it is a deoxyribose.

Figure 3. Bial’s Test

According to Study Moose (n.d), for the Phosphate test to give out a positive result, it should
theoretically show a yellow precipitate which means that a phosphodiester bond exists between DNA and
RNA between the 3’ Carbon atom and the 5’ Carbon atom of the ribose sugar. To compare it with the
group’s results, the unhydrolyzed (see Figure 4- left) has white precipitates while the hydrolyzed (Figure
4-right) showed an off-white solution with brown and dark yellow precipitates. This indicates that the
hydrolyzed sample is positive and there exists a phosphate and phosphodiester bond.

Figure 4. Test for Phosphate

V. CONCLUSION

1. Reducing sugars, purine bases, pentoses and phosphate ions are present in DNA.

2. Acid hydrolysis causes depurination, cleaving the Purine N-glycosyl bonds present in DNA. This allows
for the liberation of the purine bases (Adenine and Guanine) in DNA. The unhydrolyzed sample did not
undergo this chemical reaction, resulting in the inability of some test to produce positive outcomes.

VI. RECOMMENDATIONS

1. Rather than a hot plate for each group, gathering a warming region in the research facility is
increasingly effective. A zone that has hot plates on the designated temperatures and is prepared
for the understudies to utilize will be a lot more efficient than the current methods used to heat
samples.
2. The right number of hardware must be prepared for each group. The sharing of materials to work
with may cause contamination among the samples since vital equipment like blenders are shared
and used by all groups conducting the experiment. It is also the common cause as to why groups are
not able to perform the task in the allotted time.
 REFERENCES

Lincoln High School. (n.d). Chemical Tests to identify Biomolecules. Retrieved from https://sites.google.
com/a/wrps.net/cns-ontl/cns-2nd-semester-weblinks/unit-7-resources---lab/chemical-tests-to
-identify-biomolecules

Harper College. (n.d). Bial’s Test. Retrieved from http://dept.harpercollege.edu/chemistry/chm/100/

dgodambe/thedisk/carbo/bial/bials.htm

Characterization of Nucleic Acids. (2016, Jul 27). Retrieved from

https://studymoose.com/charaterization
-of-nucleic-acids-essay
 EXPERIMENT 4B

ANALYSIS OF NUCLEIC ACIDS

BSN-1C

GROUP 3

Danao, Louise Victoria

Dumanon, Leila
Dy, Charisse Maryjoy
Elesis, Jenlove Vincent
Galabin, Wendy Maze
Ilagan, Trianah Marie
Ituhat, Hugh Klied
Joaquin, Clint Jr.