Nursing Biochemistry

Laboratory Report

Nucleic Acids
Activity No. 11

Amiruddin, Algenne Marhee P.

Daud, Ayesha Munira A.
Del Rio, Gianella B.
Locker No.31; NurBio Lab C
1st Semester, SY 2019-2020
Nursing Biochemistry
Laboratory Report

RATIONALE

Nucleic acids are very much important in the life print of an individual. They carry specific
function and information and thus give instruction as to how these are expressed in the human body.
Nucleic acids, by directing the process of protein synthesis, they determine the characteristics of every
living thing. These are also capable of being broken down to yield phosphoric acids, sugars, and nitrogen
bases (Roberts, 2019). The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA). These nucleic acids have their distinct characteristics that work together hand on
hand in order to accomplish a specific function in the body.

In terms of function, the DNA is responsible for the storage and transmission of the genetic
material (College of Science and Information Technology, n.d). It is the master blueprint for life and
constitutes the genetic material of in all free-living organisms. On the other hand, RNA is involved in the
manufacturing of proteins (College of Science and Information Technology, n.d). It also contains the
genetic material for certain viruses, but is also found in all living cells.

In terms of their sugar composition, the DNA contains the D-deoxyribose while RNA has D-
ribose (College of Science and Information Technology, n.d). The pentose sugar in DNA differs that of
the RNA by the absence of a hydroxyl group (-OH) and the 2’ carbon of the sugar ring. Without an
attached phosphate group, the sugar attached to one of the bases is called as the nucleosides (Roberts,
2019).

In terms of their pyrimidine base composition, DNA contains thymine while RNA contains uracil
(College of Science and Information Technology, n.d). Each nucleic acid contains four of five nitrogen-
containing bases: adenine, guanine, cystosine, thymine, and uracil. In this case, adenine and guanine are
purines, while cytosine, thymine, and uracil are pyrimidine.

In terms of their structure, DNA is double helix while RNA is made up of single strand (College
of Science and Information Technology, n.d). DNA is mostly composed of two polynucleotide chains
twisted around each other in the form of double helix. The nature of the DNA strands are complementary,
that is why, if it is inverted, it looks superficially the same. On the other hand, the RNA structure differs
with DNA in three aspects: (1) the backbone of RNA contains ribose rather than deoxyribose, (2) RNA
contains uracil in place of thymine in DNA, and (3) RNA is found as a single polynucleotide chain except
for the cases of some bacteria (The Structures of DNA and RNA, 2012).

In this laboratory experiment, the objectives are:

1. To isolate RNA from yeast
2. To determine the presence of phosphate group
3. To determine the presence of pentose
4. To determine the presence purines

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

RESULTS AND DISCUSSION

Table 1. Qualitative Test (Filtrate)

Test Reagent Results

1. Nucleoproteins 10% NaOH, 1% CuSO4 Violet layer on the surface

2. Mild Acid Hydrolysis 10% H2SO4 Light yellow to light orange

a. Presence of 10% HNO3, 5% ammonium Yellow color with brown

Phosphate molybdate particles; white ppt

b. Presence of Ribose Bial’s Orcinol Reagent #1, #2, and #3: light yellow to
#1 (Solution for Acid colorless
Hydrolysis)
#2 (0.1% Ribose)
#3 (0.1% Glucose)

c. Presence of Purines 10% NH4OH, 5% AgNO3 Pink/white ppt color

Documentation for Isolation of RNA from Yeast

Discussion for Isolation of RNA from Yeast

In this process, the yeast and the white sand were grinded in a mortar in order to produce a
powdered-like substance. After having the powder-like substance, NaOH was added in order to create a
smooth creamy paste. The beaker that contained the mixture was covered with a watch glass in order to
avoid evaporation. This process involved heating with NaOH for 30 mins in a 90˚ C. The reaction of

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

mixture with the NaOH served to disrupt the cell membrane and lyse the cell extracting the nucleic acids.
It is also through the NaOh that increased the pH level of the solution, thus the denaturation of
contaminent proteins; inactivate nucleases which can degrade RNA. Also through heating, the cell
membrane loosens by increasing the kinetic energy of the lipid molecules, therefore releasing more RNA.
After the heating process, the mixture was filtered thrice through the use of cheesecloth and once through
the use of filter paper. The filtrate was used to perform the qualitative tests.

Test no. 1 Discussion (THEORETICAL)

Nucleoproteins are formed from the conjugation of proteins and nucleic acids. The protein
components of nucleoproteins are simple proteins, albumins, histones and protamines (Nucleic Acid,
2011). In the experiment for test for nucleoproteins, the filtrate was added with the reagents NaOH and
CuSO4. The combination of these two reagents is also known as the Biuret test. The test is a general test
for compounds that contain peptide bonds (Karki, 2018). Peptide bonds are covalent bonds formed
between amino acids. When biuret is treated with dilute copper sulfate in alkaline condition, a
purple/violet colored compound is formed (Karki, 2018). The formation of purple colored product is also
due to the reduction of Cu2+ to Cu+ made by the detected reducing sugar (Balda, 2011). This is the basis
of biuret test widely used for identification of proteins and amino acids. The test sample produced a violet
layer on the surface which indicates for the presence of peptide bonds. This is because nucleoproteins
contain proteins in its hydrolyzed form.

Test no. 2 Discussion (THEORETICAL)

Boiling 10% H2SO4 results in hydrolysis of nucleic acids to mononucleotides. Further hydrolysis
of pure nucleotides will breakdown N-B-glycoside bonds and ester bonds which releases free purine
bases, ribose, and phosphate (Villamor, 2014). The product of Hydrolysis is further used for other
necessary qualitative tests such as for the presence of Phosphate, presence of Ribose, and the presence of
Purines. Equation for Mild Acid Hydrolysis:

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

Test no. 2a
Presence of Phosphate

Discussion (THEORETICAL)

The yellow precipitate indicated in the table shows a positive results. The reaction of the
ammonium molybdate solution with the concentrated nitric acid will show a yellow precipitate due to the
presence of phosphate or a crust of phospo-ammoniummolybdate (Wikipedia, 2019). The equation for the
presence of phosphate:

Test no. 2b
Presence of Ribose

Discussion (THEORETICAL)

The purpose of the Bial’s Orcinol Test is use to distinguish pentoses from hexoses (Karki, 2018).
In this test, test tube number 2 should theoretically give a positive result – a blue green colored complex.
Note that ribose solution or the ribose itself is a pentose which means that it contains five carbon chains.
The test reagent consists of orcinol, hydrochloric acid, and ferric chloride (Karki, 2018). The reaction
depends on the conversion of ribose to an aromatic aldehyde (furfural) which then reacts with orcin (3,5-
dihydroxy toluene) to give the blue green color (All Medical Tests, 2016). For test tubes 1 and 2, they are
expected to give a negative result because there is no presence of ribose. For test tube no. 3, it gave a
negative result because glucose is a hexose which means that it contains six carbon chains.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

Test no. 2c
Presence of Purines

Discussion (THEORETICAL)

The purpose of this test is to detect the presence of purine bases – adenine and guanine. The
positive result for this test is shown by the white precipitate that is formed. When a white ppt is formed,
there is a presence of purines. These purine bases are readily known to be soluble in dilute acids.
According to Kerr and Seraidarian (1945), “free purine bases may be separated from the purine
nucleosides by precipitation with silver nitrate in dilute acid solution. When the solution is treated with
sufficient excess of NaOH to precipitate a little silver oxide, the nucleosides are quantitatively
precipitated.”

ANSWERS TO QUESTIONS
1. Name the purine bases found in nucleic acids.

a. Purines consist of a six-membered and a five-membered nitrogen-containing ring that is

fused together. Adenine and guanine are the two purine bases found in the nucleic acids.
Adenine is a white crystalline purine base, having a molecular weight of 135.15 daltons
and a melting point of 360 to 365 C. On the other hand, guanine is a colorless, insoluble
crystalline substance, having a molecular weight of 151.15 daltons (BiochemDen, n.d).

2. Account for the formation of the precipitate in the test for purines.

a. The formation of a white precipitate indicates the presence of purines. The hydrolysis of
glucosidic bonds between purine bases and nucleic acids results in the release of purine
bases, adenine and guanine, caused by NH4OH. The formation of foamy gelatinous
substance is caused by the Ag+ ions (San Juan, 2013).

3. What is the basis of Bial’s Orcinol test?

a. The purpose of this test is to check and confirm the presence of pentose. In regards to the
test for the presence of Ribose, the Bial’s Orcinol reagent was used. The sugars found in
the nucleic acid are pentoses, thus the utilization of the Bial’s Orcinol test (All Medical
Tests, 2016).

4. What is ATP? Why is it important in the cell?

a. The Adenosine Triphosphate is the primary energy carrier in all living organisms on
earth. Microorganisms capture and store energy metabolized from food and light sources
in the form of ATP. When the cell requires energy, ATP is broken down through
hydrolysis. The high energy bond is broken and a phosphoryl group is removed. The
energy released from this process is used to drive various cellular processes. ATP is
constantly formed and broken down as it participates in biological reactions and it is

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

central to the health and growth of all life. Without it, cells could not transfer energy from
one location to another, making it impossible for organisms to grow and reproduce
(Donaher, 2016).

CONCLUSION

Nucleic acids play a great role in the lives of every individual. These serve as blueprint to every
trait and information that the body has. Through DNA, it encodes the sequence amino acid
residues in proteins, using the genetic code. The role of nucleic acids is not merely limited on
genetic information; rather in metabolic processes and protein synthesis as well. Based on the
objectives that were laid upon, the first one was not fully accomplished. There came a problem in
the process of filtration, and thus it affected the rest of the qualitative tests. For the second
objective, theoretically, the phosphate group was identified. As for the third and fourth
objectives, they were identified also through theoretical results. Although the objectives of the
experiment were not fully accomplished by firsthand experience, through the theoretical results,
the knowledge grew deeper and was further developed.

REFERENCES

All Medical Tests. (2016, October 6). Bial’s Test: Principle, Reagents, Procedure and Results. Retrieved
from http://allmedtests.com/bials-test/

Balda, K. (2011). Report Exp 6 and 7 (DNA and RNA). Retrieved from

https://www.slideshare.net/kevbalda/report-exp-6-and-7-dna-and-rna

BiochemDen. (n.d). Basic Components of Nucleic Acids – Purines and Pyrimidines. Retrieved from
https://www.biochemden.com/basic-components-nucleic-acids-purines-pyrimidines/

College of Science and Information Technology. (n.d). Nurbio lab: biochemistry laboratory manual.

Zamboanga City, Zamboanga Del Sur: Ateneo de Zamboanga University. Retrieved from
https://lookaside.fbsbx.com/file/Protein%20Precipitation%20procedure%20and%20data%20sheet
.pdf?token=AWzytXh-
KQl9SVSaSlJf175XFrVBjeHmEfR35JuIARDxhXHJDnw7hqNd84s2K1hnEXCC5iRU8GwvLN
R6vvCmSfdppzMj7T-
RNUVjZJbL8RprII38BU1xuhUOjAaMvFWw54grdqb7VFuWskq6fZXmUzr2hij-cCVAAx-
2jddirTbTjw

Donaher, A. (2016, October 19). What Is ATP and What Does It Do? Retrieved from
https://www.luminultra.com/what-is-atp-and-what-does-it-do/

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

Karki, G. (2018). Biuret test: Principle, requirements, Procedure and Result. Retrieved from

https://www.onlinebiologynotes.com/biuret-test-principle-requirements-procedure-and-result/

Karki, G. (2018). Tests for specific carbohydrates: Seliwanoff’s test, Bial’s test and Iodine test. Retrieved
from https://www.onlinebiologynotes.com/tests-for-specific-carbohydrates-seliwanoffs-test-bials-
test-and-iodine-test/

Kerr, E. & Seraidarian, K. (1945, March 15). The separation of purine nucleosides from free purines and
the determination of the purines and ribose in these fractions. Retrieved from
http://www.jbc.org/content/159/1/211.full.pdf

Nucleic Acids. (2011). Retrieved from

https://www.slideshare.net/ayesexy/nucleic-acids-9522668?next_slideshow=2

Roberts, R. (2019, August 7). Nucleic acid. Retrieved from

https://www.britannica.com/science/nucleic-acid

San Juan, G. (2013, February 10). [Prezi Slideshow]. Retrieved from https://prezi.com/_3artlknnoye/jan/

The Structures of DNA and RNA. (2012). Retrieved from

http://biology.kenyon.edu/courses/biol63/watson_06.pdf

Villamor, J.J.T. (2014, March 11). Nucleic Acids. Retrieved from https://www.scribd.com/document/2117

70717/Nucleic-Acids

Wikipedia. (2019, April 21). Phosphate test. Retrieved from https://en.wikipedia.org/wiki/Phosphate_test

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University