Laboratory Exercise # 8

Nucleic Acid Isolation and Analysis

WORKSHEET

Name: Almira Carla S. Prologo Date: 7-23-19

Year and Section: BSMT 2-C Group No: 7

RESULTS

Table 1. Color change upon addition of diphenylamine

Sample Color Remarks

Extract Light Greenish Blue color Positive Test for DNA , blue color indicates presence of nitrates
Ribose ( did not perform the ribose) Diphenylamine does not react with the ribose sugar
No color reaction
Glucose Green Presence of simple sugars, the blue solution changes color to
green and yellow depending on the amount of sugar.

DISCUSSION:
 Explain the differences/similarities of the color reactions of the extract, ribose, and glucose.

OBTAINING CHEEK CELLS

Figure 1: shows the 1ml 8% NaCl Figure 2: shows the 1ml 8% NaCl
placed on the 55 degree C water bath

Figure 3: shows the water Figure 4: mixture of heated

with cheek cells obtained from NaCl with the cheek cells
the mouth
RELEASING DNA FROM INSIDE THE CHEEK CELLS

Figure 4: shows the mixture of Figure 5: shows the solution

cheek cells with liquid shampoo added with 95% ethyl alcohol

According to Bite Size Bio Blog (n.d.), ethanol

precipitation is a commonly used technique for
concentrating and de-salting nucleic acids (DNA or
RNA) preparations in aqueous solution. The clouds of
white strands are the DNA. The DNA is not soluble in
ethyl alcohol, so it precipitates where the two liquids
meet. Soap bubbles from the "cheek" solution will get
tapped in the DNA strands. Based on our activity the
Figure 6: shows the DNA white strand on the tube can be seen in a transparent
precipitate liquid on the upper part of the mixture. While the lower
part of the tube is white in color.

COLLECTING THE DNA

Figure 7: shows the Figure 8: shows the Figure 9: DNA dissolve

collected DNA in a 1ml decanted mixture DNA in a sterile distilled
ethyl alcohol leftover water.
DNA ANALYSIS

Figure 10: shows the Figure 11: shows the solution

solution added with was heated in a boiling water
Diphenylamine reagent bath

Figure 12: shows the color Figure 14: shows the color
of solution turned light of solution turned with
greenish Blue glucose turned green

Blue color appears in diphenylamine test because of the reaction of nitrates. One major difference
between DNA and RNA is their sugar: DNA contains deoxyribose, whereas RNA contains ribose. DNA can be
identified chemically with the Dische diphenylamine test. The reaction between the Dische reagent and 2-
deoxypentose results in the development of a blue color. The intensity of the blue color is proportional to the
concentration of DNA. Dische reagent does not react with the ribose sugar in RNA and does not form a blue-
colored complex (Science Buddies, 2013). In our activity the extract was color blue because the extract contains
deoxyribose from the DNA in our cheek cell forming a blue color solution. The DNA we use from our cheek
cell has low concentration which is why the solution has a low intensity of blue color forming light greenish
blue. Since there is no ribose performed in the activity and the Diphenylamine reagent does not react with the
rbose sugar then it would not produce a blue colored solution. In the glucose solution, from yellow colored
solution turning to green because when the solution was heated there is a reduction reaction in which causes the
solution to change color from yellow to green.
GUIDE QUESTIONS:

1. What is the purpose of using detergent in this experiment?

Based on the Science Buddies Article (2013), to get the DNA from a cell, scientists typically rely
on one of many DNA extraction kits available from biotechnology companies. During a DNA extraction,
a detergent will cause the cell to pop open, or lyse, so that the DNA is released into solution. Then alcohol
added to the solution causes the DNA to precipitate out. In our experiment we used the same process to
pop the DNA of the cheek cells. We used the liquid soap solution so that we can visually see the DNA
strands and by using the ethyl alcohol causes the DNA to precipitate. In addition to that the detergent is
insoluble to the alcohol that is why the alcohol merged at the upper portion of the tube where the DNA
strands can be seen, while the soap solution submerge at the bottom of the tube.

2. What property of the nucleic acid makes it insoluble in ethyl alcohol?

According to Bite Size Bio (n.d.), water is a polar molecule – it has a partial negative charge near
the oxygen atom due the unshared pairs of electrons, and partial positive charges near the hydrogen
because of these charges, polar molecules, like DNA or RNA, can interact electrostatically with the water
molecules, allowing them to easily dissolve in water. Polar molecules can therefore be described as
hydrophilic and non-polar molecules, which can’t easily interact with water molecules, are hydrophobic.
Nucleic acids are hydrophilic due to the negatively charged phosphate (PO3-) groups along the sugar
phosphate backbone and ethanol has hydroxyl groups (-OH), but their carbon chain portion is non-polar
which make them hydrophobic. The molecule increasingly becomes overall more nonpolar and therefore
less soluble in the polar water as the carbon chain becomes longer. That is why nucleic acid is insoluble
with ethyl alcohol.

3. What differentiates the DNA from RNA molecule?

According to Helmenstine (2019), DNA contains the sugar deoxyribose, while RNA contains the
sugar ribose. The only difference between ribose and deoxyribose is that ribose has one more -OH group
than deoxyribose, which has -H attached to the second (2') carbon in the ring. Based on our laboratory
manual activity 9, DNA is a very long thread like molecule made up of a very large number of
deoxyribonucleotides joined together. DNA is a linear double stranded polymer made up of
deoxyribonucleotides while RNA is a linear polymer in which nucleotides are linked together by means
of phosphodiester bridges. It does not form a double helix like DNA.DNA is stable under alkaline
conditions while RNA is not stable. DNA is responsible for storing and transferring genetic information
while RNA directly codes for amino acids and as acts as a messenger between DNA and ribosomes to
make proteins. DNA and RNA base pairing is slightly different since DNA uses the bases adenine,
thymine, cytosine, and guanine; RNA uses adenine, uracil, cytosine, and guanine.

Helmenstine also made a chart on the comparison of DNA and RNA:

Comparison DNA RNA

Name Deoxyribonucleic Acid Ribonucleic Acid
Function Long-term storage of genetic Used to transfer the genetic code from the
information; transmission of genetic nucleus to the ribosomes to make proteins. RNA
information to make other cells and new is used to transmit genetic information in some
organisms. organisms and may have been the molecule used
to store genetic blueprints in primitive
organisms.
Structural Features B-form double helix. DNA is a double- A-form helix. RNA usually is a single-strand
stranded molecule consisting of a long helix consisting of shorter chains of nucleotides.
chain of nucleotides.
Composition of deoxyribose sugar ribose sugar
Bases and Sugars phosphate backbone phosphate backbone
adenine, guanine, cytosine, thymine adenine, guanine, cytosine, uracil bases
bases
Propagation DNA is self-replicating. RNA is synthesized from DNA on an as-needed
basis.
Base Pairing AT (adenine-thymine) AU (adenine-uracil)
GC (guanine-cytosine) GC (guanine-cytosine)
Reactivity The C-H bonds in DNA make it fairly The O-H bond in the ribose of RNA makes the
stable, plus the body destroys enzymes molecule more reactive, compared with DNA.
that would attack DNA. The small RNA is not stable under alkaline conditions,
grooves in the helix also serve as plus the large grooves in the molecule make it
protection, providing minimal space for susceptible to enzyme attack. RNA is constantly
enzymes to attach. produced, used, degraded, and recycled.
Ultraviolet Damage DNA is susceptible to UV damage. Compared with DNA, RNA is relatively
resistant to UV damage.

4. What are the four basic nucleotides that make-up the DNA molecule?

DNA is made up of molecules called nucleotides. Each nucleotide contains a phosphate group, a sugar
group and a nitrogen base. The four types of nitrogen bases are adenine (A), thymine (T), guanine (G) and
cytosine (C).

5. Draw the structure of the sugar found in DNA.

Figure 14: shows the

structure of DNA where
sugar is also found

Figure 15: shows the structure

of sugar (color orange part) in
the DNA
Summary and Conclusion:

All living organisms have DNA, which is short for deoxyribonucleic acid; it is basically the blueprint for everything
that happens inside an organism’s cells. Overall, DNA tells an organism how to develop and function, and is so important
that this complex compound is found in virtually every one of its cells. DNA is vital to a human body since it an element tha
makes up the basic unit of life which is the cell and gives

References:

Bite Size Bio Blog. (n.d.). Ethanol Precipitation of DNA and RNA: How it works. Retrieved from
https://bitesizebio.com/253/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/ on July 28 2019

Helmenstine,A. (2019). DNA vs. RNA – 5 Key Differences and Comparison. Retrieved from https://www.technology
networks.com/genomics/lists/what-are-the-key-differences-between-dna-and-rna-296719 on July 28, 2019

Science Buddies. (2013). Squishy Science: Extract DNA from Smashed Strawberries. Retrieved from
https://www.scientificamerican.com/article/squishy-science-extract-dna-from-smashed-strawberries/ on
July 28 2019
 Laboratory Exercise # 9
Biochemistry of Nucleic Acids

WORKSHEET

Name: Almira Carla S. Prologo Date: 7-26-19

Year and Section: BSMT 2-C Group No: 7

OBSERVATIONS and GUIDE QUESTIONS:

RNA Analysis

1. What color results? When CuSO4 was added, the tube formed several layer of different color The uppermost is
blue color, then followed by purple color and at the middle after the purple is orange color and lastly at the bottom
part is color yellow, however when the solution was mixed it turned the tube comprises mostly purple color.

Figure 1: shows the color Figure 2: shows the color

before solution was mixed after solution was mixed

2. A positive biuret test would indicate the presence of what type of molecules in the filtrate?

The biuret test is a chemical assay that detects the presence of proteins in a sample. The normal color of biuret
reagent is blue. The reagent turns violet in the presence of peptide bonds -- the chemical bonds that hold amino
acids together. The test relies on a color change to confirm the presence of proteins. If proteins are found, the
sample will turn violet. In our activity, the filtrate from yeast and white sand contains nucleic acids from
Saccharomyces cerevisiae, also known as baker's yeast which indicates that the presence of peptide bonds in
nucleic acid formed the purplish color which result to a positive test in biuret.

3. What type of molecule present in the suspension is hydrolyzed by the mild acid treatment?

Base on Wikipedia (n.d.) RNA hydrolysis is a reaction in which a phosphodiester bond in the sugar-phosphate
backbone of RNA is broken, cleaving the RNA molecule. This means that the RNA molecule is hydrolyzed by mild
acid treatment. RNA is susceptible to this base-catalyzed hydrolysis because the ribose sugar in RNA has a
hydroxyl group at the 2’ position. This feature makes RNA chemically unstable compared to DNA, which does not
have this 2’ OH group and thus is not susceptible to base-catalyzed hydrolysis.
 Figure 3: shows the
hydrolysate

4. What are the products of hydrolysis?

In the hydrolysis of yeast RNA, the product of hydrolysis separates the components of the nucleic acid into the base
pairs, pentose sugar, and phosphate group.

Purines analysis

5. What happens after addition of silver nitrate?

The role of silver nitrate/basic condition is to precipitate purines via its interaction with the purines nitrogens. A
white precipitate indicates the presence of purines (Karaman, 2016).In our activity when we added
ammonium hydroxide to the hydrolysate it formed two layer and when mixed, the solution turned into pale
yellow color. Upon adding silver nitrate the solution turned cloudy or white turbid which indicates the
presence of purine in the solution.

Figure 5: Shows the Figure 6: shows the Figure 7: shows the

hydrolysate added with result after adding NH4 solution after added with
ammonium hrdroxided OH AgNO3

6. Name the Purines in the nucleic acid from yeast:

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. They are the most widely occurring nitrogen-
containing heterocyclic in nature. The Purines in the nucleic acid are the GUANINCE AND ADENINE.
Ribose Analysis
7. What color is produced after boiling? A dark brown solution with a shade of turbid straw yellow is produced after boiling
and contains black particles or in the solution.

Figure 8: shows the solution added

with bials Figure 9: shows the solution after heated

8. Result with test on 0.1% Ribose: Clear straw yellow or pale yellow color of the solution.

Figure 10: Shows the solution with ribose Figure 11: shows the solution after
heating

9. Result with test on 0.1% Glucose: same color with the reaction in ribose, pale straw yellow solution

Figure 12: shows the Figure 13: shows the

solution added with color of solution after
glucose boiled.
 10. What component of RNA reacts to this test? The pentose sugar of the RNA reacts positive to the reagent used for
the test.

Phosphate Analysis

11. What color results after heating of the tube? Clear liquid and slightly turbid at the bottom with yellow green
precipitate or molecules in the liquid

Figure 15: Shows the solution in the Phosphate analysis,

contains yellow green precipitate.

12. What compound of phosphorus is found in nucleic acids?

Phosphate groups are found in nucleic acids. They are composed of nucleotides, which are the monomers made of
three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. If the sugar is a compound
ribose, the polymer is RNA (ribonucleic acid); if the sugar is derived from ribose as deoxyribose, the polymer is
DNA (deoxyribonucleic acid) (Wikipedia n.d.).

13. What is ATP?

According to Helmenstine, (2019) adenosine triphosphate or ATP is often called the energy currency of the cell
because this molecule plays a key role in metabolism, particularly in energy transfer within cells. The molecule acts
to couple the energy of exergonic and endergonic processes, making energetically unfavorable chemical reactions
able to proceed. Based on body building articles (2018) ATP is required for the biochemical reactions involved in
any muscle contraction. As the work of the muscle increases, more and more ATP gets consumed and must be
replaced in order for the muscle to keep moving.

14. Why is ATP important in the body?

According to Norman (2017) ATP is important to cells because to them ATP is like a “striking a match” that
releases just the right amount of energy every time and exactly when a cell needs that energy. It is efficient, too —
very little energy leaks away as heat. In general, ATP is vital in the human body because it involves most of the
energy conversion in our body to keep us alive and be able to do things properly.
Summary and Conclusion:

Nucleic acid is an important class of macromolecules found in all cells and viruses. The functions of nucleic acids
have to do with the storage and expression of genetic information. The main role of nucleic acids is to store information that
is used to make proteins. Nucleic acids come in two main forms: deoxyribonucleic acids, also known as DNA, and
ribonucleic acids, also known as RNA. The main function of DNA is to store the genetic information that cells in the body
need to function. Together, they keep track of hereditary information in a cell so that the cell can maintain itself, grow,
create offspring and perform any specialized functions it's meant to do. Nucleic acids thus control the information that
makes every cell, and every organism, what it is. Understanding how genes are read by the cell and used to create proteins
creates enormous opportunities for understanding disease. Genetic diseases occur when errors are introduced into the
genes that DNA carries; those errors create faulty RNA, which creates faulty proteins that don't function the way they're
supposed to.

References:

Helmenstine,A. (2019). DNA vs. RNA – 5 Key Differences and Comparison. Retrieved from https://www.tech
nologynetworks.com/genomics/lists/what-are-the-key-differences-between-dna-and-rna-296719 on July
28, 2019

Karaman,R. (2016). Test for purines. Retrieved from https://www.researchgate.net/post/What_is_the_role

_of_ammonium_hydroxide_and_Silver_nitrate_in_the_test_for_purines on August 8, 2019

Norman (2017). Cell Respiration. Retrieved from https://www.quora.com/Why-is-ATP-important on August 8,

2019

The Editors of Encyclopedia Britannica. (n.d.). Adenosine Triphosphate. Retrieved from https://www.britan
nica.com/scienceenzyme on August 4, 2019

Wikipedia. (n.d.). RNA Hydrolysis. Retrieved from https://en.wikipedia.org/wiki/RNA_hydrolysis August 4,

2019