EXPERIMENT 5: ISOLATION OF DNA FROM BOVINE SPLEEN

2011-35493, 2011-85007
Biochemistry 34.1, HEJ, Sir Avvin Pelovello
I. Abstract

Nucleic acids are polymers of nucleotides, composed of nitrogenous bases, sugar, and phosphate groups, that carry
genetic information and other important cellular processes. There are two kinds of nucleic acids; deoxyribonucleic
acid (DNA) and ribonucleic acid (RNA). In this experiment, extraction of DNA was performed from spleen. Four
qualitative tests were used to confirm the presence of specific nucleotide components, such as the deoxy-sugars,
purine bodies, and phosphate groups. Both the Schiff test and diphenylamine test are confirmatory for deoxyribose.
Theoretically, purines form insoluble salts with silver ions, while a phosphomolybdate yellow complex is formed by
the phosphate group in the molybdate test. Experimental results showed that the extracted DNA and HDNA are
positive in deoxyribose through the Schiff test and diphenylamine test, as well as in phosphate group through the
molybdate test. Test for purines obtained inconsistent results from the positive controls.

II. Keywords: deoxyribonucleic acid, supercoiling, purines, deoxyribose, phosphate group

III. Introduction
Nucleic acids are long linear polymers that
carry genetic information that can be passed down
from one generation to the next. The two basic kinds
of nucleic acids are deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA). These biological molecules
consist of linked monomeric units called nucleotides.
Nucleotides have diverse functions in cellular
metabolism. Each nucleotide contains three primary
components which are the phosphate group, pentose
sugar, and nitrogenous base. Phosphates and sugars
which make up the structural backbone in nucleic
acids are linked together by phosphodiester bonds.
Sugars and nitrogenous bases linked together by N-
glycosidic bonds form nucleosides. Nitrogenous bases
are categorized as purines (adenine and guanine) that
are composed of two fused rings incorporating two
nitrogen atoms in each ring, and pyrimidines (cytosine,
thymine, and uracil) that are composed of a single ring
with two nitrogen atoms. The orderly sequence of
bases along a nucleic acid can encode genetic
information (Berg, Tymoczko, & Stryer, 2012).
DNA are found in chromosomes of cells. They
are carrier of information that makes all the functional
macromolecules of the cell, even DNA itself (DNA
replication). They interact with basic proteins called
histones to form nucleosomes. DNA double helix is the
fundamental structure of DNA in which two
polynucleotide strands wound together forming a long,
slender, helical molecule. RNA, on the other hand,
consist only of one polynucleotide strand. Various
forms of RNA are involved directly in protein synthesis
(transcription and translation). RNA contain the
pentose D-ribose, whereas 2-deoxy-D-ribose is found in
DNA. Both DNA and RNA have adenine (A), guanine
(G), and cytosine (C) nitrogenous bases. However,
thymine (T) is only found in DNA while uracil (U) only
occurs in RNA. In accordance to Chargaffs rules and
Watson and Cricks rules, these bases have unique
complementary base pairing relationship: A and T, C
and G (Garrett & Grisham, 2010).
The experiment was done with the intent of
isolating high molecular weight DNA from bovine
spleen. In addition, it was performed for the purpose
of identifying the mode of action of each of the
reagents or chemicals used in bovine spleen DNA
extraction. Effective purification and isolation of DNA
is imperative in the characterization of fragments of
DNA and confirmation of its major components.
IV. Experimental
In the isolation of DNA from tissue, fresh
bovine spleen was initially cleaned and 40 g of it was
weighed. Using a blender, the tissue was
homogenized and for every gram of the spleen, 3.0 mL
of ice-cold sodium-citrate saline buffer was added.
This was done for 10 minutes at high speed. Then, the
homogenized tissue was centrifuged at 6000 rpm for
10 minutes. The supernatant was discarded while the
pellet was suspended in 14 mL of 2.0 mL NaCl. It was
vigorously stirred for a minute. 1.0 mL of SDS solution
was then added. The next step which involves 10-
minute incubation in 40 water bath was omitted. For
a second time, it was centrifuged at 6000 rpm for 10
minutes. The pellet was discarded instead of the
supernatant. To the supernatant, an equal volume of
cold absolute ethanol was slowly added by means of
letting the solution pass along the sides of the test
tube. A glass rod was carefully inserted all the way to
the bottom of the tube. Then, it was stirred in small
circles in a clockwise direction in order to collect the
fibrous DNA. The collected DNA fibers were washed
with 70% ethanol. 1 mL of the DNA fibers was Table 1. Qualitative results for the positive and
resuspended in 10 mL of sodium citrate saline buffer. negative controls.
This was labelled accordingly.
(+1) 1% (+) 1% (-)
In DNA hydrolysis, 5 mL of the dissolved DNA Test
DNA HDNA dH2O
sample was mixed with an equal amount of 10% of
H2SO4. Then, the test tube, with marble as cover, was Schiff cloudy faded pink clear
placed in a boiling water bath for 15 minutes.
Afterwards, cool it down. Concentrated NH4OH was deoxyribose deep blue deep blue turbid
added to the tube for neutralization. This was
confirmed by using a litmus or pH paper. The purines turbid turbid clear
hydrolyzed DNA was labelled properly as HDNA.
Four qualitative tests were done in the yellow yellow
phosphates clear
analysis of both the crude DNA and the hydrolyzed precipitate precipitate
DNA (HDNA). There are three controls for every
qualitative test. Two for the positive controls: 1% DNA Spleen was used as the main source of DNA
and 1% HDNA. For the negative control, distilled water in the experiment. The following visible results were
was used. obtained.
The first test is the Schiffs Test. This was
initially done by adding 10 drops of Schiffs reagent to Table 2. Qualitative results for the DNA and hydrolyzed
10 drops of the DNA and HDNA samples in different DNA extracted from spleen.
test tubes. The tubes were covered and were set to
Test DNA HDNA
stand for 10 minutes. Color of the solution was
observed and was noted down.
Test for deoxyribose is the next test. In Schiff cloudy clear
different test tubes, 10 drops of DNA and HDNA were
deoxyribose light blue dark blue
placed. Then, 1.0 mL of diphenylamine reagent was
added. It was mixed by utilizing a vortex mixer and was purines clear light bronze
set in a boiling water bath for 10 minutes. The color of
the resulting solution was observed and recorded. yellow yellow
phosphates
The third qualitative test is the test for precipitate precipitate
purines. 10 drops of DNA and HDNA were placed in
different test tubes. 10 drops of concentrated NH4OH The spleen or the thymus contains a high
was added and 2 drops of 0.1 M AgNO3 was added concentration of DNA, which is why both are preferred
afterwards. Formation of white precipitates was as the main source of the material in laboratory
observed and noted. experiments.
Lastly, the test for phosphates was carried The basic structure of deoxyribonucleic acid
out by incinerating 0.5 mL of the DNA and HDNA (DNA) is in the form of a double helix; that is, two
samples in different porcelain crucibles for 15 polynucleotide chains are wrapped around each other.
minutes or when only residue is left. The residue was Nucleotides are monomeric units of nucleic acids. It is
then cooled. Next, 3.0 mL of distilled water was added made up of three parts; a nitrogenous base (purines
to the residue. Thorough mixing was done. and pyrimidines), a sugar (ribose, deoxyribose), and a
Subsequently, 0.5 mL of 10% (NH4)2MoO4 and 2 drops phosphoric acid residue. These three components are
of concentrated HNO3 were added. The solutions were covalently bonded to each other in a molecule
then subjected to heat for 2 minutes. They were (Campbell & Farrell, 2012). Nucleotides have a variety
allowed to stand and the formation of a yellow of roles in cellular metabolism. They are energy
precipitate was observed. currency in metabolic transactions, the essential
chemical links in the response of cells to hormones
V. Results and Discussion and other extracellular stimuli, and the structural
components of an array of enzyme cofactors and
The following visible results were obtained metabolic intermediates. As mentioned, these
from the four qualitative tests after the isolation of molecules are constituents of DNA and RNA
DNA from spleen: (ribonucleic acid), which are molecular repositories of
genetic information (Nelson & Cox, 2013).
 DNA supercoiling is important in a number of
biological processes, such as compacting DNA.
Topoisomerases are also able to change DNA topology
to facilitate functions in replication and transcription.
Due to the increased packaging of DNA during nuclear
division, DNA must undergo supercoiling and
segregation from daughter cells (Nelson & Cox, 2013).
It can be observed from the qualitative results
that the extracted DNA from the spleen are consistent
with the positive controls in the tests for deoxyribose,
phosphates, and the Shiff test.
The Schiff test is a chemical test that is
generally used to detect the presence of organic
aldehydes. A dye is present in the reagent used, also
by the same name, which can either be basic fuchsin,
new fuchsin, or pararosanaline. This test can be used
to confirm the presence of deoxyribonucleic acid
through the presence of deoxyribose. When
hydrolyzed in acid, the deoxyribose creates an
aldehyde that readily combines with the Schiff
reagent, consequently staining the nuclei with a
magenta color. This test cannot be used with RNA,
which contains ribose, which is unable to form an
aldehyde when hydrolyzed (Wenk, 2014). This is
readily observed from the results of the positive
controls. The positive DNA control produced a cloudy
result, while the hydrolyzed DNA produced the faded
pink result from the reaction of the aldehyde
production with the dye. Only the extracted DNA
produced consistent results with the positive control,
while the hydrolyzed extracted DNA did not produce
the expected magenta color. This may be due to the
failure of the hydrolysis of the extracted DNA that is
needed to produce the aldehyde from the
deoxyribose.
The test for deoxyribose involves the addition
of the diphenylamine reagent. This test, also known as
the Dische diphenylamine test, can confirm the
presence of DNA in samples. The reaction of the
reagent with 2-deoxypentose results in the
development of a blue complex, which is a
consequence of the conversion of the pentose to an
aldehyde (Paikara, et. al, 2014). From the results of
the experiment, both the DNA and HDNA extracted
from spleen obtained consistent results with the
positive controls.
The test for purines involves ammoniacal
silver nitrate solution. In principle, purines form
insoluble salts with silver ions. The white precipitate is
the result of the purine bodies in the form their silver
compounds after the precipitation of phosphates
occurs. The precedence of the phosphate
precipitation is due to the basic conditions of the
solution (Hawk, 2013). The turbid solution is not a
highly conclusive qualitative result for the
confirmation of purines in the solution. It is possible,
however, that the formation of white precipitate
produced the turbid solution, indicating the presence
of purines from the positive controls. The results from
the extracted DNA and HDNA are inconsistent to the
positive controls. This may due to the absence of
purine bodies from the extracted DNA.
Finally, phosphate ions react readily with
ammonium molybdate to produce a yellow precipitate.
This is due to the formation of a phosphomolybdate
complex. Results from the positive DNA and HDNA
controls confirmed the presence of phosphate groups
from the DNA. Both the extracted DNA and HDNA are
also consistent with the results.
From these qualitative tests, DNA can be
pinpointed from RNA and other present compounds.
Theoretically and in principle, the Schiff test and
diphenylamine test are highly conclusive tests for the
presence of DNA. As discussed above, these tests can
easily detect the presence of DNA through its
deoxyribose that is converted to aldehyde and giving
specific, observable results.
There are other methods that can be used to
detect the presence of DNA in samples. One example
is the Killer-killani test where a brown coloration is
observed. Feulgen staining, which is also related to
the Schiff test, can also be used extensively to locate
DNA in a biological samples. This is done by means of
degrading RNA with hydrochloric acid at 60oC while
the deoxyribose is converted into an aldehyde. The
acidic environment does not allow the Schiff
coloration to occur (Wenk, 2014).
VI. Conclusion and Recommendation
The extraction of DNA from spleen was
confirmed through a series of qualitative tests. Both
the Schiff and test for deoxyribose involves the
opening up of the pentose sugar to an aldehyde,
forming the necessary complexes for a positive result.
Test for purines follow the principle of insoluble salt
formation with silver ions, forming white precipitate.
Test for phosphates is due to the formation of a
phosphomolybdate yellow complex. The experiment
showed that the extracted DNA and HDNA are
consistent with the positive controls except for the test
for purines. This confirms the successful extraction of
the molecule from spleen.
It is recommended to use other qualitative
methods in confirming the presence of DNA in
samples, such as the Feulgen staining and Killer-
killani test. Different sources for extraction can also be
used to determine and compare the presence of DNA.

VII. References

Berg

Campbell, M., Farrel, S. (2012). Biochemistry (8th

edition). USA: Cengage Learning.

Garrett

Hawk, P. (2013). Practical physiological chemistry a

book designed for use in courses in practical,
physiological chemistry in schools, of
medicine and of science. London: Forgotten
Books.

Nelson, D., Cox, M. (2013). Leningher principles of

biochemistry (6th edition). USA: W.H. Freeman
and Company.

Paikara, D., Kumari, S., Gayatri, L., A, Y. (2014).

Isolation, qualitative and quantitative
estimation of DNA from various sources. India:
Department of Biotechnology and
Microbiology Bihlai Mahila Mahavidyalaya.

Wenk, P. (2014). A most bodacious stain an official

publication of the Michigan society of
histotechnologists. USA: Tech Points.