Experiment No.

5: ISOLATION OF DNA FROM SPLEEN

2015-129722015-48305
Submitted on 20 October 2017
I. ABSTRACT
DNA isolation is the first step in the study of specific DNA sequences within a complex DNA population, and in analysis of
genome structure and gene expression. In most research laboratories and industries, there is an increasing demand for a
high-molecular weight DNA for analysis. However, the DNA from most cells is usually found in close association with basic
proteins called histones, and their integrity is affected by several factors such as pH, temperature, ionic strength, cellular
conditions and mechanical stress. In order to understand the structure of DNA, biochemical preparations are designed to
obtain a pure DNA from its natural source. Thus, selecting a source for DNA isolation is an essential step considering that
its quantitative ratio and sequence of bases vary with the source of DNA. In this experiment, a spleen from pig was chosen
as source, and it was homogenized and centrifuged to make the DNA available and susceptible for isolation. After isolating
the DNA, a portion of it was subsequently hydrolyzed using 10% H 2SO4. Both the hydrolyzed (HDNA) and unhydrolyzed
DNA were subjected to qualitative tests such as Schiffs test, test for deoxyribose and phosphates, and spectrophotometric
analysis. Results showed that only the HDNA sample tested positive for Schiffs test which can be attributed to its hydrolyzed
or depurinated form to which the Schiffs reagent can react. For the tests for deoxyribose and phosphate, both the DNA and
HDNA samples showed positive results implying the presence of deoxyribose and phosphate groups in both samples. The
spectrophotometric analysis showed that absorbance of the sample was 0.796 at 260nm and 0.472 at 280nm obtaining a
ratio of 1.68. This ratio of the obtained absorbances is significantly lower than the accepted value; thus, it is inferred that is
has protein contaminants. With these results, the isolation of DNA from porcine spleen is, therefore, successful.
Furthermore, it can also be concluded that the qualitative tests used were effective to detect presence of DNA and its
functional groups. Spectrophotometry, moreover, is also an effective technique for determining the presence of
contamination in the isolated DNA.

II. Keywords: DNA isolation, spleen, sodium citrate buffer, hydrolysis, Schiffs test, test for deoxyribose, test for
phosphates, spectrophotometric analysis

III. Introduction
Nucleic acids are linear polymers made up of
nucleotides as its monomeric units. Nucleotides are
biological molecules possessing a heterocyclic
nitrogenous base, a five-carbon sugar (pentose),
and phosphate as principal component of its
structure (see Figure 1). They have numerous
biochemical roles in participating as essential Figure 1. General structure of nucleotides. This is a
intermediates in virtually all aspects of cellular ribonucleotide. In deoxyribonucleotides the OH group
on the 2 carbon (in red) is replaced with H. (Nelson &
metabolism. They serve as energy currency in Cox, 2008).
metabolic transactions which essentially link the
response cells to hormones and other extracellular When nitrogen base and aldopentose are
stimuli. Moreover, they are also structural combined via an N-glycosidic bond, the product is a
components of an array of enzyme cofactors and nucleoside. Whereas, when a phosphoryl group
metabolic intermediates and, most importantly, they (PO2
3 ) is added to a hydroxyl group on the
constitute nucleic acids, namely, the carbohydrate, it becomes a nucleotide (Boyer,
deoxyribonucleic acids (DNA) and ribonucleic acids 2012). The nitrogenous base of nucleotides comes
(RNA) which are molecular repositories of genetic in two types: pyrimidine bases and purine bases
information (Nelson & Cox, 2008). (see Figure 2). Pyrimidine bases are single-ring
1|Biochemistry 34.1 Isolation of DNA from Spleen
aromatic compounds and they commonly occur as
cytosine, thymine, and uracil. Purine bases, on the
other hand, are double-ring aromatic compounds
occurring as adenine and guanine, both of which are
found in DNA and in RNA. These nucleotides are
held together by 3,5-phosphodiester bonds (see
Figure 3) (Campbell & Farrell, 2012).

Figure 2. General structures of pyrimidine and purine

(Nelson & Cox, 2008).

In 1869, DNA was first isolated from biological

materials; however, during this time, it was not yet
Figure 3. Covalent structure of DNA showing the
known of being involved in the transfer of genetic phosphodiester backbone linking deoxyribose through
information until mid-1940s. For this reason, it was the 3 and 5 hydroxyl groups (Boyer, 2012).

subjected to physical, chemical, and biological

investigations leading to Watson and Cricks
elucidation of the three-dimensional structure of
DNA by X-ray diffraction analysis in 1953. Because
of their work, DNA is now envisioned to be a double
helix which is recognized as a significant form of
native DNA (see Figure 4) (Boyer, 2012).
2|Biochemistry 34.1 Isolation of DNA from Spleen
DNA constitutes a small percentage in cells and it
is localized in a defined part of the cell. In
prokaryotes, their DNA is localized in the nucleoid
that is not separated from the rest of the cells
cytoplasm by a membrane. Whereas in eukaryotes,
the bulk of DNA is localized in the nucleus, the
organelle that is separated from the cytoplasm by a
membrane (Surzycki, 2000). The DNA of most cells
is usually found in close association with basic
proteins called histones. In order to understand the
structure of DNA, biochemical preparations are
designed to obtain a pure DNA from its natural
source. Thus, selecting a source for DNA isolation is
an essential step considering that the quantitative
ratio and sequence of bases vary with the source of
the DNA (Burtscher, 2007). Several methods are
available for the isolation of high-molecular-weight
DNA. Generally, all methods involve removal of cell
membrane and nuclear membrane from around the
DNA and the separation of DNA from other cell
components such as cell membrane debris,
proteins, lipids, or RNA, without affecting the
integrity of DNA (Katoch, 2011). However, their large
size and fragile nature make it difficult to isolate them
in a completely intact, undamaged form. For this
reason, there are several factors that must be
considered in the isolation of DNA: pH, temperature,
ionic strength, cellular conditions and the
mechanical stress on the DNA. These factors greatly
affect the various structural aspects of an intact
DNA. With the understanding of these factors,
general procedures were outlined (Boyer, 2012). In
this experiment, it was sought to isolate a high
molecular weight DNA from a porcine spleen
through alcohol precipitation. Identification of the
mode of action of each reagent used in the DNA
extraction was also done in this experiment.
Figure 4. (A)The Watson-Crick double helix. (B) Pairing
of A-T and G-C bases in the DNA double helix. Note that
the two strands are antiparallel; their and phosphodiester
bonds run in opposite directions. (Boyer, 2012).
3|Biochemistry 34.1 Isolation of DNA from Spleen
III. Methodology
ISOLATION OF DNA FROM PORCINE SPLEEN
Twenty (20) grams of fresh and cleaned porcine
spleen was diced and homogenized for 10 minutes
using a cold blender with ice-cold sodium citrate
saline buffer. The homogenate was centrifuged at
6000 rpm for 10 minutes and the pellet was kept
while the supernatant was discarded. The pellet was
suspended in 40 mL 2.0 M NaCl and was stirred
vigorously for one minute. One (1.0) mL sodium
dodecyl sulfate (SDS) was added to the solution and
was then incubated for 10 minutes in a 40oC water
bath. Solution was again centrifuged at 6000 rpm for
10 minutes and this time collecting the supernatant
and discarding the pellet. Equal amount of cold
absolute ethanol was added to the solution. DNA
fibers were collected by inserting a glass rod all the
way to the bottom and stirring in small circles.
Collected DNA fibers were washed with 70%
ethanol. DNA fibers were resuspended in 10 mL
sodium citrate-saline buffer at 4C.
HYDROLYSIS OF DNA
Approximately half of the DNA sample was
mixed with an equal amount of 10% H2SO4 in a test
tube. It was then placed in a boiling water bath for
15 minutes. After cooling, concentrated NaOH was
used to neutralize the solution.
QUALITATIVE TESTS
Schiffs Test
Ten (10) drops of DNA and HDNA sample were
collected and were separately mixed with 10 drops
Schiffs reagent. Solutions were let to stand for 10
minutes and the change in color was observed
afterwards.
Test for Deoxyribose
Ten (10) drops of DNA and HDNA sample were
placed in two separate test tubes. One (1.0) mL
diphenylamine reagent was added to each test tube.
It was then subjected to vortex and was heated for
10 minutes. Color of resulting solution was observed
afterwards.
Test for Phosphates
In a crucible, 0.5 mL of the DNA and HDNA
samples were incinerated, until no more liquid is left.
The residue was then cooled and 3.0 mL dH2O was
added to it, mixing it thoroughly. The solution was
filtered, discarding the residue and collecting the
filtrate, wherein 0.5 mL 10% (NH4)2MoO4 and 2
drops concentrated HNO3 were added. The solution
was heated for two minutes and was allowed to
stand after.
Spectrophotometric Analysis
The DNA sample and the sodium citrate-saline
buffer solution (blank) were placed in separate
quartz cuvettes. Using a UV/Vis spectrophotometer
(PerkinElmer Lambda 25), the absorbance of the
solutions was read at 260 nm (excitation
wavelength) and 280 nm (emission wavelength).
The ratio of the absorbance was calculated.
IV. Results and Discussion
DNA ISOLATION FROM PORCINES SPLEEN
By definition, nucleic acids are biomolecules
known of storing genetic information in cells and
transferring this information from old cells to new
cells. There are two groups of nucleic acids: DNA
and RNA (ribonucleic acid). DNA codes for the
functioning of the cell and it is located mainly in the
4|Biochemistry 34.1 Isolation of DNA from Spleen
nucleus of the cell and with a small amount in the
mitochondrion of eukaryotic cells. Inside a non-
dividing cell, the DNA present are usually loosely
packed and are unraveled as chromatin during
interphase. This loosely packed DNA are the ones
undergoing the transcription process and are found
in all non-dividing cells (Kuensting, 2004 & BioNinja,
n.d). As cell begins to cell divide, these loosely
packed DNA begin to condense coil with the help of
histones, a special protein that facilitates the
packaging of DNA. The histone-coiled DNA folds
over itself many times further making its packaging
more condense and compact. This compact DNA
again coils itself forming a spring-like supercoil
(Kuensting, 2004). This supercoiled DNA are now
called chromosomes. The reason why DNA undergo
such transformation is that in this form, the DNA can
now easily be divided during cell division or mitosis
thus forming two daughter cells which are identical
to the parent cell (Kuensting, 2004 and BioNinja,
n.d.). Since the DNA in the daughter cells are still
compact, it will not participate in the transcription
process but it will after the DNA decondenses after
the telophase process (BioNinja, n.d.).
Although almost all cells contain DNA, the
amount present in some tissues is usually quite
small and, moreover, contain high
deoxyribonuclease (DNase) activity that breaks
down DNA into smaller fragments. Therefore, a
good source of DNA is that with high quantity of the
material and have low DNase activity. With these
criteria, lymphoid tissues are good in this respect
and thymus is the best source, with spleen as a good
alternative (Bisen, 2014). For this reason, spleen
from pig was chosen for DNA isolation.

Figure 5. Porcine spleen used as the source of DNA
(left) showing negative result; spleen homogenate
(right).
DNA is easily denatured when subjected to
mechanical stress and extreme physical and
chemical conditions. Furthermore, it is
vulnerable to nucleases, which must be inhibited. To
address the dilemma about nucleases, sodium
citrate was used as homogenizing buffer which will
bind with Ca2+ and Mg2+ that are cofactors for DNase
and stages until the removal of protein is carried out
as fast as possible in cold condition. The prepared
sodium citrate-saline buffer was also prepared at pH
7.4 because at this ionic strength,
deoxyribonucleoprotein is insoluble and separates
well from other proteins. Conducting
homogenization process at cold condition also
causes minimal activity of residual DNase (Bisen,
2014).
The obtained homogenate was subsequently
subjected to differential centrifugation at 6000 rpm at
which, according to Meah and Westhead (2012),
isolates the nuclear fraction and other large
organelles from the homogenate as the pellet. The
collected pellet was suspended to NaCl solution and
sodium dodecyl sulfate (SDS) was added. SDS is a
denaturing detergent which emulsifies lipids and
proteins, and breaks cellular membrane
disrupting the noncovalent interactions that hold the
5|Biochemistry 34.1 Isolation of DNA from Spleen
cell membrane together (see Figure 6) (Goldman &
Green, 2009).
Figure 6. Use of SDS in lysing plasma membrane
(Retrieved from https://www.slideshare.net/Geet_singh).
In the case of this experiment, the SDS will break
also
the nuclear membrane which will allow the genomic
DNA to be released from the nucleus. Prior to the
addition of SDS, the pellet was suspended to NaCl
solution for the purpose of making the DNA available
for precipitation by suspending it to the supernatant.
This is possible because each nucleotide of the DNA
molecule possesses a negatively charged
phosphate group. Therefore, DNA molecules are not
the
attracted to each other but are repelled by the like
the charges they possess. By adding NaCl to a solution
containing DNA neutralizes the negative charges of
the DNA molecule, making the separate DNA
molecules more likely to collect together and
become visible (see Figure 7). NaCl is able to do this
because the sodium and chloride ions separate in
solution and the positive sodium ions (Na+) are
attracted to the negative charges of the DNAs
phosphate groups, thus neutralizing the negative
charge (Bisen, 2014).
by
 acid polymer are so diminished that inter-helical
interactions are possible, allowing the nucleic acids
to aggregate (Pikur & Rupprecht 1995).

The ethanol was added slowly on the side of the

test to avoid mixing and then the alcohol will form its
own layer on top of the NaCl solution layer. This
method of isolating the DNA from the system is
called spooling. It is done carefully placing a glass
stir rod though the alcohol layer until it touches the
bottom of the tube (see Figure 8). By slowly spinning
the rod between the fingers, while watching the
Figure 7. Sodium cations neutralizing a nucleotide interface between the two layers, the DNA present
(Zumbo, 2014).
will clump together at the interface between layers to
form a milky translucent mass (McIntosh, 2017).
Since the DNA is already suspended in the NaCl
solution, the supernatant from the 6000rpm
differential centrifugation was collected. To isolate
the DNA suspended in the supernatant, it was
precipitated using absolute ethanol. Ethanol is the
most commonly used alcohol component in
precipitations of DNA and RNA. Due to the structural
differences between ethanol and water, ethanol has
a much lower dielectric constant than water does.
By lowering the dielectric constant of a solution
containing nucleic acids and monovalent cations, Figure 8. Spooling aggregated DNA strands using a glass
stirring rod (Retrieved from
the Coulomb force of attraction increases between http://chemconnections.org/general/chem106/Tech%20P
rep/DNA-2016.html).
the cations and the negatively charged nucleic acid
backbone (that is, the resistance from the solvents
electric field sufficiently diminishes to permit efficient The collected aggregate of DNA strands was

interaction; the solvation shells surrounding the then stored in sodium citrate-saline buffer at 4C.

solutes charges depletes). When the cations and Saline-sodium citrate buffer is used as a

negatively charged nucleic acid backbone interact, hybridization buffer, to control stringency; thats is, to

nucleic acids are neutralized, therefore no longer require all bases of one polynucleotide to be paired

dissolve in water and precipitate out of solution. with complementary bases on the other (Heslop-

Also, ethanol induces conformational changes to the Harrison & Schwarzacher, 2006).

nucleic acid structure; the repulsive forces between

the negatively charged phosphates with a nucleic
6|Biochemistry 34.1 Isolation of DNA from Spleen
 Figure 9. Isolated impure DNA from spleen.
HYDROLYSIS OF DNA
Approximately, half of the dissolved DNA was
mixed with an equal amount of 10% H2SO4 in order
to allow hydrolysis of the phosphodiester bonds.
DNA can actually be hydrolyzed without the
presence of acid since it is thermodynamically
favored (G = -5.3 kcal mol ); however, it
-1
kinetically very slow so it usually acid-catalyzed
(Fekry & Gates, 2009). Based on the study of Watts,
et al. (2009), DNA oligonucleotides are vulnerable to
depurination and cleavage under acidic conditions.
Depurination in DNA is a chemical reaction of purine
deoxyribonucleosides, deoxyadenosine and
deoxyguanosine, in which the -N-glycosidic bond is
hydrolytically cleaved releasing a nucleic base,
adenine or guanine.
7|Biochemistry 34.1 Isolation of DNA from Spleen
Figure 10. Mechanism of acid-catalyzed depurination
and cleavage in DNA, shown for adenine base but which
can also occur with guanine (Watts et al., 2009).
Due to the protonation at N-7 of the purine, it
allows breakage of the N-glycosidic bond. The
resulting structure is in equilibrium with an open-
chain aldehyde form and can undergo -elimination
(Watts, 2009). Moreover, heat was applied in the
solution to facilitate the bond breaking among the
phosphodiester bonds.
SCHIFFS TEST
Both the DNA and HDNA samples were tested
using Schiffs reagent. Upon addition of the Schiffs
reagent, color change was observed in the HDNA
sample, that is, it turned into a magenta colored
solution. Whereas, no visible color change was
observed in the DNA sample.
Figure 11. DNA sample (left) showing negative result;
HDNA sample (right) showing positive result for Schiffs
test.
 Schiffs test, basically, is used for detecting
aldehydes and for staining biological tissues. Schiffs
reagent is an aqueous solution of basic fuchsin or
pararosaniline that is decolorized by sulfur dioxide
and it is commonly prepared by addition of
hydrochloric acid to a dye solution containing a
metabisulfite or bisulfite salt (Wenk, 2014).
Figure 13. Structure of the "decolorized" Schiff reagent
(Mack, 2013).

The reaction of the Schiff reagent with aldehydes

is complex that several research groups reported
multiple reaction products. The currently accepted
mechanism is that the pararosaniline and bisulfite
combine to yield the "decolorized" adduct with
sulfonation at the central carbon (see Figure 13).
The free, uncharged aromatic amine groups then
Figure 12. Reaction of pararosaniline with an acid
react with the aldehyde being tested to form two
yielding the Schiff reagent (Wenk, 2014).
aldimine groups. These electrophilic aldimine
Biological materials that tested positive usually groups then react with further bisulfite, and the Ar-
underwent Feulgen reaction during the Schiffs test NH-CH(R)-SO3 product (and other resonance-
which is a cytochemical reaction for the deoxyribose stabilized species in equilibrium with the product)
moiety of DNA. The DNA has to be subjected first to give rise to the magenta color of a positive test
a mild acid hydrolysis and the it is treated with (Robins, et al., 1980).
Schiffs reagent. In the hydrolysis, RNA is removed
leaving only the DNA; however, the purines in the
DNA are removed (depurination) at the level of
purine-deoxyribose glycosidic bond, thus,
unmasking the aldehyde groups of the deoxyribose
(see Figure 10). When these aldehyde groups react
with the Schiffs reagent, it gives a magenta color.

Figure 14. Reaction of Schiffs reagent with the

aldehyde groups forming the magenta-colored complex
(Wenk, 2014).

8|Biochemistry 34.1 Isolation of DNA from Spleen

 This discussion confirms that the HDNA sample diphenylamine yielding the blue-colored complex
will, indeed, give a positive result since upon (Saradamba, 2012).
hydrolysis, it will have an aldehyde group to which
the Schiffs reagent will react. On the other hand, the
DNA sample is expected to have a negative result
since it not hydrolyzed, thus, it has no aldehyde
group. However, other results showed that the DNA
sample has a positive test in Schiffs test. This result
can be attributed to the presence of free-aldehyde
groups in the sample since the isolated DNA is
impure.

TEST FOR DEOXYRIBOSE

Diphenylamine was added to both DNA and
HDNA samples and both samples produced a blue
color solution which is a positive result for the
presence of deoxyribose.

Figure 16. The reaction mechanism of diphenylamine

Figure 15. DNA sample (left) and HDNA sample (right) reacting with a deoxyribose sugar (Plummer, 1998).
showing positive results for the presence of deoxyribose.

This is consistent with the experimental results

Addition of diphenylamine, also called as Dische
obtained that both DNA and HDNA samples will test
test, is commonly used for estimation of DNA.
positive for the presence of deoxyribose. Although
Diphenylamine reacts with the deoxyribose sugar
the DNA sample was not hydrolyzed prior to this test,
producing a blue-colored complex which has a sharp
it was hydrolyzed upon the addition of
maximum absorption at 595nm. When the DNA
diphenylamine reagent since the reagent contains
sample is heated under acidic conditions, it will
hydrochloric acid and sulfuric acid and heating was
undergo depurination followed by dehydration giving
also done. This makes it possible for the DNA to
rise to a highly reactive -hydroxylevulinylaldehyde
yield -hydroxylevulinylaldehyde to which the
(see Figure 16). Under acidic condition, -
diphenylamine will react.
hydroxylevulinylaldehyde will react with
9|Biochemistry 34.1 Isolation of DNA from Spleen
TEST FOR PHOSPHATE
The samples were incinerated to destroy or to
weaken the phosphate bonds thus resulting in the
formation of water soluble phosphate ions (Berg,
Tymoczko, & Stryer, 2012). Filtration was done to
separate the large components (e.g. impurities) of
the solution. After filtration, ammonium molybdate
and nitric acid were added to the solution.
Afterwards, the solution was heated to catalyze the
reaction. Ammonium molybdate reacts
phosphate under acidic condition to form ammonium
phosphomolybdate which is observed as a yellow
precipitate in water. Shown below is the balanced
equation of the reaction,
12 (NH4 )2 MoO4 + H3 PO4 + 21 HNO3
((NH4 )3 PMo12 O40 + 21 NH4 NO3 + 12 H2 O
Figure 17. Reaction of ammonium molybdate with
phosphate ion to form ammonium phosphomolybdate
(Retrieved from https://bitesizebio.com/7214/ask-a-
chemist-how-colorimetric-assays-work/).
Since both DNA and HDNA contain phosphates,
theoretically both will test positive by providing a
yellow precipitate. As shown in Figure 18, both the
DNA and the HDNA are yellow; however, only the
DNA sample has a visible precipitate. One of the
possible reasons for the invisible precipitate in the
HDNA sample would be the low amount of
10 | B i o c h e m i s t r y 3 4 . 1 I s o l a t i o n o f D N A f r o m S p l e e n
phosphate. Nevertheless, the test results were in
line with the theoretical results.
with
Figure 18. Positive results for phosphate test; DNA
sample (left) with precipitate; HDNA sample (right)
without precipitate.
SPECTROPHOTOMETRIC ANALYSIS
When isolating DNA, contaminants like proteins
are also isolated that is why a test for purity must
also be done. One way of testing the purity of a
nucleic acid is through spectrophotometry. In the
test for purity, two wavelengths were used. The first
wavelength is 260 nm. This wavelength
corresponds to the maximum wavelength at which
nucleic acids absorb UV light (Thermo Fischer
Scientific, 2009). The second wave length used was
280 nm corresponding to the maximum wavelength
proteins absorb UV light (Caprette, 1995).
The ratio of absorbance at 260 nm versus
absorbance ate 280 nm can be used to determine
the purity of a nucleic acid. The acceptable ratio of a
pure DNA would be ~1.8 while for a pure RNA would
have a ration of ~2.0. A ratio that is below the
accepted value, could mean that a lot of protein
contaminants are present or that the nucleic acid
concentration is very low while a ratio that is above
the accepted value could mean high residual RNA
contamination (Teare, et al., 1997).
 The absorbance of the sample was 0.796 at
260nm and 0.472 at 280nm obtaining a ratio of 1.68.
The ratio is a bit lower than the accepted value
indicating a possible medium amount of protein
contaminants.
With these qualitative analyses performed, the
presence of DNA was ascertained by
colorimetry. Changes in the original color of the
samples indicates presence of functional groups. In
this experiment, application of multiple tests makes
it possible to detect the presence of DNA since each
determines the presence of every functional group
present in DNA.
Other methods than can be conducted to confirm
the isolation of DNA from biological samples are gel
electrophoresis, polymerase chain reaction (PCR)
using primer, and Southern hybridization analysis. In
DNA gel electrophoresis, an electric field is applied
to a gel matrix made of agarose, and within the gel,
charge particles will migrate and separate based on
their size. The negatively charged phosphates of the
DNA backbone cause DNA fragments to move
toward the anode - a positively charged electrode
(JoVE Science Education Database, 2017). For
PCR, it is a useful method of detecting even low
concentrations of DNA by amplifying the DNA
fragments using primers that are designed to
precisely bind to the sites within the strand of DNA.
Lastly, it is said that the best method confirming the
presence of DNA is the Southern hybridization
analysis in which uses electrophoresis in gel,
blotting onto a membrane, and hybridizing to a
labeled fragment of DNA which is complementary to
the DNA introduced (Kempken & Jung, 2010).
11 | B i o c h e m i s t r y 3 4 . 1 I s o l a t i o n o f D N A f r o m S p l e e n
V. Conclusion an Recommendation
DNA was successfully isolated from spleen
using differential centrifugation, addition of sodium
citrate to uncoil the DNA from proteins, addition of
SDS to uncoil the DNA itself, and alcohol
precipitation. Half of the extracted DNA was kept
while the other half was hydrolyzed. Both these
the
sample were subjected to three qualitative tests
namely Schiffss test, and tests for the presence of
deoxyribose and phosphate groups. The results of
the tests are all in line with the theoretical results
wherein only the hydrolyzed DNA would test positive
for the Schiffs test, and both the DNA and HDNA
tested positive for the presence of deoxyribose and
phosphate groups. The isolated DNA sample was
also subjected to spectrophotometric test to
determine its purity and results showed that the
isolated DNA is only contaminated by a small to
medium amount of protein contaminants implied by
the obtained ratio of the obtained absorbances.
In the isolation of DNA, a fresh and appropriate
source must be secured. The source must be
appropriate to ensure high amount of DNA will be
isolated. It should also be fresh so that degradation
of DNA will be prevented. The isolated DNA should
also be subjected to other qualitative tests such as
Keller-Killiani test to determine the presence of
pentose, Murexide to test for purines, Bials orcinol
test to confirm isolation of DNA, and agarose gel
electrophoresis to purify the isolated DNA.
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I hereby certify that I have given substantial

contribution to this report.

________________________
DINO, Allen Jasper T.

________________________
SOLOMON, Marc Ralph M

13 | B i o c h e m i s t r y 3 4 . 1 I s o l a t i o n o f D N A f r o m S p l e e n