Isolation and Characterization of DNA

Go, L.J., Gonzaga, N.C., Gonzalez, T.A.Y., Gumarao, A. & Iglesias, M.S.
Group 5, 2C-Medical Technology, Faculty of Pharmacy

ABSTRACT
Isolation of DNA is usually a key preliminary step to subsequent molecular analysis. The
experiment aimed to isolate DNA from a plant source, which is an Onion, and characterize DNA
after acid hydrolysis. The minced onion was weighed out 25g and was mixed with 50 mL of
homogenizing solution then undergone heating to 60°C and was added with papain where it was
further heated and then placed in an ice bath. The homogenate was filtered and was mixed with
ice cold EtOH then it was suspended in 5 mL of TE buffer. The precipitate was further used for
acid hydrolysis, where it was mixed with 1M HCl and was heated at 100°C for 60 minutes and
then neutralized with 1M KOH. In chemical characterization, 3 tests were performed namely;
dische test, where diphenylamine was added with the hydrolyzed DNA solution and with the
standard deoxyribose solution, murexide test, where concentrated HNO3 and 10% KOH were
introduced to the hydrolyzed solution and standard solutions of adenine and guanine, and wheeler-
johnson test, where an excess of bromine water and excess Ba(OH)2 was added to the nucleic acid
solution and standard solution of uracil and cytosine. A white thread-like precipitate was attained
after the isolation of DNA, where it formed a sepia-colored solution in dische test, colorless in
murexide test and white precipitate in colorless solutions with test for pyrimidines. DNA are
composed of nitrogen bases (guanine, adenine, cytosine and thymine) and a sugar called D-
deoxyribose.

INTRODUCTION incorporating two nitrogen atoms in each

Nucleic acids played different key
roles in the molecular processes associated
with life, especially the storage and transfer
of genetic information. Nucleic acids are
molecules found in comparative abundance
in the nucleus of cells, where they constitute
5% to 15% of the dry weight of the cell. Also,
this are of two types, namely; DNA and RNA
which are polymer of nucleotides. A
nucleotide is a combination of a nitrogen
base, a 5-carbon sugar, and a phosphoric acid.
The nitrogen bases fall into two
categories: purines and pyrimidines. Purines
are composed of two fused rings
rings and part of these are adenine and
guanine. On the other hand, pyrimidines are
composed of a single-ring with two nitrogen
atoms in the ring structure and part of these
are cytosine, thymine and uracil. The 5-
carbon sugars found in the nucleic acids are
D-ribose and D-deoxyribose in which the
main difference is the absence of oxygen
atom on carbon atom number 2 of the latter
sugar.
DNA, which forms a right-handed
double helix, consists of two polynucleotide
strands wound around each other. The
structure of DNA is so distinctive that it is
referred as the double helix. Each nucleotide
monomer in DNA is composed of
nitrogenous base, a deoxyribose, and
phosphate. The mononucleotides are linked
to each other by 3’,5’-phosphodiester bonds
which join the 5’-hydroxyl group of the
deoxyribose of one nucleotide to the 3’-
hydroxyl group of the sugar unit of another
nucleotide through a phosphate group.
Hydrogen bonds are formed between the
nitrogen bases which are oriented toward the
helix interior are due to the antiparallel
orientation of the two polynucleotide strands.
General procedure for nucleic acid
isolation usually contains 3 steps. The first
one is the disruption of the cell wall and
release of the DNA into a medium in which
it is soluble and protected from degradation.
This calls for the use of an enzyme, such as
the commercial papain or meat tenderizer, or
lysosome to disrupt the cell wall. The
medium for solution of DNA is a buffered
saline solution containing EDTA. DNA is
more soluble and stable in salt solution than
in distilled water since it is ionic. Divalent
metal ions are binded by EDTA so that it
could form salts with the anionic phosphate
groups of DNA and also, EDTA inhibits
deoxyribonucleases that have a requirement
for Mg2+ or Mn2+.
In the second step, dissociation of the
protein DNA complexes occur. To solubilize
the inner membrane and disrupt the ionic
interactions between positively charged
histones and the negatively charged
backbone of DNA, detergents are commonly
used. Sodium dodecyl sulfate (SDS), an
anionic detergent, bind to protein and gives
them extensive anionic character. It also acts
as a denaturant of deoxyribonucleases. A
high concentration of a salt (NaCl) is added
to ensure complete dissociation of the DNA-
protein complex and to remove bound
cationic amines. It acts by diminishing the
ionic interactions between DNA and cations.
Lastly, separation of the DNA from
other soluble cellular components is done.
The solution must be deproteinized before
DNA undergoes precipitation. This is
brought about by treatment with chloroform-
isoamyl alcohol followed by centrifugation.
Chloroform causes surface denaturation of
proteins while isoamyl alcohol reduces
foaming and stabilizes the interface between
the aquaeous phase and the organic phases
where the protein collects. An ice-cold
ethanol can precipitate DNA out and make all
the other components of the mixture stay in
the solution.
When DNA undergoes acid
hydrolysis, it causes depurination. Purine N-
glycosyl bonds are cleaved, thus adenine and
guanine are liberated and apurinic site
remains. It is used to determine the base
composition of the nucleic acids.
In Dische Test, the reaction between
the dische reagent and 2-deoxypentose
results in the development of a blue color.
The reaction depends on the conversion of
pentose to 2-hydroxylaeyulinic aldehyde
which then react with diphenylamine to give
a blue-colored complex. The intensity of the
blue color is proportional to the concentration
of DNA. For Murexide Test, the addition of
conc. HNO3 which is color yellow then
turned red upon adding of 10%KOH. The
principle behind this is the oxidation of
purine by concentrated HNO3 forming
dialuric acid and alloxan, then condensation
reaction leading to formation of alloxanthan.
Neutralization by 10% KOH leads to
formation of murexide or ammonium
purpurate. In the test for pyrimidine, the
sample produces a green coloration when the
sample is treated with bromine water. The
addition of barium hydroxide will turn the
liquid purple. The principle behind this is the
formation of dialuric acid by neutralization.
The objectives of the experiment are
isolation of DNA from plant source, which is
the onion, and characterizing the DNA after
undergoing acid hydrolysis. Different
chemical test are used for characterization
namely: Dische test, Murexide test and
Wheeler-Johnson Test.
METHODS
The sample for the experiment is an
onion, which has low starch content thus
allowing the DNA to be seen clearly. The
sample was minced and weigh out 25g. It was
mixed with 50-ml of homogenizing solution
, blended for 45 seconds and was heated to
60°C in a flask over a hot plate. Commercial
papain or meat tenderizer was added to the
solution and was kept in the 60°C water bath
for another 10 minutes. Afterwards, it was
placed in an ice bath for 5 minutes then the
homogenate was filtered through 4 layers of
cheesecloth or through a filtered paper into a
250-ml beaker. With the use of a pipette, 15-
20 mL of ice cold ethanol was added. The
precipitate formed was spooled by using a
pipette and was transferred in to another test
tube where it was resuspend with TE buffer.
After isolation, the DNA sample
undergone acid hydrolysis. A 1M HCl was
added to the sample and was heated to 100°C
for 60 minutes while the tubes were covered
with marbles. The hydrolyzed DNA was then
used for chemical characterization.
For Dische Test or Test for
Deoxyribose, 1.5 mL of the hydrolyzed DNA
was added with 3.5 mL of the diphenylamine
reagent in a test tube. The same was done
with 0.5 mL of the standard deoxyribose
solution. Both tubes were placed in a boiling
water bath for 10 minutes and was observed
after.
In the Test for Purines or Murexide
Test, 5 – 10 drops of the nucleic acid solution
was added with few drops of concentrated
HNO3 in a small evaporating dish. The same
step was done with standard guanine. Both
were evaporated using a water bath and the
residues formed was moistened with 10%
KOH. After further heating, few drop of
water was added then the color of the
solutions were noted. The solution was again
evaporated and the color of the residue was
observed.
In the Test for Pyrimidines or
Wheeler-Johnson Test, 0.5-mL of the DNA
solution was treated with an excess of bromie
water until the solution turned yellow in a test
tube. The same procedure was done for the
standard cytosine and uracil solution. The
excess were removed by boiling the solutions
until it turned light yellow or colorless. An
excess Ba(OH)2 was added then the solution
was tested with lithmus paper. The solution’s
color was noted after.
RESULT AND DISCUSSION
After performing all the methods, the
results were written on the given table in the
data sheets. The table below shows the
physical description of the plant DNA
(onion).
Table 1: Physical Description of the Plant
DNA (Onion)
DNA Physical
Description
Plant DNA (Onion) White thread-like
The precipitation of DNA was due to
the addition of ice-cold ethanol to the
solution. DNA have an ionic nature which
makes it insoluble in an aqueous medium
which was made less polar by addition of an
organic solvent. Therefore, a white thread-
like precipitate was formed.
The DNA was characterized by using
different chemical test. The table below
shows the result of the chemical tests done in
the experiment.
Table 2: Results of the Chemical
Characterization Tests
Based on table 2, the DNA changed
color into a brown solution in the test for
deoxyribose and it was same with what
happened to the standard solution, which
indicated a positive result. Meanwhile, in the
test for purines, the DNA color turned from
brown to colorless while the standard
guanine solution turned from purple to brown
residue which shows a negative result. In the
test for pyrimidine, both the standards formed
a purple precipitate while the DNA formed a
white precipitate in a colorless solution
which indicates a negative result.
The onion DNA showed a positive
result in the test for deoxyribose because the
sugar found in the DNA is D-deoxyribose
which reacts with the diphenylamine reagent.
However, the visual result should be a blue
colored solution. On the other hand, the DNA
must have a positive result with the test for
purines and pyrimidines because the DNA
are composed of four bases which are
guanine, thymine, adenine and cytosine.

Chemical Standard Onion DNA CONCLUSION

Test DNA reacts in acid hydrolysis and
Test for Brown Brown less reactive in basic hydrolysis since it is
Deoxyribose solution colored negatively charged because of the phosphates
solution surrounding it, which means that its
Test for Guanine: Before KOH: isoelectric pH is above 7 making it basic.
Purines Purple to Brown DNA is ionic in nature making it insoluble in
brown residue less polar aqueous medium. As a result, DNA
residue After adding: precipitated out in the presence of ice-cold
Colorless ethanol because all the other components of
Test for Uracil: Colorless the mixture stayed in the solution except for
Pyrimidines Purple solution with DNA.
Precipitate a white
Cytosine: precipitate The erroneous color results of the
Purple standards in the chemical characterization
Precipitate was because of the reagents used. They might
be contaminated or were unused for a very
long time. Because theoretically, a blue
solution must be seen as a positive result in
dische test, a red residue in the murexide test,
and a purple coloration in the wheeler-
johnson test. Thymine cannot be determined
through the test for pyrimidines since it has a
methyl group which prevent its interaction
with excess of bromine water. Meanwhile,
the error in the DNA was because of the
improper execution of the methods by the
experimentalists.

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