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 , KL Hagiwara, CBDL Javier, AMF Labajo, AP Lansangan
Group 5, 2CMT, Faculty of Pharmacy, UST

 

The experiment aims to isolate DNA from microbial, plant and animal sources, it also aims to determine
the purity of isolated DNA and characterize DNA following acid hydrolysis. DNA was isolated from the
plant source, onion. Absorbance was measure under the wavelengths 260nm and 280nm which was
followed by hydrolysis using hydrochloric acid after which was subjected to different qualitative color
reaction: Dishe Test, a test for deoxyribose, Test for Phosphates, Murexide test, a test for Purines and
Wheeler-Johnson Test, a test for Pyrimidines. The computed absorbance ratio is 0.56. Dische Test
produced a blue solution, Test for Phosphates produced a yellow precipitate, Murexide test yields a
yellow to red residue while Wheeler-Johnson test yields a pale yellow turbid solution.

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 twisted about each other in a double helix. Both
 chains are right handed however, since each
Nucleic acids are the biological strand has both free 5͛ hydroxyl group at one
molecule essential for life. They make up the end and a free 3͛ hydroxyl on the other end,
most important macromolecules. They are high each strand has a polarity or directionality. The
molecular weight biopolymers of polarity of the two strands of the molecule is in
mononucleotides. The backbone of a opposite directions and thus, DNA is described
nucleic acid is made of alternating sugar and as anti-parallel structure.
phosphate molecules bonded together in a long
chain. Each of the sugar groups in the backbone
is attached to a third type of molecule called
a nucleotide base. Though only four
different nucleotide bases can occur in a nucleic
acid, each nucleic acid contains millions of bases
bonded to it. The order in which these
nucleotide bases appear in the nucleic acid is
the coding for the information carried in
the molecule.

Two structural classes occur in cell: DNA

and RNA. These are macromolecular structures
composed of regular repeating polymers
formed from nucleotides. These are the basic
building blocks of nucleic acids and are derived
from nucleoside which has two components: a
five-membered pentose carbon sugar and a
nitrogenous base.
DNA or deoxyribonucleic acid is the hereditary
material in humans and almost all other
organism. Most DNA is located in the cell
nucleus but a small amount can also be found in
the mitochondria. DNA is a two stranded
structure consisting of two polynucleotide chain
Figure1. The double
helical structure of DNA
The nucleotide bases of the DNA
molecule form complementary pairs: The
nucleotides hydrogen bond to another
nucleotide base in a strand of DNA opposite to
the original. The Purines bases adenine (A) and
Guanine (G) are found in both RNA and DNA, as
is the Pyrimidines Cytosine (C). The other
Pyrimidines are each restricted to one type of
nucleic acid: Uracil (U) occurs exclusively in
RNA, whilst, Thymine (T) is limited to DNA. This
bonding is specific, and adenine always bonds
to thymine (and vice versa) and guanine always bath until the solution reached 60oC. 25 g of
bonds to cytosine (and vice versa). This bonding minced onion was weighed out and was added
occurs across the molecule, leading to a double- to the pre-heated homogenizing solution which
stranded system was left seated for 5 minutes with eventual
stirring in the water bath. 1.5 g of papain (or
4ml of meat tenderizer solution) was added and
 was kept in the 60oC water bath for another 10
 minutes after which was immediately placed in
 an ice bath for 5 minutes. The solution was
 swirled gently to allow even cooling. After, the
 contents of the flask were poured in a blender
 and were homogenized for 45 seconds. The
 homogenate was filtered through 4 layers of
 cheesecloth into a 250 ml beaker. The solution
 was cooled on ice. Using a pipette, 20 ml of ice
 cold ethanol was immediately added, and was
 allowed to slowly drip down the sides of tube.
 The tube was left to stand for 5 minutes
 without disturbing it. The DNA was spooled by
 snagging it with a Pasteur pipette with a hook
bent on the tip. The DNA was transferred into a
Figure2. GC base pairing with 3 hydrogen bonds clean test tube and was resuspended in TE
(below) and AT base pairing with 2 hydrogen buffer.
bonds (upper).
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DNA is also capable of replicating. Each 
strand of DNA in the double helix can serve as a A 0.5 ml aliquot of the DNA solution was
pattern for duplicating the sequence of bases. dissolved in 4.5 ml of TE Buffer. The solution
This is critical when cells divide because each was transferred to a quartz cuvette and the
new cell needs to have an exact DNA copy absorbance was determined at 260nm and at
present in old cell. 280 nm. The buffer solution was used as the
blank reagent. The A260/A280 ratio was
The Nucleic Acid isolation and calculated.
procedures involve three steps. 1) Disruption of
cell membrane and membranes of sub cellular V



 
nucleus to release nucleic acids. 2) 
Disassociation from nucleoproteins and For Acid Hydrolysis, DNA sample was mixed
denaturation of proteins and 3) separation of with 1M HCl. The mixture was heated at 100oC
DNA from other soluble cellular component. for 60 minutes with occasional agitation. The
tubes were covered with marbles. After, the
 test tube was carefully cooled under running
 
 water and was neutralized with 1M KOH. The
 pH was adjusted to 4-6 with glacial acetic acid
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 using pH paper. The mixture was centrifuged
 and was decanted into clean test tubes for
To isolate DNA from its source, onion, 50 ml of chemical characterization
homogenizing solution was placed in a 125 ml
Erlenmeyer flask and was heated in a water V 




 solution turned yellow. The same was done
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 " to the standards cytosine or uracil. Excess
 were removed by boiling the solution until
3.5 ml diphenylamine reagent was added to it turned light yellow or colorless. Excess
1.5 ml of hydrolyzed DNA solution. The Ba(OH)2 solution was introduced. The
same was done to the 0.5 ml standard solution was tested with litmus paper
deoxyribose solution. Both tubes were
placed in a boiling water bath for 10   
 

minutes and were immediately cooled. 
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 In isolation of DNA from source, onion a first
A volume of 1ml conc. H2SO4 was added to step is homogenization. Homogenization media
1 ml nucleic acid solution and standard includes: 1) SDS (Sodium Dodecyl Sulfate) A
phosphate solution. Both tubes were biological detergent which causes the cell
heated over a small flame and were shaken membrane to break down further and
frequently until the contents of the tube emulsifies the lipids and proteins of the cell by
turned brown. The mixture was cooled after disrupting the polar interactions that hold the
which, 0.5 ml conc. HNO3 was introduced. cell membrane together. The detergent forms
The solution was heated until white fumes complexes with these lipids and proteins
appeared and the solution was colorless. 1 causing them to precipitate out of the solution.
ml of water was added to the colorless SDS is the major ingredient in laundry
liquid and was heated for 5 minutes in a detergent. 2) EDTA (Ethylenediamine tetracetic
boiling water bath. The mixture was cooled acid) weakens the cell by binding the divalent
after. Introduced to the mixture was 1 ml cations (Mg++ and Ca++) which are needed for
10% (NH4)2MoO4 solution. The solution was membrane stability. This further aids in
mixed well and was diluted to 10 ml with breaking open the cells of the onion and lastly,
water. It was left to stand for 5 minutes. 3) NaCl (Sodium chloride) enables nucleic acids
 to precipitate out of an alcohol solution
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 of DNA causing the strands to come closer
Into a small evaporating dish, 5- 10 drops of together and coalesce.
nucleic acid solution was placed. A few
drops of conc. HNO3 were introduced. The Homogenization involves heating and
same was done to the standards adenine or blending the onion tissue in order to break
guanine solution. The mixture was carefully down the cells. The heat treatment softens the
evaporated in a water bath until dry. The phospholipid in the cell membrane and
residues formed were moistened with 10% denatures the DNAse enzymes which if present,
KOH and was further heated. The changes would cut the DNA into small fragments so that
in color were noted upon addition of KOH. it would not spool. The onion tissue is mixed in
A few drops of water were introduced to a blender with homogenization media, which
the mixture and was warmed. The residue breaks down the cell wall, cell membrane and
was evaporated and the color was noted. nuclear membrane allowing the release of DNA.

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 The next step is followed by
h " deproteinization. Deproteinization involves
A 0.5 ml of nucleic acid solution was treated adding a protease enzyme Papain, a common
with an excess of bromine water until the enzyme used to clean soft contact lenses. This
will denature the proteins clinging to the DNA Table2. Results of Qualitative Color Reaction of
making the molecule flexible and easy to spool. Acid hydrolyzed DNA
Precipitation of DNA involves adding ethanol h
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alcohol which causes every component in the Dische Test Light Blue Blue solution
filtrate to stay in solution except DNA. solution
Test for Formation of Formation of
Table1. DNA isolation Phosphates Yellow yellow
  $ 
 precipitate precipitate

%
 Murexide Formation of Formation of
Plant DNA Light Yellow Test red precipitate yellow to red
turbid Solution precipitate
Wheeler- Formation of Pale yellow
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 johnson Test Purple turbid
 precipitate solution
The measurement of the absorbances
allows measurement of the DNA *V h 


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concentration and provides information 
about the contaminant levels. DNA absorbs DNA can be identified chemically with the
light most strongly at 260nm so the Dische diphenylamine test. The reaction
absorbance value at this wavelength (called between the Dische reagent and 2-
A260) can be used to estimate the DNA deoxypentose results in the development of
concentration while the absorbance at a blue color. The reaction depends on the
280nm is used as an indicator of protein conversion of the pentose to w-
contamination. A good quality DNA sample hydroxylaevulinic aldehyde which then
should have an A260/A280 ratio of 1.7-2.0 reacts with diphenylamine to give a a blue
colored complex (test tube 1 and 2). The
  intensity of the blue color is proportional to
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  the concentration of DNA.

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DNA is generally quite stable. It will resist
attack in acid and alkali solutions. However,
in mild acid solutions - at pH 4 - the beta-
glycosidic bonds to the purine bases are
hydrolyzed. Protonation of purine bases
occurs at this pH. Protonated purines are
good leaving groups hence the hydrolysis.
Once this happens, the depurinated sugar
can easily isomerize into the open-chain Figure3. Dische Test Reaction
form and in this form the depurinated (or 
apurinic) DNA is susceptible to cleavage by #V h 
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hydroxyl ions. 
In the test for presence of phosphates for
DNA, a yellow precipitate was obtained. The
ammonium molybdate solution reacted
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 with the sample which yields yellow
 crystals, phosphoammonium molybdate
which is a positive result.
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 Retrieved February 9, 2011 from:
h "
Bromine water reacted with the sample to
form 5-bromo-6hydroxyhydroxo derivative
which produces a green coloration. Upon
addition of Ba(OH)2 will give a result of
purple precipitate.
V February 9, 2011 from:
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 Isolation-From-Onion-Ultraviolet-
In the test for presence of purines, DNA is
reacted with Nitric acid since Purines are
known to be readily soluble in dilute acid.
Nitric acid oxidized it leaving a yellow
precipitate upon evaporation; however it
turned red when moistened with a base, a
positive result for presence of purine bases.
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+


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