Isolation, Purification, and

Qualitative Color Reaction

of DNA of Allium cepa
Introduction
DNA (deoxyribonucleic acid)
Biochemically defined as a polynucleotide.
A nucleotide consists of a 5-carbon sugar (deoxyribose
in DNA), nitrogenous bases, and a phosphate group.
The nitrogenous bases are purines (Adenine, Guanine)
and pyrimidines (Cytosine, Thymine).
Takes the form of a double-helix.
The backbones form phosphodiester bonds while the
nitrogenous bases form hydrogen bonds with their
specific pair.
DNA
The 2 strands of the double helix are anti-parallel.
The strands are designated as ends of the strands are
designated as 5 or 3 in classic nomenclature.
The naming is dependent upon a free phosphate group
attached to a 5 carbon or a free OH group attached to
a 3 carbon.
This gives the DNA directionality.
DNA
One of the 4 biomolecules essential to all life
including lipids, amino acids, and carbohydrates.
Blueprint of organisms.
Primary role is to store information for protein
synthesis.
The morphology and physiology of organisms are
the result of their DNA expression.
Allium cepa (onion)
Usually referred to as the bulb onion or the common
onion
Used as a vegetable, an ingredient in savory food.
Most onion cultivars are about 89% water, 4%
sugar, 1% protein, 2% fibre, and 0.1% fat.
Contains allyl propyl disulphide, which is thought to
have a similar effect to insulin in balancing blood
sugar levels.
DNA Isolation
Extraction and purification of DNA from a sample using
chemical and physical means.
Physical methods (i.e. centrifugation) and chemical
methods (i.e lysing of cell membranes with detergents)
are used.
Principal uses of DNA isolation include use in forensics
and basic research.
Objectives
To isolate DNA from Allium cepa (onion)
To determine the purity of the isolated DNA
To characterize the components of isolated DNA
after acid hydrolysis
Methodology
A. Preliminaries
50ml homogenizing
White onion
solution
- Place into 250ml - Remove first two
Erlenmeyer flask layers
- Heat in water bath - Mince the onion
until 60C - Weigh 25g
- Add onion in
homogenizing solution
- Stir
- Let sit in water bath for
5 minutes, stir ever 2
minutes
 - Transfer flask into ice bath for 5
minutes
- Swirl gently
- Pour contents into blender
- Blend for 45 seconds
- Filter the homogenate through 4
layers of cheesecloth into clean
10ml graduated cylinder
- Measure the volume of the filtrate

Residue Filtrate

- Discar
- Transfer to 250ml beaker
- Cool in ice bath
d
- Tilt beaker at 45 angle
- Slowly put the ice cold 95% ethanol
twice the volume of the filtrate
- Leave undisturbed for 2-3 minutes
until bubbling stops
- DNA will float on top
 - Place spooler in beaker
- Gently twirl the spooler in and out
of the 2 layers in one direction
- Lift therod out of the beaker
- Spool as much of the DNA
- Put on watch glass
- Air dry
- Weigh

DNA Precipitate
B. Determination of DNA Concentration and Purity

1.0 mg DNA
Precipitate
- Dissolve in 3.3 ml SSC solution
- Read absorbance at 260nm and
280nm
- Determine protein concentration
and nucleic acid concentration
using the nomograph
- Calculate absorbance ratio of the
DNA solution
- Rationalize
Nucleic Acid and purity of isolate DNA
Protein Concentration
and Absorbance Ratio
C. Acid Hydrolysis of DNA
DNA

- Add 1.0ml of 1M HCl

- Cover the medium test tube with
marble
- Heat at 100C for 60 minutes with
occasional agitation
- Remove from water bath
- Allow to cool for until room
temperature
- Add 2.5ml distilled water
- Neutralize with 1M NaOH, check
using litmus paper
- Filter hydrolyzate
- Add distilled water to 4ml
DNA hydrolyzate
D. Chemical Characterization of DNA
D.1. Test for Deoxyribose (Dische Reaction)
1ml DNA
Hydrolyzate/standard
- Add 1.5 ml of diphenylamine
reagent
- Heat for exactly 10 minutes in
boiling water bath
- Cool immediately
- Observe results
Colored solution
D.2. Test for Phosphate
1ml DNA
hydrolyzate/standard
- Add 1ml concentrated H2SO4
- Add 0.5ml of concentrated HNO3
- Add 1ml of distilled water
- Heat for 5 minutes in a boiling
water bath
- Allow to cool
- Add 1ml of ammonium molybdate
solution
- Mix well
- Dilute to 10ml of water
- Let
Yellow solution, stand for 10 minutes
yellow
- Observe the color of solution and
precipitate
precipitate
D.3. Test for Purines (Murexide Test)

DNA
Hydrolyzate/standard
- Add 3 drops in an evaporating dish
- Add 10 drops of HNO3 acid
- Evaporate to dryness, water bath in
fume hood
- Add 3 drops of water
- Warm
Red residue
D.4. Test for Pyrimidines (Wheeler-Johnson Test)

0.5ml DNA
Hydrolyzate/standard
- Treat with excess of bromine water
until solution turns yellow
- Remove excess bromine by boiling
solution until light yellow or
colorless
- Add Ba(OH)2 in excess
- Test with litmus paper
Purple precipitate
Results and Discussion
DNA Extraction from A. cepa
A. cepa was used because it contains a small amount of starch
which allows the DNA to be more visible.
Homogenization involves heating and blending the onion tissue
in order to breakdown the cells and denature enzymes such as
DNAse whose action may make it hard to spool DNA.
Papain denatures proteins attached to DNA making it easier to
spool.
Ethanol causes most other unneeded cellular components to be
dissolved in the solution except DNA effectively precipitating it.
The cheesecloth traps the precipitated cell debris while the
soluble DNA passes through.
Homogenizing solution
Made up of salt, distilled water, and detergent
To separate DNA from unneeded cellular
components and keep DNA in location where it
will not be tainted
Homogenizing solution
Sodium citrate
sodium citratebuffer stabilizes theDNAby forming a Na+ shell around the
negatively charged phosphates of theDNA
EDTA
chelating agent used to remove DNAse from the environment after the nuclear
membrane has been broken down.
NaCl
provides Na+ ions that will obstruct negative charge of DNA, permitting the ends
to come nearer so they can precipitate out of a cold solution
Detergent (sodium dodecyl sulfate)
causes break down of cell membrane by emulsification of cell proteins and lipids
disrupts polar connections that collectively holds the cell membrane
Saline-Sodium Citrate Buffer
Biological buffers
need good stability, lack of toxicity, precision in pH, pka
between 6.0 and 8.0 (where most biological reactions occur),
free of enzymatic and hydrolytic activity and minimal
participation in biological reactions
SSC Buffer - acid salt buffer solution, used as a buffering
agent to control acidity
Acid hydrolysis
Using a strong acid at high temperature to break down
the stable structure of DNA into its components.
Strong acid: HCl
Temperature: 100C
These conditions break both phosphate ester bonds and
N-glycosidic bond between deoxyribose, and purine and
pyrimidine bases.
Group DNA Percentage Yield

1 0.39%

2 0.22%

3 0.057%

4 0.55%

5 0.77%

6 0.008%

7 0.53%

8 3.128%

9 .089%
Group Absorbance ratio Protein (mg/mL) Nucleic acid
(ug/mL)
1 1.5440 0.34 57

2 0.7800 0.50 4.5

3 1.2639 0.04 2

4 1.7800 0.01 5

5 1.2749 0.04 7

6 1.2520 0.20 8

7 1.2290 0.10 10

8 1.3240 0.06 4.1

9 1.1470 0.40 19
Determination of DNA Concentration
and Purity
Nucleic Acids can be detected via several approaches, including
analytical methods that are spectrophotometric, fluorometric or
colorimetric. Though it can not readily distinguish RNA from DNA,
the ratio is commonly employed.
At 260nm, DNA absorbs light the strongest. The absorbance at
this wavelength was used to estimate the concentration of the
DNA isolate.
At a wavelength of 280nm, protein contamination can be
estimated as well. The presence of amino acid can be detected on
this wavelength.
The more light absorbed by the sample, the higher the nucleic
acid concentration in the sample.
 Absorbance ratio =

A good quality of DNA should have

approximately 1.7-2.0 absorbance level to
consider their DNA isolate to be pure.
Dische Reaction
Group Standard DNA Hydrolyzate
1 Blue turbid solution Light blue turbid solution
with precipitate
2 Blue solution Light blue solution with
white material
3 Blue solution Colorless solution
4 Grayish blue turbid Light blue turbid solution
solution
5 Bluish purple solution Faint yellow solution
6 Bluish purple solution Clear solution with white
precipitate
7 Dark blue turbid solution Light yellow turbid
solution
8 Blue turbid solution Clear light blue solution
with white material
9 Light blue solution Light blue turbid solution
Test for Deoxyribose (Dische
Reaction)
Dische Test
Used to detect presence of deoxyribose

Reagent: Diphenylamine reagent

Composition: diphenylamine, glacial acetic acid, sulfuric acid,
ethanol

Positive result: Blue solution

Test for Deoxyribose (Dische
Reaction)
Principle:
Pentose conversion to a molecule that reacts with diphenylamine
reagent
Mechanism:
Upon heating, the deoxyribose is hydrolyzed to -
hydroxylevulinyl aldehyde. Which then complexes with
diphenylamine, to produce a blue colored solution. The intensity
of the blue color depends on the concentration of DNA.
Diphenylamine does not react with the ribose sugar in RNA and
does not form a blue-colored complex.
Phosphate Test
Group Standard DNA Hydrolyzate
1 Yellow turbid solution Clear solution
2 Light yellow clear solution Clear colorless solution
3 Yellow precipitate, yellow Colorless solution
solution
4 Yellow precipitate, yellow Clear colorless solution
turbid solution
5 Yellow precipitate, yellow Clear colorless solution
solution
6 Yellow precipitate, faint Clear colorless solution
yellow solution
7 Yellow precipitate, light Clear colorless solution
yellow turbid solution
8 Yellow precipitate, yellow Clear colorless solution
material on top, clear
colorless solution
9 Yellow precipitate, clear Clear solution
solution
Test for Phosphate
Test for Phosphate
Used to detect presence of phosphodiester bond linked at C3 or C5 in the
ribose

Reagent: conc. H2SO4, conc. HNO3, ammonium molybdate

Positive result: Yellow precipitate (ammonium phosphomolybdate)
Principle:
A small amount of the sample is acidified with conc. HNO 3 to which a
littleammonium molybdateis added. The presence of phosphate ions is
indicated by the formation of a bright yellow precipitate layer (ammonium
phosphomolybdate).
Murexide Test
Group Standard DNA Hydrolyzate
1 Red residue No residue
2 Yellow orange solution, Faint yellow solution
red residue
3 Yellow orange solution, Faint yellow solution
red residue
4 Red orange residue Faint orange residue
5 Yellow orange residue Faint brown residue
6 Red residue Brownish yellow residue
7 Red orange residue Light orange residue
8 Bright red orange Orange solution, yellow
solution, red residue residue
9 Red orange solution and Faint yellow solution
residue
Murexide Test
Murexide Test
Used to detect presence purines
Purines are soluble in diluted acid

Reagent: concentrated HNO3, 10% KOH

Positive result: Red residue
Principle:
Oxidation of purine by concentrated HNO 3, forming dialuric acid
and alloxan. Condensation reaction leading to formation of
alloxanthan. Neutralization leading to formation of murexide.
Wheeler-Johnson Test
Wheeler-Johnson Test
aqualitativetestforthepyrimidinebasescytosineanduracil
Reagent: Bromine water, Ba(OH)2
Positive result: purple precipitate after Ba(OH)2
Principle:
Bromination of dialuric acid through addition of bromine water
and neutralization through addition of excess barium oxide
Wheeler-Johnson Test
Mechanism:
When uracil/cytosine is dissolved in bromine water and the
solution is treated with an aqueous solution of BaOH2 in excess,
a purple or violet-blue precipitate color is produced.
Formation of purple precipitate involves several intermediate
reactions:
Uracil and bromine water react to form dibromoxyhydrouracil (the same
compound is obtain when cytosine is treated with bromine water).
Dibromoxyhydrouracil is sensitive towards alkalis. When treated with an
excess of Ba(OH)2, the two atoms of bromine are replaced by hydroxyl
groups and isodialuric acid is formed.
Isodialuric acid undergoes a rearrangement into dialuric acid
Both isodialuric and dialuric acids give a purple precipitate with Ba(OH) 2
Conclusion
DNA was successfully isolated from Allium cepa.
Unfortunately, only one group (Group 4) was able to
obtain a pure DNA isolate.
The presence of deoxyribose was confirmed using the
Dische Test. While the presence of purines were
confirmed using the Murexide Test. The phosphate test
showed negative results due to acid hydrolysis.
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