Isolation and Characterization of

DNA from Allium cepa (white onion)

Ignacio, Richelle Angelika
Macaraeg, Angeline Claire
Rivera, Nico
Solano, Paulina Aleccia
Viernes, Gian Alfonso
3 Bio 2

INTRODUCTION

NUCLEIC ACIDS
Nucleic acids are biopolymers, or large biomolecules,
essential for all known forms of life. Nucleic acids, which
include DNA (deoxyribonucleic acid) and RNA (ribonucleic
acid), are made from monomers known as nucleotides.
There are two principal types of nucleic acids, DNA
(deoxyribonucleic acid) and RNA (ribonucleic acid).

Nucleotides
Purine or pyrimidine bases
bonded to sugars (ribose or
deoxyribose) , which in
turn are bonded to
phosphate groups

PURINE

PYRIMIDINE

DNA
Deoxyribonucleic acid
Polymer of deoxyribonucleotides
Monosaccharide is 2-deoxyribose
Nitrogenous bases are adenine, guanine,
cytosine, & thymine
Double helix structure
H bonding between the nitrogenous
bases in the center of the helix helps
hold double helix together

Figure 1. Retrieved from

http://www.terravivos.com/secure/cryovaultjoinus.htm

DNA
2 strands of DNA are complementary strands

Sequence of bases on one automatically

determines sequence of bases on another

2 strands of DNA are antiparallel

Run in opposite directions

Only when 2 strands are antiparallel can the

base pairs form H bonds that hold the
strands together

Allium cepa
Allium cepa, the onion (also called bulb onion or
common onion), is a monocot bulbous perennial
It is the most widely cultivated species of the
genus Allium

OBJECTIVES

 To isolate DNA from Allium cepa

To determine the protein and nucleic acid
concentration by UV-Vis Spectrometry and to
calculate the absorbance ratio
To separate components of DNA through acid
hydrolysis
To characterize DNA through various qualitative

METHODOLOGY

A. Isolation of DNA from onion

PRELIMINARIES
1. Heat the tip of a pipet in a Bunsen flame until it can
bend back up towards the top to form a hook
2. Cool 50 mL of 95% ethanol in an ice pack

50 mL homogenizing
solution

Whole onion

- Place in a 250 mL Erlenmeyer flask

- Heat in a water bath until the
solution reaches 60C

- Remove the first few layers and

mince the onion
- Weight 25 g of the onion

A. 50 mL of 60C

B. Minced onion

homogenizing solution
A. 50 mL of 60C homogenizing
solution and B. Minced onion
-Put in Erlenmeyer flask and stir and let sit in the water
bath for 5 minutes and stir occasionally about every 2
minutes
- Add 4 tablets of pulverized crude papain powder (after 5
minutes of adding the onion) and keep in the water bath
for 10 minutes more (stir occasionally about every 2
minutes)

- Immediately transfer the flask into an ice bath for 5

minutes and swirl gently
- Quickly pour the contents into a blender and blend for
45 seconds
-Filter the homogenate through 4 layers of cheesecloth

Residue
(discard)

Filtrate
- Measure the volume in a clean 100 mL
graduated cylinder
- Transfer to a 250 mL beaker and then cool
on ice
-Tilt the beaker at a 45 angle and then
slowly and gently pour ice-cold 95% ethanol
down the inner wall of the beaker

- Allow the mixture to sit undisturbed for 2

to 3 minutes until the bubbling stops
- Spool out as much DNA by quickly twirling
the spooler in and out of the mixture (2
layers) in 1 direction only
-Separate the remaining solution on the
spooled DNA and air dry the precipitate
(DNA)
-Weigh the air- dried DNA precipitate
Air- dried DNA
precipitate

B. Determination of DNA Concentration and Purity

1.0 mg DNA
- Dissolve in 3.3 mL Saline sodium citrate
solution
- Read absorbance at 260 nm and at 280 nm
- Determine the protein concentration and nucleic
acid concentration
- Calculate the absorbance ratio of the DNA
solution
DNA solution

C. Acid Hydrolysis of DNA

Isolated DNA
- Disperse in 1.0 mL of 1M HCl in a medium sized
test tube
- Cover the test tube with a marble then heat the
mixture at 100C for 45 minutes with occasional
agitation
- Remove the test tube from the water bath and
allow to cool to room temperature
- Neutralize with 1M NaOH and check using litmus
paper
- Add 2.5 mL of distilled water
- Filter the hydrolyzate

Residue (discard)

Filtrate
- Make up to a volume
of the filtrate of 4 mL
distilled water
DNA Hydrolyzate

D. Chemical Characterization of DNA

D.1 Dische Reaction
1.0 mL DNA Hydrolyzate/0.5 mL Deoxyribose standard
solution
- Add 1.5 mL of diphenylamine reagent
- Heat for exactly 10 minutes in a boiling water
bath
- Cool immediately and observe results
Blue colored liquid solution

D.2 Test for Phosphate

1.0 mL DNA Hydrolyzate/1 mL Phosphate standard
solution
- Add 1 mL of concentrated H 2SO4
- Add 0.5 mL of concentrated HNO3
- Add 1 mL of distilled water and heat for 5 minutes
in a boiling water bath
- Allow to cool
- Add 1 mL of ammonium molybdate solution

- Mix well and then dilute to 10 mL with water

- Let stand for 10 minutes
- Observe the color of the solution and the
precipitate formed
Colorless solution with
yellow precipitate

D.3 Test for purines/Murexide Test

DNA Hydrolyzate/Standard Guanine or Adenine
solution
- Place in a small evaporating dish
- Add a few drops of concentrated HNO 3
- Evaporate to dryness very carefully on a water bath
in the fume hood
Yellow residue
- Upon moistening with 10% KOH becomes red in
color
- Upon further heating, assumes a purplish red hue
- Add few drops of water and then warm
Yellow solution
- Evaporate
Red residue

D.4 Wheeler-Johnson Test/ Test for pyrimidines

1.0 mL DNA hydrolyzate/0.5 mL Standard
cytosine
- Treat with an excess bromine water until the
solution turns yellow
- Remove the excess bromine by boiling the
solution until the solution turns light yellow or
colorless
- Add barium hydroxide in excess (test with litmus
paper)
Clear solution with
purple precipitate

RESULTS AND DISCUSSION

A. Isolation of DNA from onion

Grp #

Weight of onion (g)

Weight of air-dried DNA ppt

(g)

% yield

25.1953

0.0543

0.2155

25.7314

0.2455

0.95

25.419

0.1382

0.54

25.6542

0.2753

1.07

25.4240

0.3739

1.47

25.1637

0.7588

3.0154

25.08

0.33

1.32

25.0979

0.0331

0.13

25.3124

0.0724

0.2460

A.Isolation of DNA from onion

Consist of 3 basic steps:

1.Homogenization
- Process of breaking up the onion tissues to separate and open
cells
- Involves the heating and blending of the onion tissues
Homogenization solution - allows the DNA to be released from
its membrane
Sodium dodecyl sulfate or SDS - is a detergent that helps in
breaking

Ethylenediaminetetraacetic acid or EDTA - inactivates

DNAses
Sodium chloride and Sodium citrate - neutralizes the
negative charges of the phosphate groups of the DNA

Heating
- Softens the phospholipid in the cell membrane and
denatures DNAse
Temperature used: 60C (Leads to denaturation of DNA
when the temperature 60C is exceeded)

Blending
- Further breakdown of cell membranes
- 45 seconds only in order to not destroy DNA

2. Deproteinization
- Process of removal of the proteins that are associated with cells
and DNA molecules
- Involves the adding of the protease enzyme papain

Pulverized papain - A proteolytic enzyme which hydrolyze the

proteins clinging to the DNA
Filtration
Material used: Cheesecloth
- Used to filter out other parts of the cell (cell walls,
membrane) effectively
- Removes any solid material, resulting in a clear cell
homogenate

3. Precipitation
- process of forcing the DNA to come out of solution via adding a very
cold alcohol into the solution
Ice-cold 95% ethanol - used because the DNA is not soluble in
alcohol
- the colder the alcohol, the less soluble the DNA
will be
- Presence of two layers

B. Determination of DNA Concentration and Purity

GROUP #

260 nm

280 nm

Absorbance
Ratio

Protein
(mg/mL)

Nucleic Acid
(g/ml)

0.188

0.186

1.01

0.12

3.5

0.196

0.192

1.02

0.12

4.5

0.118

0.187

0.63

0.17

1.0

0.084

0.082

1.07

0.08

2.0

0.374

0.381

0.982

0.34

7.5

GROUP #

260 nm

280 nm

Absorbance
Ratio

Protein
(mg/mL)

Nucleic Acid
(g/ml)

0.178

0.178

0.15

4.1

0. 134

0.131

1.02

1.0

34

0.104

0.099

1.051

0.09

2.5

0.386

0.389

0.99

0.28

8.5

B1. Determination of protein and nucleic acid

concentration
UV spectrophotometry
DNA absorbs strongly at 260
nm
Proteins absorb strongly at
280 nm
Nomogram
Source: http://cpmcnet.columbia.edu/dept/phsio/tchrplan/oniondna.html

B2. Absorbance Ratio of the

Gives the relative
DNA
Presence of RNA

measurement of DNA
and protein content in

Pure DNA

the isolated DNA

A good quality DNA
sample should give a
value of 1.8-1.9

Indicates protein
contamination

C. Acid Hydrolysis
1M HCl
- Causes dissociation of DNA into its
components
- Causes depurination
1M NaOH
- Neutralize the excess HCl
100C
To destroy:
- Hydrogen bonds above 80C, acids with
pH < 3
- Phosphodiester bonds above 90C, acids
with pH < 2

WHY NOT SUBJECT TO

BASE HYDROLYSIS?
- Pentose of DNA does
not have a 2 hydroxyl
group
- A monophosphate
intermediate does not
form (2,3 cyclic
monophosphate
intermediate)
- DNA is stable in alkaline
hydrolysis

Depurination

Source: Mahato, R. I. Biomaterials for Delivery and

Books.<http://books.google.com.ph> Accessed 16 May 2016.

Targeting

of

Proteins

and

Nucleic

Acids.

Google

Chemical characterization of
DNA

Test for Deoxyribose/ Dische

GROUP #
DNA
HYDROLYZATE
DEOXYRIBOSE
reaction
SOLUTION
(standard)
1

Turbid white solution

Clear dark plum

solution

Light blue turbid

solution

Violet solution

White turbid with

precipitate

Purple turbid solution

Light blue solution

Purple solution

GROUP #

DNA HYDROLYZATE

DEOXYRIBOSE
SOLUTION
(standard)

Clear light blue

solution

Dark violet solution

Blue-gray solution
with suspected white
particles

Dark violet solution

Clear light blue

solution with turbid
precipitate on top
surface

Clear dark purple

solution

N.D

Slightly turbid solution

Clear solution

Purple solution

Test for deoxyribose/ Dische

reaction
TEST FOR:
REAGENT:
Used to detect the

Diphenylamine

presence of deoxyribose

Source: http://www.mpbio.com/images/productimages/molecular-structure/02150971.png

POSITIVE RESULT:

PRINCIPLE:

Blue colored liquid

solution

- Dehydration of
Deoxyribose by
H2SO4(formation of 5hydroxylevulinaldehyde)
- Complexation with

Source: http://www.und.nodak.edu/dept/jcarmich/101lab/lab3/diphenyl

Diphenylamine

Test for phosphates

GROUP #

DNA HYDROLYZATE

PHOSPHATE SOLUTION
(standard)

N.D

N.D

Clear colorless solution Clear colorless solution

with precipitate

Clear colorless
Clear colorless
solution; no precipitate solution; no precipitate

Clear colorless solution Clear colorless solution

with yellow precipitate

GROUP #

DNA HYDROLYZATE

PHOSPHATE SOLUTION
(standard)

Clear colorless
solution; no precipitate

Colorless solution; no
precipitate

Turbid colorless
solution; no ppt

Clear colorless
solution; no precipitate

Clear solution with

white ppt

Colorless solution
without precipitate

N.D

Clear colorless solution

Clear colorless
Clear colorless
solution; no precipitate solution; no precipitate

Test for Phosphate

TEST FOR:
To

determine

REAGENT:
the

presence of phosphate

HNO3
H2SO4
Ammonium molybdate

Positive result:

Principle:

yellow precipitate

Precipitation
Phosphate

(ammonium
phosphomolybdate)
H3PO4 + HNO3 + 12(NH4)2MoO4

(NH4)3PO4.12MoO3

of

Murexide Test/Test for Purines

GROUP #

DNA HYDROLYZATE

GUANINE SOLUTION
(standard)

N.D

Red residue

(unfinished)
2

N.D

N.D

N.D

Red residue

(unfinished)

GROUP #

DNA HYDROLYZATE

GUANINE SOLUTION
(standard)

after drying:
light yellow residue
after moistening:
light yellow solution

after drying:
yellow residue
after moistening:
red color of ppt/solution
after 2nd drying:
red residue
after addition of water:
orange solution

(unfinished)

Brown residue

Dark orange residue

(unfinished)

(unfinished)

GROUP #

DNA HYDROLYZATE

GUANINE SOLUTION
(standard)

N.D

N.D

N.D

N.D

N.D

N.D

N.D

yellow solution with red and

yellow precipitate

(unfinished)

Test for Purines/Murexide Test

TEST FOR:
Presence of purines

REAGENT:
Concentrated HNO3
10% KOH

POSITIVE RESULT:

PRINCIPLE:

Red residue

- Purine in DNA gets

oxidized by HNO3
(forms alloxan &
dialuric acid)
- Alloxan condenses to
form alloxanthin
- Neutralization yields
murexide

Test for Purines/Murexide Test

Reaction
mechanism:

Wheeler-Johnson Test/Test for

GROUP #
DNA HYDROLYZATE
STANDARD SOLUTION
Pyrimidines
1

Clear, colorless Solution

Turbid, dark purple

solution

N.D

N.D

N.D

N.D

Clear solution
White Precipitate

Yellow Solution
No Precipitate

N.D

N.D

GROUP #

DNA HYDROLYZATE

STANDARD SOLN

N.D

N.D

Clear Solution
White Precipitate

Clear Solution
Dark Purple Precipitate

N.D

Clear Solution
White Precipitate

N.D

No Liquid
Precipitate Only

Test for Pyrimidines/Wheeler-Johnson

Test
POSITIVE RESULT:
PRINCIPLE:
Purple Precipitate

- Oxidation of
pyrimidine to form
Dialuric Acid
- Neutralization of
Dialuric Acid by

TEST FOR:

REAGENT:

Presence of pyrimidines

Bromine water

(Cytosine and Thymine)

Ba(OH)2

Test for Pyrimidines/Wheeler-Johnson

Test
Reaction mechanism:

CONCLUSIONS

DNA from white onion was isolated wherein the groups obtained a 0.13
3.0154 % yield.
Using UV spectrophotometry, the protein concentration and nucleic
acid concentration values gathered ranged from 0.09-0.34 mg/mL and
1-34 g/mL, respectively. The absorbance ratio values gathered were
less than 1.8, hence, the DNA isolated by each group indicated protein
contamination.
The DNA components were separated through acid hydrolysis yielding
phosphate groups, nitrogenous bases, and deoxyribose sugar.
Various chemical tests were used in determining the components of
DNA where:
- 5 out of 9 groups tested positive for deoxyribose in Dische test
- 2 out of 9 groups tested positive for phosphate in Test for phosphates
- None of the groups were able to complete the Murexide and WheelerJohnson test

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