TITLE

EXTRACTION OF DNA FROM ONION

INTRODUCTION

DNA is the abbreviation form of deoxyribonucleic acid, a nucleic acid that contains the
hereditary materials- the genetic instructions in all known living things including humans. DNA
is a double-stranded, helical nucleic acid molecule capable of replicating and determining the
inherited structure of a cell’s protein (Boyer, 2006). As stated by Hartwell et. al , 2011 “DNA
molecule stores biological information in units known as nucleotides” (p. 1). In this context,
nucleotides are the building blocks of DNA which consists of three chemical groups for a single
strand of DNA: one of four types of nitrogenous base, a phosphate group and a deoxyribose
sugar. Albright (2014) highlighted the function of DNA, which serves as a chemical instruction
to specify the organism or living creature be it in its development, reproduction as well as the
function of an organism (p. 5). However, in 1860s when DNA was first discovered by a Swiss
chemist named Johann Friedrich Miescher, the role of DNA being the material of heredity was
not emphasized (The Discovery of DNA, 2016).

In today’s world, analysing DNA from a sample, whether a sample of blood, saliva or
even hair roots entails only a short amount of time for scientists with the help of extraction or
isolating process of DNA to segregate DNA from unwanted substances to avoid contamination
in order for DNA to be analysed. Rice (2017) further described that DNA extraction is the
removal of deoxyribonucleic acid from any cells or even viruses in which it normally resides
which is used in early diagnostic processes to diagnose viruses and bacteria in an environment
and diagnosing ailments and genetic disorders.

Goodwin, Linacre and Hadi (2007) stated that the general principles of DNA extraction
that can be classified into three stages, which includes the process of cell lysis as the first step
by disrupting the cellular membranes in detergent and salt solution, protein denaturation as
the second step and finally, the separation of DNA from the denatured protein and other
cellular components (p. 27).
OBJECTIVES

1. To be able to extract DNA without using a kit.

2. To be able to explain the basic principle of DNA extraction procedure.
MATERIALS

Onion, blender, beaker, cheesecloth, glass rod, knife, cutting board, cylinder (20ml), Falcon
tube, forcep, mortar, analytical balance

APPARATUS

Lyse solution, isopropanol, TE buffer

METHODOLOGY

1. Cloves were worn during isolating and handling DNA.

2. An onion was chopped into two portion and each portion was weighed 5g using
analytical balance.
3. The 5g of onion of each portion was mince once again using mortar until it homogenate
and each portion was transferred into two beakers labelled A and B containing 25 ml
of onion lysis solution that contains sodium Lauryl sulphate, sodium chloride, sodium
citrate, and ethylenediaminetetraacetic acid (EDTA).
4. The mixture in each beaker was stirred using spatula. Next, the mixtures were placed
in a water bath at 60° for 10 minutes.
5. Both beakers were removed from water bath and placed in an ice bath for 1-2 minutes.
The contents in the beakers were gently swirled until it feels cool to touch.
6. After 1-2 minutes, beaker A was taken out from the ice bath while beaker B remained
in the ice bath.
7. The mixture in beaker A was combined with several other groups’ mixtures into a
blender and blended for 30-60 seconds at low speed until foams were observed.
8. The blended mixture was poured into a large (1L) beaker in the ice bath and mixture
was allowed to cool to touch.
9. A liquid began to form at the bottom of the beaker which indicates the mixture was
ready.
10. The cheesecloth was folded into several layers and onion mixture was poured through
it using a funnel into a 250 ml beaker.
11. 10 ml of onion liquid was poured in a clean tube where the blended mixture and non-
blended mixture were separated.
12. DNA from both mixture was collected using a forcep.
13. The DNA from both mixture were observed under a microscope and the observation
was described.
14. The DNA was stored in TE buffer for next practical class.
RESULTS

A) BLENDED DNA OF ONION UNDER MICROSCOPE

Figure 1.0: Microscopic view under 4x magnification of blended DNA of onion.

 B) UNBLENDED DNA OF ONION UNDER MICROSCOPE

Figure 2.0 : Microscopic view under 4x magnification of unblended DNA of onion.

Figure 2.1 : Microscopic view under 10x magnification of unblended DNA of onion

Figure 2.2 : Microscopic view under 40x magnification of unblended DNA of onion
C) BLENDED AND NON-BLENDED DNA MIXTURE WITH 10ml OF ISOPROPANOL
DISCUSSION

Based on the result of this experiment, the extracted DNA from onion between the blended
DNA and non-blended DNA exhibited differences in general appearances under microscope
where the blended DNA mixture potrays a disrupted structure, by means a not complete
structure while the non-blended DNA potrays a complete structure. As stated by Butler (2005),
DNA molecules must be isolated to allow the extraction of DNA from other cellular materials
including cellular proteins that carry the responsibility to package and protect the DNA in the
environment of the cell as these can inhibit the ability to analyse DNA after DNA extraction is
done (p. 42). In this case, the ruptured structure of blended DNA mixture was caused by the
process of blending. The blending process causes the cell wall to break and the cell contents
to come out, making it is easier for DNA to be extracted. The transparent string-like reflects
the structure of DNA has been disrupted, where the two strands of DNA are no longer attached
together to form a double-helix, helical molecule of DNA. Besides, the blended DNA of onion
can be seen clearly under microscopic view, unlike the non-blended DNA because the cell
membranes in non-blended DNA are still in a complete structure. Singh, Dasgupta and Tripahti
(2004) further described “Onion is a diploid species with 14 chromosomes per cell, its nuclear
DNA content per cell (referred to the genome size) is very high” (p. 241). In this experiment,
onion (Allium cepa) is used in this experiment because of its low starch content, allowing DNA
to be seen clearly and easily able to distinguish DNA from the rest of the solution.

According to Makkar and McSweeney (2005), cell lysis is the most important step that
needed to be carried out to achieve efficient DNA extraction where cell lysis can be achieved
by the process of freeze-thaw or enzymatic disruption of cell wall and membrane by enzymes
such as proteinase K and detergents such as sodium dodecyl sulphate and sodium Lauryl
sacrosine (p. 83). Bruns, Ashwood and Curtis (2007) further described cell lysis as an initial
step in DNA extraction protocols whereby the cellular and nuclear membrane envelopes are
disrupted (p. 40). In this experiment, onion lyse solution act as a homogenizing solution
containing sodium Lauryl sulphate, sodium chloride (NaCl), sodium citrate (NaC6H5O7) and
ethylenediaminetetraacetic acid (EDTA) was used. Sodium Lauryl sulphate is an ionic
detergent-based lysis to aid cell lysis in order to break down the cell walls. Ionic detergents
are more reactive compared to non-ionic detergents such as Triton X-100, whereby sodium
Lauryl sulphate can lead to the denaturation of many protein (Walker and Rapley, 2002).
 In the other hand, EDTA serves two purposes in lysis solution. Firstly, EDTA binds to
the divalent metal ions Ca2+, Mg2+, Mn2+ that could form salts with the anionic phosphate
groups of the DNA to maintain the integrity of cell membrane. In this case, the EDTA weakens
the cell membrane stability. Besides, EDTA also inhibits DNAses (deoxyribonuclease), an
enzyme that hydrolyzes DNA, by forming a complex (chelating) with metal ions, speficially
magnesium ion (Mg2+) and manganese (II) ion (Mn2+) which is the cofactors of nucleases
(Boyer, 2012). The purpose of adding NaCl in the lysis solution is to prevent from ionic
interactions between the negative phosphate ends in DNA and cations. This in turn cause the
strands to unite and come together, allowing DNA to precipitate when alcohol is added.
However, before undergoing the extraction of DNA, the onion is minced as the prelude
treatment in mechanical way for extracting its DNA is vital as this will allow more efficient
absorption of heat and solutions since the minced onion have larger surface area that helps
membrane at the surface easier to dissolve. Besides that, mincing the onion will exposed the
cell walls to allow the detergent to break them down.

In addition to this experiment, the temperature of the water bath is set to be at 60°
where the mixture with lysis solution is going to be placed. This is to ensure the lysis of onion
cell are complete, meaning the protein is destroyed as well as the celluloid material where
DNA is easier to extract. The reason the temperature is set to be exactly 60° is due to the
effect of temperature towards the DNA itself. In this case, when the temperature is set more
than 60°, it will denature the DNA. Chauhan (2008) stated that denaturation of DNA will affect
the DNA biological activity, where hydrogen bonds that holds the complementary sequences
of nucleotides will be destructed.

One of the safety measure that should be taken into account when handling DNA or
isolating the DNA, gloves should be worn to avoid any contamination that could affect the
outcoming result of the DNA. Besides that, during the process of blending the solution, the
time should be limited to 45 seconds, to ensure that the DNA are not ruptured except for the
protective barriers such as the cell walls and other cellular membranes in order to extract the
DNA. Moreover, after placing the mixture into the water bath, the mixture needs to be placed
into the ice bath. The ice bath serves a purpose to cool the solution, where it will prevent
further denaturation that will cause the DNA to be destroyed from prolonged heating.
According to Henry (2008), Keller and Manak (1989) stated that DNA must be precipitated
with by ethanol or isopropanol, in the presence of sodium chloride, sodium acetate or
ammonium acetate (Kirby, 1990;Sosa and Oliveira, 1992). In this experiment, isopropanol is
mixed to complete the process of extraction where precipitation is done. In this case, alcohol
precipitation is based on the phenomenon of decreasing solubility of nucleic acids in water.
This is due to the polar molecules that surround the DNA in aqueous solution, where the
positively charged dipoles of water interact strongly with the negative charges on
phosphodiesters groups of DNA (Surzkyci, 2003, p.7).
QUESTIONS

1. Where is the chromosomal DNA located in the cell?

The chromosomal DNA is located in the nucleus of a plant and animal cells.

2. The nucleotide is the basic unit of DNA. What are the three chemical groups that make
up a nucleotide? Draw a diagram of a nucleotide.

The three chemical groups that make up a nucleotide are phosphate group, pentose
sugar and nitrogenous base.

3. Determine the chemical composition of the cell membrane. Draw a diagram of the fluid
mosaic model of the cell membrane. Describe how the cell membrane can be
destroyed.

The chemical composition of the cell membrane are phospholipid, proteins, cholesterol
and glycolipid. The cell membrane can be destroyed with a very high or very low
temperature, due to chemicals and by physical damage.

Diagram of the fluid mosaic model :

4. Describe briefly the specific purpose of adding each of the following:
a. Lysis solution and heating this solution

Lysis solution is used usually for break open cells so that the DNA is released
to analyse the compound of the cells. Heating this solution is important to
denature the protein that protecting the DNA tightly on the chromosomes.

b. Blender

Blending helps to break down the cells component so that they are separated.
Therefore, it is easy to obtain the DNA from the cell. Blending also helps to
facilitate the released of cell content.

c. Isopropanol

Isopropanol is an alcohol which helps to extract DNA. The addition of

isopropanol will form two distinct layers, where the isopropanol will stay on top
of the onion juice. The insolubility of DNA in any alcohol will make DNA to come
out from the solution or precipitates which it will float into the alcohol layer
with its string-like substance that can be visualize clearly.

5. Describe briefly the action of DNAse.

DNAse (deoxyribonuclease) found to be tightly regulated in normal cells and is an

enzyme that hydrolizes and degrade the DNA down where it cleaves the
phosphodiester links in the DNA’s backbone.

6. Describe your observation under the microscope.

The structure of blended DNA as well as the non-blended DNA of onion were
scrutinised. The blended DNA shows a transparent string-like substances without any
helix form because it was ruptured by the blending process. However unlike the non-
blended DNA, it shows a complete structure without any ruptured because there is no
blending process undergone in this mixture.