Introduction

DNA, or deoxyribonucleic acid, is the molecule which carried the genetic information of
an organism except for some RNA viruses. (Trabuco and Villa) DNA molecules are large and
long strands of molecule which known as nucleic acids. A physician turned chemist, Levene
(1919) was the first to discover the order of the three major components of a single nucleotide
which are phosphate group, pentose sugar and nitrogenous base.

After this, scientist found that there were four types of nitrogen bases existed in
nucleotides which are adenine (A), thymine (T), guanine (G) and cytosine (C). The order of
these bases will determine the instruction of DNA or genetic code (Pray, 2008). Then, Watson
and Crick (1953) were success to get the accurate description of DNA structure, which is
double helical structure.

DNA is important for an organism since it contain information for synthesis of protein and
other molecules essential for cell functions. Therefore, DNA analysis is required to carry out
in many molecular work. DNA should be extracted carefully in order to prevent damage of the
molecules. In this practical, basic principle of extraction of DNA was studied and used to
extract DNA from onion cells.

Skills of conservation of DNA also learned from this experiment. According to Gaikwad
A.B., efficient DNA conservation is depending on storage temperatures, composition of storage
buffer, ionic strength, purity of DNA, length of DNA, presence of metal ions and other else.
TE buffer was used in this practical for storage of DNA. The content of TE buffer used are
sodium Lauryl sulphate, sodium choride, sodium citrate, and ethyenediaminetetraacetic acid
(EDTA).
Objective

1. Able to extract DNA without using a kit.

2. Able to explain the basic principle of DNA extraction procedure

Apparatus and material

Apparatus: blender, beakers, cheesecloth, glass rod, knife, cutting board, falcon tube, glove
and cylinder.

Materials: onions, lyse solution and isopropanol

Procedure

1. Glove was wear before isolating and handling DNA.

2. 1 or 2 onions were chopped into 1cm3 cube, 2 portions and 5g of onions were weighted
and transferred each portion into 2 beakers which contained 25ml onion lysis solution
respectively. The solutions were mixed thoroughly by shaking.
3. The mixtures were placed in a water bath at 60℃ for 10 minutes.
4. The mixtures were removed from water bath after 10 minutes and placed in ice bath.
The beakers were swirled gently until it feels cool to touch.
5. One of the mixtures was combined with other group’s mixtures into a blender. The
mixture was blended for 30 to 60 seconds at low speed until foams were observed.
6. Another mixture in the ice was allowed to continue to step 7 without blending.
7. The blended mixture was poured into a large beaker (1L) in the ice bath, the mixture
was allowed to cool to touch.
8. A cheesecloth was folded into several layers and the onion mixture was poured through
it by using a funnel into a 250ml beaker.
9. 10ml of onion liquid in the 250ml beaker was poured into a clean tube. The blended
mixture and the non-blended mixture were separated and the tubes which contained
the mixtures were labelled.
10. 10ml of isopropanol was added slowly into the mixtures and the tubes were caped.
The solution was then mixed gently by rocking the tubes back and frothed until white,
stringy solids (DNA) were formed. Then, the different between the two treatments
were observed.
11. The DNA were collected by using a glass rod.
12. The DNA were observed under a microscope and the observation was described.
13. The DNA was stored in TE buffer for next practical class.
Result

Figure 1: Falcon tube A which contain blending onion mixtures mix with cold isopropanol

Figure 2: Falcon tube B which contain non-blending onion mixture mix with cold
isopropanol

Figure 3: DNA molecules observed under microscope without cover slip at 10x
magnification
 During the process of extraction of DNA, the blended mixtures which poured into falcon
tube was labelled as A while the non-blended mixture was labelled as B. It is clearly observed
that the white and stringy solid which appeared in mixture of tube A were more than that of
tube B.

A small part of DNA from tube A was collected and observed under microscope. There
were a long and large chain of strip. The large strip is the DNA which mixed with others cell
components such as RNA. There are also some black spots observed under microscope. The
black spots are cell debris which caused by blending process. Since the DNA was observed
under electric microscope which have not enough magnification, the structure of DNA cannot
observe clearly in this experiment.

The onions DNA in tube A and B were then stored in the TE buffer for next practical usage.
The purpose of TE buffer was to solubilize DNA to protect them from degradation (Atiken A.).
Discussion

The early methods for isolating the DNA were discovered by Julius Marmur (Marmur, 1961)
which in turn were developed from classic work of Oswald Avery, Maclyn MaCarty and Colin
MacLeod, who was the first scientist showed that DNA was the genetic materials in 1940s.
During the year 1986s, simpler methods of extraction of DNA were derived (Helms, et al,
1986). This method was then modified by scientists and researches and subsequently made
their way into specialist school biotechnology projects (Rasmussen and Matheson, 1990). The
simple methods of extracting DNA is only need cheap materials and apparatus therefore it can
be carry out easily and affordable for undergraduate students.

In this experiment, extraction of DNA from onion cells was carried out in order to observe
the structure of DNA under microscope. The onions were divided into 2 portion, one of the
portion was undergo blending procedure while another did not. There are differences of
observation between blended and non-blended mixtures. In tube A, which contained blended
mixtures, showed that there were more white and stringy solid while in tube B, which
contained non-blended mixture, there were less white and stringy solid appeared.

Blending help to break the onions cells and increase the surface area exposed to detergent
based chemical (Nuffield Foundation). The lyse solution were added to both blended and non-
blended mixtures. It was function to degrade the onion’s cell membrane by trapping the lipid
and protein which were the major component of plasma membrane. Therefore, onion mixtures
of tube A which was blended will release more DNA compare to mixture of tube B which do
not undergo blending process. Thus, the white, stringy solid (DNA) observed in tube A was
more than tube B. Moreover, the white and stringy solids observed were clump together. This
was because DNA molecules which is a polar molecules preferred a polar environment
compare to a non-polar environment. Therefore, it may attach to polar materials like itself.

The structure of DNA of tube A was observed under microscope. There were a large and
long chain of DNA strip which may mixed with others cell components like RNA. This is because
RNA also insoluble in isopropanol and it may precipitate with DNA. Moreover, there were also
some black spots observed clearly under microscope. The black spots were cell debris which
may be the fragment of cell wall and cell membrane due to blending process. The structure
of DNA cannot observe clearly since the magnification of microscope used was not enough for
observation of small DNA molecule.
 According to Michael W. Davidson, who was the curator of the National High magnetic
Field Laboratory at Florida State University, many scientists used electron, scanning and
atomic force microscope to observe individual DNA molecules. However, DNA only appeared
as a string rather than being resolvable into the individual units (nucleotide) from which it is
composed. Davidson also said that chromosomes, which is high coiled and tightly condensed
DNA were easily visible under a microscope with high enough magnification. (Claiborne Ray,
2008).

From this experiment, it is clearly show that the chromosomal DNA is located in nuclease
of cells. The DNA is a long chain of molecules which made by nucleotides. Each nucleotide
consists of 3 components which are phosphate group, pentose sugar and a nitrogenous base.
The bases define types of nucleotides. The two types of bases are purines and pyrimidines.
The two main purines in DNA are adenine (A) and guanine (G) while cytosine (C) and thymine
(T) are classified as pyrimidines (Trabuco and Villa).

There are some precautions which needed to carry out in this practical. One of the
precautions are heating the solution for 10 minutes after adding lysis solution into tube A and
tube B which contained blended and non-blended mixtures. Since the heating process increase
the temperature, the cell membrane will be easier to break down by lysis solution. The cell
membrane may become more fluid when temperature increase. The fatty acid tail of the
phospholipids become less rigid and allow more movement of proteins and other molecules
in and out of membrane. This can change the permeability of cell and thus make lysis solution
destroy the cell membrane easier compare to room temperature.

Furthermore, isopropanol which added into the onion mixtures need to remain at low
temperature. Isopropanol is alcohol which dissolved the onion cells’ components except DNA
and RNA molecules. The isopropanol which has poorer ability to attract positive ions compare
to water allow DNA molecules to precipitate easily by reacting with positive ions present in
the solution. From the other hand, low temperature helped to protect the DNA by slowing
down the activities of enzymes which may break down DNA such as DNAse. DNAse usually
protect cells by destroying the DNA of viruses which entering the cell. Therefore, cold
isopropanol helped to prevent damage of DNA molecules (Nuffield Foundation).
 In this practical, TE buffer was used to store onions DNA after observation of DNA done.
TE buffer, also called as Tris-EDTA buffer, derived from its component: Tris, a common pH
buffer, and EDTA, a molecule which chelatesd cations such as Mg2+ (Atikan A.). Tris
(hydroxymethyl) aminomethane, is an effective buffer between pH 7 and 9. Its neutral range
is suitable for storage of DNA. Moreover, Tris also interact with LPS (lipopolysaccharide) in
cell membrane, which make it unstable. EDTA (ethylenediaminetetraacetic acid) can bind with
divalent cations such as Ca2+ and Mg2+. These reactions will destabilise the cell membrane
because the the divalent cations always play role to maintain the integrity of the cell
membrane (Tan A., 2018).

In summary, extraction of DNA is necessary in order to observed and study the structure
of DNA. Some techniques are required to minimise the damage of DNA molecules. To avoid
RNA and proteins mix with DNA, protease and RNAse can be used to break down these
molecules.
Conclusion

All in all, DNA molecules are able to extracted by using a kit. To carry out DNA extraction,
onions cubes need to be mix with the lyse solution under 60℃ for few minutes. The, it need
to be cooled down to touch and blending it. After this, cold isopropanol need to be added in
order to precipitate the DNA molecules. So, the white and stringy solid appeared means that
the DNA molecules in the cells precipitate.
Exercise

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

The chromosomal DNA located in the nuclease of cell.
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 main composition of the cell membrane are phospholipid bilayer, cholesterol,
membrane protein, glycoprotein and glycolipids. The cell membrane can be destroyed
when detergent or detergent base chemical is added to the solution contain cells.
Detergent contain sodium laurel sulphate, which can remove fats and protein. Since
major component of cell membrane are fat and protein, the cell membrane is broken
when detergent is added.
4. Describe briefly the specific purpose of adding each of the following:
a) Lysis solution and heating this solution
Lysis solution used in experiment is detergent base chemical. It will cause physical
disruption of cell membrane which consists majority lipid and protein. The lysis
solution breaks down lipid barrier of cell membrane by solubilising protein and
disrupting lipid and protein. The lysis solution causes lipid and protein precipitate
out of the solution. Heating process increase temperature of water bath, it will
soften the phospholipid in cell membrane and denatures DNAse enzyme which
function to cut DNA into small fragments. Heating process also help to increase
rate of lysis process of cell.
b) Blender
Blender allow cell tissues to be broken up in order to make lysis solution take effect
and break the cell membrane of onion cell easier.
c) Isopropanol
Since DNA is not soluble in alcohol, when cold isopropanol is added, DNA will
appear as white precipitate. Isopropanol has poorer ability to keep positively
charged compare to water, therefore it is easier for DNA which is negatively
charged attract positive ions to form stable precipitate. Moreover, Isopropanol also
form hydrogen bond with water and it will increase concentration of DNA and
precipitate.
5. Describe briefly the action of DNAse.
DNAse, also called deoxyribonuclease, is an enzyme which involve in hydrolysis of
degrading DNA. When white blood cells accumulate in mucus, it will break down and
release DNA which make the mucus stickier, DNAse is needed to catalyse the process
of breaking down DNA in order to clear the mucus. Moreover, DNAse also needed to
destroy DNA of viruses which entering the cell to protect cells from disrupt by the
viruses.
6. Describe your observation under microscope.
There are large and long chain of strip which are onion cells’ DNA observed under
microscope. There are also some small dark spots surrounding the DNA molecules
which are cell debris.
Reference

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