EXTRACT

DNA FROM
FRUITS
MADAM NOOR HAYATI
BT SALEH

AFIQAH NU’AIMI BINTI

ZAKARIA
MS 09175212897
H7 P37a
 HOW TO EXTRACT DNA FROM
FRUITS
G. Carboni, January 2007
Text editing by Donald Desaulniers, Ph.D.

INTRODUCTION
In recent years, it is not uncommon to read articles on DNA in both scientific and popular
magazines. DNA is regularly mentioned in the news and is often featured in TV detective or
crime-scene investigation dramas. DNA, also known as Deoxyribonucleic Acid, is a long
molecule that holds the genetic information for all living beings, be it vegetable, animal or a
simple microorganisms. It is capable of copying itself and can synthesize RNA (Ribonucleic
Acid). In more evolved or complex forms of life, DNA is contained in the nucleus of the
cells. Except for the red blood cells of mammalians, which are devoid of a nuclei, all cells of
a living being have their own DNA. The cells of an organism use certain parts of the DNA
molecule, or genes, to produce the proteins they need to function. For a more detailed
description of DNA including its structure, its functions and the mechanism by which
proteins are produced, the reader should consult the texts listed [1] the Reference section of
this paper, which are well written and contain excellent illustrations. In this article, I describe
a simple experiment that will allow you to extract a bit of DNA from a banana, however, you
can also try it using other fruits and even vegetables. It is an experiment that can be
performed both at home and in a school laboratory.

PROCEDURE
SUMMARY OF THE PROCEDURE
The procedure described below exploits the fact that the external membrane of cells and that
of their nuclei are composed of fatty substances that can be broken down using a simple
detergent. The first operation in this procedure is to break-up the fruit into a pulp or mush so
that the cells are separated each from other as much as possible thereby exposing them to the
action of the detergent. Secondly, the detergent is added to the pulp of the fruit so as to
release the DNA from the cell membranes, which encapsulate it. Thirdly, it is necessary to
filter the mixture to separate the nucleic acid from the remains of the cellular membranes.
Finally, the DNA is precipitated in alcohol where it becomes visible. The DNA you obtain
using this procedure can be observed with a microscope and can be used for other
experiments like electrophoresis or other experiments.
OBJECTIVES
• Develop an understanding of DNA by modeling the process of DNA extraction.
• Practice laboratory techniques to carry out an investigation.
• Use critical thinking skills by understanding the concept of DNA in cells and the
extraction procedure.

PURPOSE/HYPOTHESIS

DNA is present in all life. In this project, you will extract DNA to see what this molecule
looks like.
DNA is twisted inside the cell nucleus. A cell's nucleus also contains proteins and other
substances. To see the DNA, you will have to separate out the DNA from all the cell's other
molecules. You will first liquefy the substance and separate the cells by blending it.
Detergent or soap will break apart the cell's outer and inner membrane, in much the same way
that soap loosens dirt and grease. The cell's membranes are made of a fatty substance that
contain proteins. Detergent contains a substance that pulls apart the fats and proteins, freeing
the DNA.
The DNA in the nucleus is wound up with proteins. To isolate the DNA from these proteins,
you will use an enzyme, a protein that quickens a chemical reaction. Meat tenderizer contains
enzymes that cut away the proteins. Adding alcohol will then allow you to see the DNA.
DNA is not soluble in alcohol. DNA precipitates, or separates out of the solution, in alcohol,
moving away from the watery part of the solution and rising towards the alcohol. Proteins
and other parts of the cell will remain in the bottom watery layer.
PRELIMINARY OPERATIONS

MATERIALS
- pot;
- thermometer;
- plastic salad bowl;
- ice cubes;
- 50 cc of 70 - 95 % 70-95% isopropyl or denatured alcohol (ethanol) in a closed bottle;
- rags and absorbent paper tissues.

METHOD

- The day before the experiment, prepare some ice cubes;

- at least 2 hours before, place in a freezer a sealed vapor-tight plastic bottle with 50 cc of 70-
95% isopropyl or ethyl denatured alcohol. This container must to be closed tightly to prevent
alcohol vapors from being released since they are flammable;
- 15 minutes before starting the procedure, warm a pot of tap water to 60°C;

Before starting the experiment, it is important

to perform the preliminary operations described here

PREPARATION OF THE EXTRACTION SOLUTION

As mentioned previously, the DNA is held inside the nucleus of the cells of the fruit we are
using. To free the DNA, it will be necessary to breakdown the membranes of the cells as well
as those of the nuclei. As these membranes are made up of phospholipids, which are
molecules rich in fats, we will dissolve them by using a simple household detergent. We will
also use a little table salt, which helps to eliminate the proteins, called histones, on which the
DNA is wrapped.
MATERIALS
- 100 cc of distilled water but tap water can also be used ;
- a scale to weigh few grams (if possible);
- 3 g of table salt (a half teaspoon);
- 10 cc of liquid detergent;
- a 10 cc syringe without needle;
- a 100 cc beaker;
- a glass rod.

METHOD
- Pour 3 g of salt and 80 cc of distilled water in a 100 cc beaker;
- mix until the salt is completely dissolved;
- with the syringe, take 10 cc of liquid detergent and add it to the solution;
- add distilled water until you obtain a total volume of 100 cc;
- while avoiding to produce bubbles, mix to homogenize the solution;
- the extracting solution is ready.

Preparation of the extracting solution (Distilled

water, table salt, detergent, syringe, 100 cc beaker and spoon).
PREPARATION OF THE PULP
This operation serves to separate the cells from each other and to better expose them to the
action of the extraction solution.
MATERIALS
- 100 g of banana (or: kiwi, apple, pear, kaki, peas, onion, etc.);
- balance;
- knife;
- chopping board and fork;
- 250 cc beaker;
- a teaspoon.

\
METHOD
- Place 100 g of banana (without peel) on a chopping board and crush it with a fork until you
obtain a pulp. If you use an onion, with a knife obtain cubes of about 5 mm of side or less.
You can also use a mortar or a blender. If so, do not shred the pulp too much;
- pour the mush in a 250 cc beaker.

Preparation of the fruit pulp

EXTRACTING THE DNA
The aim of this operation is to breakdown the membranes of the cells and their nuclei to free
the DNA. The pulp will heated to 60°C to speed up and help the process of breaking down
the membrane walls. Heating the pulp also helps to deactivate certain enzymes like DNase
that can degrade the DNA. However, if the pulp is held at an elevated temperatures for too
long a time the DNA may begin to fragment du to the heat exposure. For this reason it is
advised to cool the pulp after approximately 15 minutes in a bath of chilled water.

MATERIALS
- thermometer;
- pot with water at 60°C;
- salad bowl with tap water and ice cubes.
METHOD
- Pour the extracting solution on the mush;
- place the beaker in a bain-marie in the pot with water at 60°C;
- mix the mush so to distribute the extracting solution and to make the temperature uniform;
- after 15 minutes, place the beaker in a bain-marie in the water with ice cubes;
- mix the mush to make the temperature uniform;
- after 5 minutes, remove the beaker from the cold water and prepare yourself for the
filtration.

Figure 5 - Pour the extraction solution in the Figure 6 - The pulp should be kept at 60°C for
pulp and mix. no more than
15 minutes and then chilled to about 0°C for 5
minutes.
FILTRATION
The filtration process is used to collect the liquid rich in DNA and to separate it from the
cellular remnants and the other tissues of the fruit, which will be discarded.

MATERIALS
- sieve with a diameter of about 12 cm;
- coffee filter paper (laboratory filter is too much thick). Kitchen absorbent tissue paper can
also be used, provided that it does not have any visible holes;
- bowl.

METHOD
- place the sieve on the bowl;
- take a filter paper, soak it and place it in the sieve;
- pour a little pulp on the filter, taking care that is goes through the filter paper ;
- mix with care to help the filtration and avoid ripping the filter paper;
- the filtered liquid you will obtain is rich in DNA.

Filtering the pulp using a sieve, filter paper and a bowl.

REMOVING THE PROTEINS (optional)
With this additional operation it is possible to obtain a purer DNA extract, but it it is not
essential if you want to observe the DNA. Because DNA is wrapped on proteins called
histones is will be necessary to remove these proteins to obtain a DNA extract of higher
purity. To remove these histones, you can use proteolytic enzymes like Protease. While you
can purchase protease in a shop that sells chemical products, you can also substitute it with a
substance that is much easier to find; it is found in the juice of the pineapple which that
contains Bromelain, a substance able to breakdown proteins into the amino acids of which
they are composed.

MATERIALS
- Proteolytic enzyme (ex: Protease or pineapple juice);
- a 5 cc syringe without needle.
METHOD
- In a tube, pour 5 cc of the filtered solution;
- add 1 cc of pineapple juice and mix;
- wait 2 - 3 minutes to let to the bromelain react.

Figure 9 - In a tube, pour 5 cc of filtrate and 1

Figure 8 - Obtaining the pineapple juice.
cc of pineapple juice.
PRECIPITATING THE DNA
DNA is quite soluble in water and invisible, while it is insoluble in alcohol wherein it
precipitates and becomes visible. By adding alcohol to the DNA filtrate solution in the tube,
the DNA is rendered visible.

MATERIALS
- Some tubes for the possible repetition of the operation;
- tubes holder;
- icy alcohol (kept in a freezer).
METHOD
- Slowly, pour in the tube of the previous step some icy alcohol by avoiding it mix with the
filtrate;
- the volume of the alcohol has to be about the same of that of the solution;
- let the tube rest for 5 minutes to allow to the DNA to precipitate and accumulate in the tube.
Now, at the interface between alcohol and the filtrate you should be able to see a milky
substance, which tends to increase in volume as time progresses. This milky substance is the
DNA of the banana. Unfortunately, inside this milky little mass, you may observe numerous
tiny bubbles. The presence of these bubbles is due to the property that the solubility of gases
in a cold liquid is higher than in a warm one. While alcohol was in the freezer it likely
absorbed some gases that are expelled as the liquid is warmed up.

Figure 11 - Tube with DNA of the banana

Figure 10 - Very slowly, pour some ice cold
alcohol into
the tube and avoid mixing the alcohol with the
filtrate.
mixed with a numerous tiny air
bubbles freed from the alcohol which is
warming up. In Figure 1, there
are less bubbles and the DNA is observed as a
milky substance.
OBSERVATION THROUGH THE MICROSCOPE (optional)
MATERIALS
- some clean microscope slides;
- hook made with a long metal wire;
- dye to stain the nucleus (ex: Toluidine, Methylene Blue, Aceto-Orcein);
- dropper;
- microscope.
METHOD
- with a long metal wire ending with a hook, extract a sample of DNA from the tube and
place it on a clean microscope slide;
- level the mass on the slide and stain it with a nuclear dye;
- if necessary, add a little water and mount the coverslip.
By observing this preparation under the microscope, do not expect to see the well-known
double helix ladder structure of the DNA. You cannot see it even with an electronic
microscope. What you will see are clumps or flocks of DNA material which look like a
tangled mass of protein strands as illustrated Figure 12.

Sample of banana DNA at about 100 X

(stained with a 1% solution of Toluidine).

CONCLUSION
This experiment was not so difficult to carry out after all, was it not?. The aim of this simple
experiment was to provide you with an introduction to the procedures that are used in
molecular biology. Often, the techniques used in modern microbiology laboratories are based
on simple operations like this one. In other cases the procedures can be quite complex and
may involve more sophisticated manipulations and equipment. In all cases a sound
knowledge of biology and chemistry is essential to understanding how DNA is used in the
fields of life sciences and health sciences. If this experiment has sparked an interest in
pursuing future explorations.