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Acosta, Cartier

Ancheta, KC
Bacus, Joey

Extraction of DNA and Protein from Commercially Branded


Pineapple Samples and Microscopic Analysis and Observation
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.

PRELIMINARY OPERATIONS
MATERIALS

METHOD

- 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.

- 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 60C;

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
protein,
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 PULP


This operation serves to separate the cells from each other and to better expose them to the action of the
extraction solution.
MATERIALS

METHOD

- 100 g of banana (or: kiwi, apple, pear, kaki,

- Place 100 g of banana (without peel) on a

peas, onion, etc.);


- balance;
- knife;
- chopping board and fork;
- 250 cc beaker;
- a teaspoon.

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.

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 60C 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

METHOD

- thermometer;
- pot with water at 60C;
- salad bowl with tap water and ice cubes.

- Pour the extracting solution on the mush;


- place the beaker in a bain-marie in the pot with
water at 60C;
- mix the mush so to distribute the extracting
solution and to make the temperature uniform;
- after 15 minutes, place the beaker in a bainmarie 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.

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

METHOD

- 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.

- 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.

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

METHOD

-Proteolytic enzyme (ex: Protease or pineapple


juice);
- a 5 cc syringe without needle.

- 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.

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

METHOD

- Some tubes for the possible repetition of the


operation;
- tubes holder;
- icy alcohol (kept in a freezer).

-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.

OBSERVATION THROUGH THE MICROSCOPE (optional)

MATERIALS

METHOD

- some clean microscope slides;


- hook made with a long metal wire;
- dye to stain the nucleus (ex: Toluidine,
Methylene Blue, Aceto-Orcein);
- dropper;
- microscope

- 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.