1.

Abstract

Deoxyribonucleic acid, or better known as DNA, is a molecule within the cell nucleus,
holding genetic information. Overall, DNA tells an organism in what manner to progress and
function, and is so vital that this complex compound is set up virtually every one of its cells
(Squishy Science: Extract DNA from Smashed Strawberries, 2013). To extract the DNA out
of it, weve to gently break open the cell, the nuclear membrane, then separating DNA from
proteins and causing it to precipitate out of a solution. This is accomplished by means of
several chemicals, based on the structure of membranes, DNA and its electronegativity.
Sodium chloride (NaCl), or any other compounds with sodium in it, are used to stabilize DNA
after its being stripped of its proteins and later on will help in precipitation. Basically the
process started with lysis of DNA, followed by removal of membrane lipids and lastly,
precipitation of DNA. The result that we had is a thick, white clump. For a better result, we
should extend the period of leaving alcohol and mixture of oranges.

2.0

Introduction

DNA is the blueprint of life. Every cell in each living things has DNA as the prove of their
existence. It determines the genetics of the individual organisms. DNA extraction is a routine
step in many biological studies including molecular identification, phylogenetic inference,
genetics, and genomics (Chen, Rangasamy, Tan, Wang, & Siegfried, 2010). It is one of the
most up-to-date in the biological sciences. One of the purpose is to diagnose numerous
medical illnesses and also be used for genetic engineering of plants and animals.
To extract the DNA from the specified organism (in this experiment, oranges was used), the
cells need to be separated first by the means of physical separation; blending. Addition of
salt help to break up protein chains that bold nucleic acids together and help the final
appearance of DNA strands to stick together. Adding amount of detergent into the thick,
paste-like mixture causing the cell membrane layer and nucleus to break open. This
combination of salt and detergent addition together help releasing DNA strands. Layer of
alcohol on top of thick paste help the DNA to float. At this moment DNA strands appear as
clumps of white stringy stuff where the water and alcohol layers meet.

3.0

4.0

Objectives

To study the DNA extraction method from specified fruit (orange).

To extract, isolate and observe the DNA of orange.

Theory

DNA is a long polymer made of nucleotides. Nucleotides are repeating structural unit of
nucleic acids (Brooker, 2012). They are linked together linearly to form either DNA or RNA
(so-called molecular cousin of DNA as they only differ in their coding). The AT/GC rule shows
that most organisms has equal amount of A and T, G and C. It states that that purines always
bond with pyrimidines; which keep the width of the double helix relatively constant (Brooker,
2012). For the DNA to be identified as genetic material, it must meet four criteria;
information, transmission, replication, and variation.

Figure 4.1: Basic structure of DNA

One of the easiest way to generally extract DNA from its nucleus is by DNA extraction
method used in this experiment. An ideal extraction technique should optimize DNA yield,
minimize DNA degradation, and be efficient in terms of cost, time, labour, and supplies. It
must also be suitable for extracting multiple samples and generate minimal hazardous waste
(Chen, Rangasamy, Tan, Wang, & Siegfried, 2010). DNA extraction is used to enhance our
understanding on how a complex work of gene coding can act as building block of cell,
protein, our flesh and blood. Its a process of extracting DNA (removing DNA from the cell or

virus in which it normally resides) from a sample using both chemical and physical
processes.
First, the cell disruption or cell lysis. Motion and physical force coming from grinding the fruit
with aid of a blender, which will eventually break down the fruits cell wall. Addition of salts
along the grinding process make the DNA extraction easier. The positively-charged sodium
ions (Na+) protect the negatively-charged phosphate group that run along the DNAs
backbone. Sodium chloride (or commonly known as table salts) aid in removing protein
bounded to DNA. Adding NaCl also helps in keeping the proteins dissolved in the aqueous
layer, ensuring only DNA, not protein, will precipitate (solidify and appear) in alcohol.
Secondly, addition of detergent or surfactants. The blending action only separated the fruits
cell, but as we know each cell is bounded by a cell membrane layer. And inside that layer,
theres another one called nucleus which hold the DNA that wed like to extract. So, to break
those layer open, addition of detergent is truly necessary because it helps to dissolve the cell
layers (which are a lipid bilayer). DNA is released once the membrane is disrupted. This
detergent ability is due to its structure; hydrophilic head and hydrophobic tail (Klug,
Cummings, Spencer, & Palladino, 2012). They are amphipathic molecules. The mechanism
that break the membrane layer is similar to soap-and-grease mechanism; which the when
they (the molecules of soap and grease) come in contact, theyll form a greasy soapy ball
(as shown in Figure 4.2).

Figure 4.2: A greasy soap ball formed when soap and grease come in
contact

Same situation applied to detergent and membrane layer. Detergent molecules that come in
contact with membrane layer will captures the lipids and proteins.

Figure 4.3: Detergent molecules in contact with membrane

layer and releasing DNA

Lastly, addition of ice-cold alcohol which causing the DNA to precipitate. DNA is soluble in
water but insoluble in the presence of salt and alcohol. When DNA comes out, it tends to
clump together, which make it visible to our naked eyes. DNA normally stays dissolved in
water, but when salty DNA comes in contact with alcohol it becomes undissolved. This is
called precipitation. The physical force of the DNA clumping together as it precipitates pulls
more strands along with it as it rises into the alcohol. You can use a wooden stick or a straw
to collect the DNA. If you want to save your DNA, you can transfer it to a small container
filled with alcohol.

5.0

Material and apparatus

i.

Alcohol

ii.

Detergent

iii.

Salt
4

iv.

Toothpick

v.

Orange

vi.

Dropper

vii.

Glass rod

6.0

Procedures

i.

A cup of alcohol was placed in a container filled with ice for about 30 minutes.

ii.

The selected fruit (orange) was peeled and cut into small pieces.

iii.

Then, pieces of orange were mixed with warm water and teaspoon of salt.

iv.

The mixture then was blended until it formed a thick, pourable liquid.

v.

The thick mixture of orange was then pour into a glass jar, with aid of coffee filter.

vi.

Two tablespoon of liquid dish soap was added and stirred. Caution was taken to
ensure theres no bubbles formed.

vii.

The cooled alcohol is taken out from container with ice, and slowly dropped into the
filtered mixture. Only small amount of alcohol was used, just to form a thin, visible
layer on top of filtered orange mixture. Aid of dropper is necessary.

viii.
ix.

Next, the mixture is left for about 10 minutes.

As the result, formation of long white strand is visible on top of the alcohol.

Figure 6.1: Mixture of orange pour

with help of coffee filter

Figure 6.2: Squeezing the fruit

7.0

Results

However, theres slight problem on getting the long white, visible DNA strand extracted from
the cell of fruit. The strand doesnt appear as thick white long strand, but instead it appears
as a white clump.

Figure 7.1: White clump that seems cloudy

Figure 7.2: View of thick, white clump

8.0

Discussion

Theres white clump found in the layer between alcohol and fruits mixture. It supposed to be
thick and long white strand. This is due to the period of leaving alcohol with fruits mixture is
short (less than 15 minutes). DNA appears as white clump because its very long. Each cell
of our body comprises of six feet of DNA, with only one-millionth of an inch wide. To fit all
length of these DNA into our cells, it first must be packed well. DNA will tightly twist itself,
making appearance of clumping together. So when its in the nucleus it used to be in folded
pattern, therefore when you extract it, it unfolds.
Some may get a stringy-threads form of extracted DNA. Its actually a single-stranded DNA.
The nitrogenous base of DNA double helix is held together by a very strong hydrogen bond.

When alcohol (i.e.: polar protic organic solvent) is added to fruits mixture, the H-bond of
bases pairs break and reform with alcohol in certain parts.
In the experiment, sodium was used for precipitation of DNA. Generally, all nucleic acids are
polar. Talking about DNA, the phosphate groups come with high polarity conveying negative
charges. The characteristic also applied for water solubility, since water is also polar. Water
with positive charge interact with negative charge of DNA, combining and resulting a
solution. For further testing, it requires sodium because sodium provide stronger positivelycharged ion in the solution (because water has relatively weak positive charge) (Taylor, n.d.).
Itll the providing temporary attraction between sodium and backbone.
The salt used is sodium chloride (NaCl) while the detergent used containing sodium laurel
sulfate (NaC12H25SO4). Sodium chloride aids in removal of proteins around DNA and keeping
those proteins dissolved in the aqueous layer (ensuring they do not precipitate in alcohol
along with DNA). Sodium chloride also used to neutralize the charge of DNAs sugar
phosphate backbone, thus making the DNA less soluble in water (less hydrophilic). In the
sodium chloride solution; sodium (Na) is an ion with positive charge. Sodium chloride
molecule was separated into its element; sodium and chloride. Since DNA have high
negative charge (because each nucleotide of the DNA molecule holds a negatively-charged
phosphate group), they were repelled due to their similar negative charge. So its neutralized
by the Na+ ions in the NaCl solution, making the separate DNA molecules became together
(attracted with each other) and become visible. Sodium chloride possesses this ability due to
separated Na and Cl ions. The Na+ will attracted to negatively-charged of DNAs phosphate
group. Addition of sodium chloride also allowing DNA to precipitate in alcohol. Without salt
addition, the negatively-charged DNA will not be precipitated.
Its usually the addition of detergent that causes DNA to expand away and cause the
viscosity of the solution to increase (Robertson, Ross, & Burgoyne, 2002). Commonly,
adequate concentrations of mild (i.e.: nonionic) detergents compromise the matrix of cell
membranes, in that way assisting lysis of cells and removal of soluble protein, regularly in
innate form (Detergents for Cell Lysis and Protein Extraction, n.d.). Detergent contains
sodium laurel sulfate, which in cleaning dishes, it removes fats and proteins. As in DNA
extraction method, it removes lipid and proteins (the one that make up the membranes
surrounding the cell and nucleus) by pulling them apart. In detail, the detergents interrupt the
lipid wall by solubilizing proteins and disrupting the lipid-lipid, protein-protein and protein-lipid
interactions.

When they (these membranes) are broken, the DNA were released. The

quantity of detergent required for ideal protein extraction be determined by the CMC (critical

micelle concentration), aggregation number, temperature and nature of the membrane, and
the detergent (Detergents for Cell Lysis and Protein Extraction, n.d.).

9.0

Conclusion

In a nutshell, it can be concluded that were able to extract, isolate and observe the fruits
DNA. The membranes of the cells have been successfully broken down and their nuclei
released to free the DNA. A good extraction method for DNA isolation should produce
adequate and intact DNA of reasonable purity. Extraction is the most important step in
isolating different types of bioactive compounds from fruits and vegetables (Yanga, Jianga,
John, Chenc, & Ashrafd, 2011). The procedure should also be rapid, simple and economical
(UKWUBILE, 2014). The white clump can be deduced as an almost-success result, though
its a bit cloudy. Extending time of alcohol and mixture together can improve the results
appearance. Another caution that need to be taken is note that high concentrations of NaCl
may hinder enzyme activity; therefore, DNA solution purified by means of this method should
be deposited and washed with 70% ethanol (generally, its an alcohol with certain
concentration) to remove residual salt (Cheng, Guo, Yi, Pang, & Deng, 2003).

10.0

Recommendations

i.

Run the same experiment using different fruit to observe the difference in their DNA

ii.

appearance (if theres any).

Strawberry can also be used because it yields more DNA than any other fruits and
they are octoploid; having eight copies of each type of DNA chromosome. (Human

iii.

cells are generally diploid; having two sets of chromosomes.)

Allocate more time for each step. Ensure that the detergent sit for at least five
minutes before adding alcohol. Let mixture of oranges and alcohol sit for more than

iv.
v.

half an hour.
Dont add too much water to fruit mixture. The cell soup should be opaque.
We can run few tests to determine the concentration and quality of the obtained
DNA. For instance, optical density readings taken by a spectrophotometer can be
used to determine the concentration of DNA (Biotech Learning Hub, 2009).

11.0

References

Biotech Learning Hub. (2009, June 18). Retrieved

http://biotechlearn.org.nz/themes/dna_lab/dna_extraction

from

DNA

extraction:

Brooker, R. J. (2012). Genetics: Analysis and Principles (4th ed.). New York: McGraw-Hill.
Chen, H., Rangasamy, M., Tan, S. Y., Wang, H., & Siegfried, B. D. (2010). Evaluation of Five
Methods for Total DNA Extraction from Western Corn Rootworm Beetles. PLoS
Journals, 6.
Detergents for Cell Lysis and Protein Extraction. (n.d.). Retrieved from ThermoFisher
Scientific:
https://www.thermofisher.com/my/en/home/life-science/proteinbiology/protein-biology-learning-center/protein-biology-resource-library/pierceprotein-methods/detergents-cell-lysis-protein-extraction.html
Genetic Science Learning Center. (2016). Retrieved from Why Am I Adding Detergent?:
http://learn.genetics.utah.edu/content/labs/extraction/howto/detergent/
Klug, W. S., Cummings, M. R., Spencer, C. A., & Palladino, M. A. (2012). Concepts of
Genetics (10th ed.). California: Pearson.
Robertson, J. R., Ross, A. M., & Burgoyne, L. (2002). DNA In Forensic Science: Theory,
Techniques And Applications. CRC Press.
Squishy Science: Extract DNA from Smashed Strawberries. (2013, January 31). Retrieved
from Scientific American: http://www.scientificamerican.com/article/squishy-scienceextract-dna-from-smashed-strawberries/
Taylor, S. (n.d.). Why Is Sodium Used in DNA Extraction? Retrieved from eHow:
http://www.ehow.com/about_6504902_sodium-used-dna-extraction_.html
UKWUBILE, C. A. (2014). Genomic DNA extraction method from Annona senegalensis Pers.
(Annonaceae) fruits. African Journal of Biotechnology, 750-753.
Yanga, B., Jianga, Y., J. S., Chenc, F., & Ashrafd, M. (2011). Extraction and pharmacological
properties of bioactive compounds from longan (Dimocarpus longan Lour.) fruit A
review. Food Research International, 18371842.

12.0

Appendices

10