DNA Extraction of Ananas comosus (Pineapple)

Republic of the Philippines

POLYTECHNIC UNIVERSITY OF THE PHILIPPINES
Office of the Vice President for Academic Affairs
College of Science
Department of Biology

DNA Extraction of Ananas comosus

(Pineapple)
Glenver O. Arbuis
Angelic Kayte Cabacungan
Cristina Marie M. Fadrilan
Judy Anne V. Galiza
Janelan M. Martin
Rejoice Santelices

ABSTRACT
All living things, plant or animal, contain a molecule called DNA or deoxyribonucleic acid

which can be found inside the nucleus of a cell. DNA contains the genetic information for the

development and growth of an organism. DNA extraction is one of the most basic techniques to

isolate the DNA in the cell’s nucleus. Ananas comosus (Pineapple) was used for the extraction

of its own DNA. The procedures of this experiment is divided into three steps namely extraction,

dilution and spectrophotometry. With the results, separate layer was formed on top of the

pineapple liquid after adding 95% cold ethanol. After a minute of observation, white fluffy cloud

at the interface between the two liquids became visible. This white precipitate was the DNA

extracted from pineapple. On the other hand, DNA purity were observed using

spectrophotometer. Results have shown that Group A has the lowest absorbance ratio (ABS

Ratio) of 0.8097 while Group D obtained the highest ABS ratio of 1.0577. In terms of DNA and

protein concentration, Group A obtained the highest value of 7.2650 and 456.83, respectively,

while on the other hand Group E has the lowest value of 1.9596 and 49.785, respectively.

Additionally, higher A2 (280 nm) can yield to higher protein concentration, less purity, and more

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contaminated (Groups A, B, C). In that, less amount of salt was added during the laboratory

activity since majority of the groups got high protein concentration. With regards to the purity of

the DNA samples, Group D and E have a good extracted sample because the ABS ratios

recorded were close to 1.7-2.0.

INTRODUCTION
Deoxyribonucleic acid is a molecule made out of two chains that coil around one another

to form a double helix structure. DNA is present in all living organisms, plant or animal, and is

located inside the cell’s nucleus. It plays an important role in synthesis of protein and it carries

genetic information for the development, growth and proliferation of every single known

organism and viruses (Albert et al., 2002).

DNA isolation is one of the most essential techniques in studying the DNA itself. DNA

extraction and purification plays important role for the advancement of biotechnology and

forensics (Adnan, 2010). Those are the prior steps in analysis of DNA used by the scientist to

detect genetically inclined disorder, produce finger DNA by the means of biometrics, and create

genetically modified organism that can provide beneficial products such as antibiotics, insulin,

and hormones (Jie, 2018).

According to Alaska BioPREP Virtual Textbook (n.d.), basic steps of DNA extraction are

1) lysis, 2) precipitation, and 3) purification. Lysis uses detergents and enzymes such as

Proteinase K to free the DNA and dissolve cellular proteins. In addition, precipitation separates

DNA from this cellular debris and alcohol plays an important role for DNA to precipitate out of

the aqueous solution because it is not soluble in alcohol. And for purification, it is the process of

rinsing with alcohol to remove any remaining unwanted material and cellular debris.

In this laboratory experiment, the students used Ananas comosus (pineapple) as a

sample for the extraction of DNA. Furthermore, they were also able to test the purity and

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DNA Extraction of Ananas comosus (Pineapple)

concentration of A. comosus’ DNA using a spectrophotometer. This experiment aimed the

students to know how to isolate DNA in a plant specimen and also to attain knowledge

regarding the importance of DNA in all living organisms.

METHODOLOGY

This chapter will discuss the procedures taken by the students aiming to extract DNA

from pineapple using the given methods and measure the absorbance and its ratio, the DNA

concentration, and protein concentration of the dissolve DNA.

Materials

The tools and equipment were borrowed from the school laboratory storage and other

supplies were bought by the students. The table below shows the materials needed to execute

the experiment.

Table 1. Supplies and its Function

SUPPLIES FUNCTION

Pineapple Fruit to be extracted

5-7ml of joy dishwashing liquid Destroys the plasma membrane

250ml Distilled water Medium

100ml of ice cold alcohol ( 95% ethanol) Enables the DNA to precipitate

1-2 table spoons of iodized salt Makes the DNA less hydrophilic

50ml of ice tubes Allows the solution to be at cold temperature

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Table 2. Tools and its Function

TOOLS FUNCTION

Beaker Storage for the medium

Handling the DNA without mixing it back to the

Inoculating loop
solution

Glass dropper Extraction of the product when it is visible

Watch glass Drying the DNA sample

Test tubes Procuring small batch of solutions

Sample holder in spectrophotometer specially

Cuvette
made from quartz glass.

Ice bucket Container for the ice bath.

Table 3. Equipment and its Function

EQUIPMENT FUNCTION

Blender To destroy the cell wall

Equipment for measuring the solutes by

Spectrophotometer (Shimadzu uv-1800) measuring the light absorbed by the solution in

the solution

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Procedures

Although the methods used for DNA extractions varies somewhat, the goals remain the

same: release DNA by destroying cell wall and cell membrane of the cell, and to precipitate

DNA out of solution so that it can be seen. Common components of a DNA extraction lab

include: Alcohol, Detergent or dishwashing liquid will do, and salt. The procedures of the

experiment is divided into three steps namely extraction, dilution and spectrophotometry.

Extraction

The students chilled the distilled water and alcohol in the freezer for later purpose.

Simultaneously, the whole pineapple was peeled off and only 2/3 will be used. It was cut

into small pieces and immediately put it in the blender together with ice and slowly blends

it until it turns to fruit mush or looks like a pineapple shake. Once done, add the extraction

solution made from 5-7ml of dishwashing liquid and a table spoon of iodized salt. Adding

salt (NaCl) to a solution containing DNA neutralizes the negative charges of the DNA

molecule, making the separate DNA molecules more likely to collect together and become

visible. Salt is able to do this because the sodium and chloride ions separate in solution

and the positive sodium ions (Na+) are attracted to the negative charges of the DNA’s

phosphate groups, thus neutralizing the negative charge. Because each nucleotide of the

DNA molecule possesses a negatively charged phosphate group, DNA is a negatively

charged molecule. Therefore, DNA molecules are not attracted to each other but are

repelled by the like charge they possess that is why the students were added salt to the

pineapple mush. In this step the detergent breaks down the cell membranes in which it

contains sodium laurel sulfate, which cleans dishes by removing fats and proteins. It acts

the same way in the DNA extraction protocol, pulling apart the lipids and proteins that

make up the membranes surrounding the cell and nucleus. Once these membranes are

broken apart, the DNA is released from the cell. The salt removes proteins that are bound

to the DNA (Fig. 1). Cold water was added to remain the temperature of the solution low.

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DNA Extraction of Ananas comosus (Pineapple)

Since using ice-cold water will increase the yield of DNA. Leave the homogenized solution

in the blender for about 20 minutes until the bubbles will make it to the surface of the

solution. Next, the bubbles were skimmed on top of the homogenized solution and the

solution was put to a beaker and then finally transferred to five (5) test tubes. Ideally, it is

better if there is a sieve to filter out the unwanted things in the solution like the unblended

stuffs of pineapple that are irrelevant to the experiment. It has also salt in it that provides a

suitable environment for the cells, begins to break apart proteins in the cell, and later,

helps the DNA separate from the other cell components to be able to see it. The chunks

that fall to the bottom of the container were proteins and other cell fragments clumping

together with the help of the salt. This makes the DNA solution a little cleaner. At this

point, the DNA and some bunch of other cell parts, proteins and lipids floating around in a

solution that is mostly water; is called an aqueous solution. Now, it is ready to precipitates

the DNA, remove the alcohol in the freezer. The students were tilted the test tube

containing the pineapple mush and carefully poured the cold alcohol inside of the test tube

because by doing this it will not mix with the solution. NOTE: at this point do not shake the

test tube. The alcohol should be at two times the volume of DNA solution (Fig. 2). After

pouring alcohol on it, the test tube was put on the ice bucket containing ice to regulate the

coldness of the solution, then after around 10 minutes, using the inoculating loop the

alcohol was stirred part of the test tube, make sure that the pineapple mush below is

undisturbed. While stirring the alcohol part, white particles start to come out and that is the

DNA of the pineapple (Fig. 3). DNA is soluble, or dissolves in water, but it is insoluble in

alcohol, so the DNA that touches the alcohol at the interface. The cold alcohol helps the

DNA precipitate (solidify and appear) more quickly. Salty water helps the DNA precipitate

(solidify and appear) when alcohol is added.

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Figure 1. Pineapple mush with Figure 2. Pineapple mush with Figure 3. DNA of pineapple

salt and liquid detergent 95% ethanol (white precipitate)

Dilution

The students used the inoculating loop to stir the alcohol part of the test tube to

separate it and utilizing the glass dropper to sip the white particles which are the DNA of

the pineapple (Fig. 4). After getting some DNA, carefully released it on the watch glass

and put on the electric fan, wait until there is no liquid on the watch glass (Fig. 5). Two test

tubes were prepared; one has the extracted DNA with 5mL distilled water and the other

test tube with 5 mL distilled water only. Then, the DNA was diluted and make sure that

there are no visible particles (Fig. 6). And lastly, it was transferred on the cuvette.

Figure 4. Sipping the DNA on Figure 5. Transferring to watch Figure 6. Test tube with mixed extracted DNA
and 5mL distilled water (left) and test tube with
test tubes glass to dry it up 5mL distilled water only (right)

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Spectrophotometry

The students poured the diluted DNA solution in a sample holder called cuvette and

poured distilled water to another cuvette as a reference blank that is also found in the

sample solution except the substance the students are trying to analyze or measure. In

this lab experiment, the students were measured the absorbance of the DNA that was

dissolve in distilled water (Figures 7 and 8). The reference blank in this case would be

water alone. Because other compounds in a solution (or the solvent itself) may absorb the

same wavelengths as the compound being analyzed, students were compared the

absorbance of our test solution to a reference blank.

Before turning ON the power switch, check to be sure that nothing is placed in the

sample compartment and cell holder. Keep the sample compartment cover closed during

measurement or 100 %T (0 Abs) correction (Figures 9, 10 and 11). Any outside light

detected on the spectrometer may interfere with accurate measurement and correction.

When the instrument power was turned ON, the UV-1800 starts executing various checks

and initializations. The students let their professor do the start up process of

spectrophotometer. The mode menu screen in the spectrophotometer was displayed.

The Bio-Method function is selected. As selected, [7. Bio-Method] in the [Mode

menu] screen, the screen for selecting a quantitation method appears. Select the item

number for DNA quantitation [DNA Quantitation]. Measurement was performed at

specified two to three fixed wavelengths and obtained the concentration of DNA and

proteins, and absorbance ratio, based on the measured absorbances. The students can

choose any wavelength for measurements, including the most commonly used

wavelengths (260 nm/ 230 nm or 260 nm/280 nm). But in this experiment the 260nm/ 280

nm was selected. The students did also set the desired parameters for calculating the

DNA and protein concentrations in samples. After it was set to desire wavelength, it is

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ready to start the measurement. The measurement screen displays the sample No.,

absorbance values A1, A2, and absorbance ratio A1/A2 as well as DNA and protein

concentrations. The students were recorded the measurements results and made a table

to compare the data with the other groups.

Figure 7. Pouring distilled water Figure 8. Diluting Solution Figure 9. Powering On the
into the cuvette Spectrophotometer

Figure 11. Reference blank sample

Figure 10. Placing the cuvette
inside the compartment in a cuvette
into the sample holder
holder

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RESULTS AND DISCUSSION

This chapter presents the observations and results of the laboratory experiment followed

by a discussion of the experimental findings. DNA was extracted from Ananas comosus

(Pineapple) and tested using a spectrophotometer. Then, the following results were obtained.

Separate layer was formed on top of the pineapple liquid after adding 95% cold ethanol.

After a minute of observation, white fluffy cloud at the interface between the two liquids became

visible as shown on Figure 1. This white precipitate was the DNA extracted from pineapple.

Figure 12. White precipitate appeared in the test tubes.

One of the methods of measuring DNA purity and concentration is absorbance (optical

density) which is measured using spectrophotometer. Data of different groups were obtained

and in terms of Absorbance Ratio (ABS Ratio), Group A has 0.8097 (lowest) and Group D has

1.0577 (highest). In addition, Group A has the highest value of DNA and protein concentration

(7.2650 and 456.83, respectively) and Group E has the lowest value (1.9596 and 49.785,

respectively).

According to promega.com, absorbance readings are performed at 260nm (A260) where

DNA absorbs light most strongly. Oxford Gene technology (2011) added that it is common for

nucleic acid samples to be contaminated with other molecules like proteins. Specifically, the

amino acids tyrosine and tryptophan have a very specific absorption at 280 nm, allowing direct

A280 measurement of protein concentration (The Protein Man, 2016). In that case, the

absorbance ratio (A260/280) will tell how purity the DNA samples are. And with the results, Groups

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D and E have considered of having a good extracted DNA samples since the ratio recorded was

near in A260/280 ratio of 1.7-2.0 which is according to promega.com, it is considered as good-

quality DNA. On the other hand, higher A2 (280 nm) can yield to higher protein concentration

less purity, and more contaminated (Groups A, B, C).

According to Sweeney (n.d.), sodium chloride helps to remove proteins that are bound to

the DNA. It also helps to keep the proteins dissolved in the aqueous layer so they don’t

precipitate in the alcohol along with the DNA. In that, less amount of salt was added during the

laboratory activity since majority of the groups got high protein concentration.

Table 1. Spectrophotometric Analysis of the DNA Samples Extracted from Ananas

comosus (Pineapple)

A1 A2 DNA Protein
Group ABS Ratio
(260.0 nm) (280.0 nm) Concentration Concentration

A 0.3940 0.4866 0.8097 7.2650 456.83

B 0.2471 0.2871 0.8607 5.2070 258.45

C 0.2834 0.3399 0.8338 5.5895 312.91

D 0.0733 0.0693 1.0577 2.1158 52.044

E 0.0687 0.0656 1.0473 1.9596 49.785

Note: HIGHLITHED WITH BLUE- Spectrophotometric Analysis of DNA samples of the Authors

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REFERENCES

Alberts, B., et al. (2002). Molecular Biology of the Cell, 4th. ed. Ch. 4. Garland Science: New
York. ISBN: 978-0815316206.

Adnan, A. (2010). DNA extraction: Procedure and Importance in Forensics. Retrieved from
https://www.biotecharticles.com/DNA-Article/DNA-Extraction-Procedure-and-Importance-
in-Forensics-389.html

Ma Wen Jie (2018). Uses of DNA extractions. Retrieved from https://sciencing.com/uses-dna-

extraction-5344428.html

The Basics of DNA Extraction. (n.d). Alaska BioPREP Virtual Textbook. Retrieved July 16, 2018
from https://bioprep.community.uaf.edu/learning-modules/2-dna-extraction-4/the-basics-
of-dna-extraction/

How do I determine the concentration, yield and purity of a DNA sample? (n.d.). Promega.
Retrieved July 14, 2018 from worldwide.promega.com
https://worldwide.promega.com/resources/pubhub/enotes/how-do-i-determine-the-
concentration-yield-and-purity-of-a-dna-sample/

Why Does Tyrosine and Tryptophan Have Effect In Protein Determination and to What Degree?
(2016). G-BIOSCIENCES. Retrieved July 14, 2018 from
https://info.gbiosciences.com/blog/why-does-tyrosine-and-tryptophan-have-effect-in-
protein-determination-and-to-what-degree

Understanding and measuring variations in DNA sample quality. (2011). Oxford Gene
technology. Retrieved July 14, 2019 from https://www.ogt.com/

Sweeney, D. (n.d.). DNA Isolation from Strawberries. Retrieved July 14, 2019 from
http://www.caseciw.org/first_light_case/horn/strawberries/strawbdnaproc.html

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