Essay On DNA Extraction from a Kiwi Fruit

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DNA Extraction from a Kiwi Fruit

Deoxyribonucleic acid (DNA) is a long molecule present in nuclei of cells every living organism. Since DNA exists inside of the cell nucleus, proteins have to be denatured in order to locate the DNA. Learning how to extract and visualize DNA from organisms is a basic procedure in molecular biology. Extracted DNA is used to establish DNA fingerprints which can be used in diagnosis of genetic diseases, establish maternity or paternity, and solve criminal cases. Extraction of DNA is also required in order to make a DNA code sequence of different living organisms (American Phytopathological Society, 2015). In this practical, a designed procedure has been followed using common household materials to extract DNA from kiwi fruit cells in sufficient quantity to be visualized using gel electrophoresis under the microscope. The aims of this practical study were to learn and use scientific methods to extract and observe DNA from a kiwi fruit, and to present the results as a scientific report.
Materials and methods
Materials
1-litre Ziploc bag
Freshly cut pieces of kiwi fruit
DNA extraction buffer
Cheese cloth
Paper towel
Tape
Hook
Ice
Fluorescent die (ethidium bromide)
70% ethanol
Transfer pipettes
Two 500 Ml beakers
1.5 mL Eppendorf tube
1 test tube
A micro-centrifuge
A heat block
Methods

Procedure for extraction of DNA

A piece of kiwi fruit was squished in a tightly sealed Ziploc bag for about 3 minutes before adding 10 mL of DNA Extraction Buffer using an auto-pipette and further squished for another 2 minutes. The mixture obtained was filtered through a Cheese cloth and funnel and collected in a conical flask. After dripping had stopped, 3 mL of the sample was transferred into a labeled tube and placed on ice before adding 10mL of cold ethanol into the sample. The tube was left on the ice for about 12 minutes as the reaction proceeded. The DNA was then removed using a hook and placed into a labeled Eppendorf tube containing 70% ethanol. The tube was placed into the micro-centrifuge set at 12000 rpm for about 3 minutes; then the supernatant carefully poured off into the discard beaker. The tube was then placed on the paper towel facing upside down, and left to drain for about 5 minutes. Using an auto-pipette, 300 L of distilled water was placed into the tube and thoroughly mixed to ensure that the DNA dissolved. Finally, the tube was placed on the heat block at a temperature of 55oC for 5 minutes, then placed on ice before loading the sample on an agarose gel.
Procedure for visualizing the DNA

An appropriate amount of gel loading buffer was added to the sample before the agarose gel was loaded by the demonstrator. The gel was then connected to the power source and then left to run until complete. The demonstrator then obtained the image of the DNA.

Results

Naked Eye
After filtering the mixture of the kiwi extract and extraction buffer, a clear yellow liquid was obtained. When alcohol was added into the tube containing a mixture of the kiwi and extraction buffer, some changes were observed; the alcohol formed a layer on the fruit extract. A white jelly-like substance was formed between the two layers. When the DNA was hooked out, it felt like a jelly, very sticky and condensed. On centrifuging the DNA, it precipitated to form a pellet at the bottom of the micro-centrifuge tube. The pellet got dissolved on addition of distilled water

Microscope

When the DNA material was observed under the objective lens at X100, it appeared as mass of spiral fibers entangled together. Using immersion oils and observing stained slides at the peripheral sides provides a clear image of the DNA material, showing beaded strings in spiral forms.
Visualization
On running the DNA extract on an agarose gel, the DNA was visualized using Ethidium bromide. The images shown below were obtained from the transilluminator.

Discussion

The kiwi fruit contain lots of cells and consequently, lots of DNA is found within the fruit cells. The gel results shown in figure 1 and 2 above show the molecular ladder of the kiwi DNA and the bands obtained. In the extraction process, an enzyme present in the detergent breaks the fatty cell wall of the fruit cells, releasing the DNA from where it is located. Adding cold ethanol to the fruit extract enables the suspension of the DNA molecules onto the surface, making it easy to collect the molecules. Everything in the fruit extract dissolves in ethanol except the DNA. The alcohol pulls water from the molecules of the DNA, making it to collapse and precipitate. The precipitate is seen as white stringy jelly-like substance. The DNA fragments are separated according to size when examined under agarose gel electrophoresis (Bowater & Yeoman, 2012). The DNA molecules migrate from the cathode to the anode due to its natural negative charge. This negative charge is as a result of sugar phosphate backbone in a DNA molecule. These DNA fragments can be clearly visualized by naked eyes or under a UV light for easier visualization. The spiral form of the molecule can be visualized using a powerful microscope. The molecular weight marker provides a logarithmic scale that can be used to estimate the size of the DNA fragments that run at apparent size on agorose gel (Gullino & Bonants, 2014).

A single enzyme cut linearizes the plasmid. The cut plasmid needs to be pulled through the holes for it to move through the gel. It is easier to pull through a smaller object than a larger one. Consequently, the linearized plasmid cannot migrate at the same rate through the gel as the non-linearized plasmid. The linearized pBluescript plasmid is expected to run at 3.0 kilobases (kb) on the agarose gel. When the plasmid is digested with an enzyme present in the pBluescript plasmid, such as Bam HI site (which is found in the multiple cloning site near the Eco RI site), the restriction enzymes will cut the plasmid into pBluescript part and the insert to form two fragments of DNA. The sizes of these fragments as determined by electrophoresis on an agarose gel shown in figure 2 are 3.6 kb for pBluescript and 1.4 kb for the insert. In conclusion, the DNA molecules were successfully extracted from the kiwi fruit. The isolation protocol used in this experiment represents the basic scientific procedure used in extraction of DNA. However, to obtain a pure DNA, further purification may be required.

References

American Phytopathological Society 2015, Classroom Activities in Plant Biotechnology, retrieved 10 December 2015,
Bowater, L. & Yeoman, K. 2012, Science Communication: A Practical Guide for Scientists, John Wiley & Sons, London.
Gullino, M. L. & Bonants, P. J. M. 2014, Detection and Diagnostics of Plant Pathogens, Springer, New York.