Analysis and Comparison of Dna Extraction from Actinidia Deliciosa and Fragaria Ananasa Fruits Using Youtube Method

Analysis and Comparison of DNA Extraction from Actinidia deliciosa and Fragaria ananasa fruits using YouTube Method

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Analysis and Comparison of DNA Extraction from Actinidia deliciosa and Fragaria ananasa fruits using YouTube Method.

Introduction

This report involves DNA extraction from Actinidia deliciosa (kiwi) which is a fruiting vine that originated from Southern China and Fragaria ananasa (strawberry) which is a hybrid of the genus Fragaria and is grown all over the world. The application of DNA technology in plants has been on the rapid rise for the past two decades. For instance, similar study on the fruits of Annona senegalensis was carried out by the Science and Development Institute of Nigeria in 2013 (Ramesh et al., 2013). They carried out various procedures im order to determine the best method of extracting DNA in the highest quality possible for purification to be done on it. Similar to this procedure, DNA extraction involved denaturing the cell membranes before centrifuging the specimens in various solutions in order to extract DNA and then performing gel electrophoresis on them. They however encountered similar setbacks in the separation of DNA from other substances such as RNA, proteins and polysaccarides in the fruit. This has been the constant problem in the extraction of DNA from plants. It creates the need for a method whereby there is step by step isolation of each component until DNA of highest quality (purity) amd quantity is obtained. The purpose of this experiment is therefore to try to extract and compare DNA of the highest and purest quality from both Fragaria ananosa and Actinidia deliciosa.

Method

For this experiment, two methods were used; the Youtube method and the salting out method. However this report shall only discuss the YouTube method (How to Extract DNA from Strawberries, 2010).

In the procedure, a sample of each fruit sample was placed in different Ziplock bags which are then totally sealed. Then the samples were squished until they became mushy for about 3 minutes. This was meant to expose the cell membranes of the inner tissues of the samples to the extraction procedure. 10 mL of DNA extraction buffer containing 9.5 mL water, 0.5 mL dishwashing agent and 2 teaspoons of table salt was pipetted into the squished samples. The purpose of the detergent was to break down the cell walls of the samples' cells in order to release DNA as well as separate polysaccharides from the DNA. The salt on the other hand provided a cheap method of preventing the polysaccharides from co-precipitating alongside the DNA during the latter stages of the experiment (Robert, 2008). The mixture was then squished for a further 2 minutes to allow the solutions to mix consistently with the samples for the reactions to take place.

Once completed, each mixture was filtered through a Chux cloth into a conical flask via a funnel. It was important to do this in order to obtain quality buffered samples while eliminating the possibly unaffected parts of the specimens which may have affected the quality of the experiment going forward. Using an auto-pipette, the samples were transferred into separate 15 mL tubes, 3 mL each. The samples were then named appropriately at this stage by labeling each tube (for example strawberry) alongside the initials of the person who conducted the procedure. 10 mL of pure, cold ethanol was then added to the samples. It had to be poured down the side of the tube and not directly above in order to ensure that it formed a separate layer on top of the sample. The new mixture was then left on ice for about 10 minutes while observations of precipitation were being made.

As much precipitated DNA as possible was carefully removed using a hook and placed into a labeled micro - centrifuge tube with a volume of 1.5 mL. This tube contained 1 mL of 70% ethanol. The tube was then placed into a micro - centrifuge which was operated at top speed (12000 rpm) for about 3 minutes. The resultant supernatant was poured into a liquid discard beaker. The purpose of this centrifuging was to precipitate the nucleic acids below the supernatant. The tube had to be then placed upside down onto a paper towel in order to evaporate the ethanol which would have otherwise prevented DNA from dissolving during gel electrophoresis. The appearance of the DNA pellets at this stage was noted and compared with those from the salting out procedure.

Using an auto-pipette, about 300 μL was added into the tube and mixed thoroughly. In case the pellets failed to dissolve, a maximum of 100 μL would be added to the mixture. The tube was then heated at 550C for 5 minutes. 10 μL of the resultant sample micro - centrifuge tube with 2 μL of 6 × loading dye containing 30% glycerol, 0.25% bromophenol blue and 0.25% xylene cyanol FF. Next the mixture was loaded into agarose gel containing 1% agarose, TAE buffer, ethidium bromide, with a concentration of about 0.5 μg / L. Every individual sample's position on the loading guide was noted as well as the appearance of the pellet. Finally, the gel was connected to a power source and observed until it completed its run. A picture of the resultant DNA image was taken for result analysis.

DNA was also extracted from strawberry and kiwi fruit using a salting out technique according to the method of the Cell and Genes Practical Manual (School of Life and Environmental Sciences (LES), 2016).”

Results

When cold 100% ethanol was poured on top of the extract, a cloudy layer of concentrated DNA formed at the interface between the fruit extract and the ethanol as shown below:

[pic 1]

Figure 1: Kiwi DNA

[pic 2]

Figure 2: Strawberry DNA

The DNA for Actinidia deliciosa appeared to be clear, clumped string-like pieces while the DNA for Fragaria ananosa appeared to be long, clear string-like pieces.

Below are the images of the gel electrophoresis for the YouTube method: [pic 3]

[pic 4]

Figures 3 & 4: Gel Electrophoresis Images for Youtube Method

When compared to the salting out method, we see that the YouTube method mostly did not provide clear results. The results for the salting out method were as shown:

[pic 5]

[pic 6]

Figures 5 & 6: Gel Electrophoresis Images for Salting Out Method

Discussion

From the results above, we see that the YouTube method did not provide clear results as compared to the salting out method. We can attribute this to the fact that the YouTube method uses a lower concentration of salt than salting out. As noted earlier, a high concentration of salt enables precipitation of DNA at the expense of polysaccharides present in the samples (Robert 2008). On addition of pure ethanol, the polysaccharides dissolve due to their increased solubility as a result of the salt. It is therefore possible that the DNA precipitated was still contaminated by the polysaccharides hence the numerous gaps on the images of the electrophoresis.