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DNA Extraction Assignment

DNA Extraction from Strawberries or Kiwi Fruit

Questions:
1. What does mushing the fruit do? (What does it break?)

When we crush the Kiwi or strawberry fruits, it helps in physical breaks down of the cell walls and helps the cytoplasm to leak out to release internal ingredients out in the suspension. Mushing helps the strawberry or kiwi to cause physical smashing of strawberry/kiwi cells and exposes a greater surface area and hence DNA isolation will be easier.

2. What does the soap do to the fruit? (What does it break?)
During mechanical mushing of fruits, cell walls was disrupted and soaps present in shampoo or detergent helps in the extraction of DNA as it broke down the fatty membrane and open up the nuclear membrane to release the DNA in the solution (as it helps dissolving lipids and proteins that hold the membranes together).

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3. What does the salt do?
The salt helps in formation of an environment which helps in neutralizing the negative charges of the DNA and the positively charged ion (Na+) from the salt interacts with the negatively charged phosphates groups of nucleic acids. Hence enables the DNA strands to gather and stick with each other while the proteins whichare also released from the cells got separated from the DNA (as it causes proteins and carbohydrates to precipitate out). Hence, it will be easier to see them in the solution.

4. Why do we use the ice water bath?
Ice water bath is used for precipitation purpose in which DNA strands goes from higher energy state i.e. dissolved state to lower energy state i.e. solid precipitate and chilled temperatures helps in making this energy transition efficient and faster than normal temperatures as well as DNA will precipitate in higher yield in cold temperature. Another reason for using ice water bath is do inactive certain temperature sensitive DNAses or other DNA deactivating enzymes which is present in the solution to yield more amount of pure DNA.

5. What is being filtered out? What is going through the filter?
Filtering helps to remove precipitate and other particles from suspension and ensure that the solution is sterile which ultimately make DNA purification easier by minimizing the risk of DNA contamination. It also helps in filtering out of precipitated protein and carbohydrates from the suspension at this point.

6. What does the alcohol do? Why should it be cold?
DNA is insoluble in ethanol and pull out water from the DNA molecule due to which it collapses with itself and precipitate out from the other cellular ingredients. The DNA will become visible as white mucous strands that can be spooled with the wooden applicator stick. After precipitation, long slimy, gross, thread-like DNA clumps will be seen at the interface of the layer of alcohol and DNA solution. Ethanol has lower dielectric constant than water and initiate ionic bond formation between the Na+ (of salt) and the phosphates (PO3-) (DNA backbone) which dictated by Coulomb's law which cause DNA to precipitate. The interaction shields the charges of both ions and cannot make hydrogen bond with water molecules. Thus, this salt formation precipitates out of the ethanol solution. Colder temperature helps in deactivation of temperature sensitive DNA deactivating cellular enzymes and helps in decrease in contamination in solution.

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7. Where does the alcohol form its layer?
Strawberry long, thread-like DNA molecules will form it layer at the interface of the layer of alcohol and DNA solution. (Basically the upper layer of the solution will be of alcohol)

8. Where in the layers does the DNA form?
The strawberry/ kiwi's DNA will rise into the top layer i.e. alcohol layer and which can be identified by whitish thread like slimy, gunky and gross clumps of DNA.

9. What does it mean to "precipitate" in this experiment and what is being precipitated?
Precipitation is the process where by using alcohol and salt, DNA will get aggregated into the visible cotton thread like precipitate. DNA is hydrophilic in nature which dissolves in water but not in the alcohol. Salt and alcohol used in the DNA extraction protocol as they makes the DNA more hydrophobic as the negative charge of the DNA interact with the positive charge of the salt, instead of getting interacted with positive charge of the water. On the other hand, the water remain interacted with the negative charge of the alcohol which also cause DNA precipitation efficiently and hence there is increase in overall yield of the DNA precipitate.

10. What happens to the DNA in the foods you eat?
When we ate strawberries or kiwi, their DNA gets broke down into smaller molecules by enzymatic reactions in our metabolic processes and these smaller molecules of digested DNA which includes purines, pyrimidines, ribose, phosphates etc. are getting utilized for growth of cells and other energy utilization needs of our body (utilized for the salvage synthesis of nucleic acid) There are many supplements present in the market which contains DNA or RNA molecules which helps in recovering from surgery, to avoid Alzheimer's, sagging skin, decrease sexual drive, etc. So there will not any side effects to our body if we eat that DNA.

11. Describe your DNA.
Isolated DNA is of whitish cloudy, slimy, gross, long thread like appearance which is present in upper layer of alcohol and can be isolated by using any woodstick or pipette.

12. Theoretically, what could you do with your DNA now?
Strawberries or kiwi contains the DNA in large amount. So by extracting the DNA from the fruits, we can commercially produce any of the significant enzyme which could be useful for human's health. It will be useful in production of transgenic strawberries (Hanhineva, 2007). By manipulating the DNA at specific sites, we can cause significant change in the physiological properties of the fruit and plant which include size of the fruit, ripening time, disease resistant etc.

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