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Assignment - DNA Extraction from Strawberries or Kiwi Fruit
Q1. What does mushing the fruit do? (What does it break?)
Answer - Crushing breaks down strawberry tissues into its constituent cells and to break the cell walls to release the cell contents into the solution. This process is often referred to as homogenisation. The smaller particle size increases the surface are that is accessible for lysis solution. This ensure a greater number of cells are lysed in the later step of the extraction process.
Q2. What does the soap do to the fruit? (What does it break?)
Answer - The soap solution containschemicals such as sodium lauryl sulfate which is a detergent. Detergents present in soap solution can solubilise lipid. The plasma membrane of cell and cellular organelles such as nucleus that contain the DNA is composed of a lipid bilayer and embedded proteins. The detergent will solubilise the lipid bilayer causing the contents of cells viz. proteins, nucleic acid, carbohydrate etc, to be released into the solution. This process is called cell lysis.
Q3. What does the salt do? (Hint: It precipitates out some macromolecules we aren't interested in).
Answer - The lysed solution containsplenty of cellular proteins including enzymes that can interfere with quality and downstream application of isolated DNA and hence needs to be removed prior to DNA is extracted from the lysed solution. Proteins are a non-electrolyticsolute. Presence of high concentration of electrolyte ions can make non-electrolytes less soluble and precipitate them out of solution. Adding salts results in increase of ion concentration in the lysate. This makes the dissolved proteins less soluble and at sufficiently hogher concentration the protein gets precipitate out of the solution. This process is often referred to as salting out and is also used for extraction of proteins. Furthermore, the phophodiester backbone of DNA has uniform negative charge that makes it difficult for DNA molecules to come together. The addition of positive ions from the salt will also neutralise this negative charge of DNA backbone thus allow them to come together making the precipitation process easier.
Q4. Why do we use the ice water bath? (Hint: This has to do with enzymes).
Answer - The cytoplasm of the cell contains many enzymes some which can lyse nucleic acids including DNA thus can potentially reduce the yield of DNA. Activity of nucleases can result production of smeared DNA which is the result of random cleavage of DNA by the enzymes. The enzyme activity is known to be significantly reduced at lower temperature. Keeping the crushed solution in ice bath lowers the temperature of the solution and inhibits the enzyme activity. This keeps the DNA intact during extraction process.
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Q5. What is being filtered out? What is going through the filter?
Answer - The processes of crushing in addition to releasing the cells will create a lot of debris in the form of cell wall, fibres etc. Therefore, the homogenate is then filtered through a filter paper. The filtered-out part contains large particles such cell wall debris, fibres etc that can interfere with extraction process. Whereas the filtrate will contain the cells that are too small to be filtered out along with any dissolved solutes including carbohydrates, proteins, pigments etc.
Q6. What does the alcohol do? Why should it be cold?
Answer - The DNA molecule is surrounded by water molecules thereby keeping it in the solution. Alcohol removes the water molecule thereby allowing DNA molecules to aggregate together and precipitate out of the solution. The lower temperature of chilled ethanol aids better precipitation as solvents solubility decreases with decreasing temperature. In addition, lower temperature can prevent activity of any residual dissolved enzymes.
Q7. Where does the alcohol form its layer?
Answer - Since water is denser than alcohol the alcohol will float on top of water. In other words, the alcohol layer will be on the top. This phenomenon is called phase separation and is usually used for extracting compounds from solution. This prevents DNA being coprecipitated with other water-soluble compounds such as carbohydrates that will stay in the bottom aqueous phase.
Q8. Where in the layers does the DNA form?
Answer - DNA can be observed forming as hazy white strands resembling cotton with slimy texture in the alcohol phase on top making it easier to be removed from rest of the extraction solution via phase separation.
Q9. What does it mean to "precipitate" in this experiment and what is being precipitated?
Answer - Precipitation is the process of forming of solid solutes from a solution. The solute molecules in solution under specific conditions aggregate together to form precipitate. In the current the DNA dissolved in the lysis solution is precipitated using alcohol. This is caused by removal of hydration shell around individual DNA molecules when alcohol is added. The exposed DNA molecule in turn will aggregate together and when their aggregate size begins to exceed the intermolecular space of water, they get excluded out of the solution to form precipitate visible as hazy white strands.
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Q10. What happens to the DNA in the foods you eat?
Answer - Nucleic acids in the food is metabolised in the small intestine by enzymes such as endonucleases, phosphodiesterases and nucleoside phosphorylase. The DNA is degraded into constituent nucleotides or even bases that are then absorbed through intestinal walls for downstream metabolism.
Q11. Describe your DNA?
Answer - Following phase separation using alcohol, the DNA appeared as hazy white cotton like strands with slimy texture in the alcohol phase that formed on the top of the extraction mix. These could then be collected from solution by spooling them using wooden chopstick and then dissolved in suitable solvent.
Q12. Theoretically, what could you do with your DNA now? Investigate some possibilities on the internet.
Answer - The obtained DNA is in solid form and hence needs to bereconstituted insuitable solvent like TE buffer (pH 8.0) to ensure stability of the isolated DNA during long term storage. The shelf life of the DNA can be improved by storing them in deer freezer of refrigerator in sealed non-reactive containers. However, prior to any downstream application the quality and quantity of DNA in the solution needs to be assessed. This can be done through spectrophotometric analysis using instruments like NanoDrop. This will allow us to estimate the yield and purity of isolated DNA sample. In case the requisite equipment is not available a small volume DNA can be subjected to agarose gel to determine the quality of DNA and whether the same has suffered any damage during isolation process. Isolated DNA can as serve as source of cloning of important genes via PCR amplification. Furthermore, PCR and Restriction enzyme based analysis can be done to detect similarities or variation between various plants.
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