Post-Lab Questions 1. The Procedure calls f or vigorous grinding of the onion ce
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Question
Post-Lab Questions 1. The Procedure calls f or vigorous grinding of the onion cells with sand. Would you be able to isolate the DNA in the cells if this step were omitted? What is the purpose of the grinding process? 2. In the experiment, how did you minimize the interaction between the DNA and histones (proteins)? 3. What compounds remain in the ethanol solution after the DNIA is removed by spooling onto a glass rod? 4. Could you do without using the detergent in the procedure? Explain your answer. 5. Is the diphenylamine reagent able to distinguish between ribose and deoxyribose? Could it also distinguish between DNA and RNA? 6. What is the structural difference between glucose and ribose? Did the diphenylamine test work for the glucose solution?Explanation / Answer
Ans. #1. The primary purpose of grinding is to disintegrate the tough cell wall of plant cells. Grinding also disrupts the membranes of cell and its organelles including nucleus. The disintegration of cell wall and membranes leads to the release of nucleic acids (DNA. RNA) and other cellular contents in the aqueous medium of grinding. Therefore, grinding facilitates extraction of DNA from cell.
# If grinding is the ONLY method of disrupting cell wall and membranes, omitting grinding would NOT lead to DNA extraction.
However, if other reagents like cellulase, proteases, SDS, etc. are used for DNA extraction, the omission of grinding still gives positive result, though with a lower yield.
#2. DNA is negative charged due to presence of phosphate groups. The histone proteins are positively charged due to presence of basic amino acids. The positively charged histones interact with negatively charged DNA with ionic (non-covalent) interactions.
Addition of high salt concertation like 5M NaCl disrupts the ionic interaction between histones and DNA and cause their dissociation. So, treatment of NaCl minimizes interaction between histones and DNA.
Addition of detergents like SDS denatures proteins. So, denaturation of histones with SDS also minimizes interaction between histones and DNA because denatured Histones don’t bind to DNA efficiently.
#3. Till the step DNA is extracted and ready to be spooled out, all the other class of biomolecules including carbohydrates proteins and lipids are removed from the cell extract. There remains only the nucleic acids (DNA, RNA) in the final solution to be spooled out.
Spooling out DNA leaves behinds RNA and smaller fragments of DNA which generally do not form precipitate upon addition of chilled ethanol.
So, after spooling, few small fragments of DNA and RNA are left behind in the solution.
#4. It depends on the procedure
Detergents has following roles- it disintegrates the lipid bilayer of cell and organelles, solvates lipids, and precipitates the proteins.
Option I: Yes: If phenol is used, the DNA extraction can be accomplished (though with lower yield) because phenol treatment removes lipids and proteins. The lipid bilayer can be disintegrated distilled water (hypotonic).
Option II: No. If neither of detergent and phenol treatment is not used, DNA can’t be extracted. Because presence of protein as contaminate would mask the DNA purification processes.
#5. Yes. Diphenylamine (Dische diphenylamine test) reacts with deoxyribose sugar forming a blue color. It does not react with ribose sugar, so the solution remains colorless.
# Yes. DNA has deoxyribose sugar whereas RNA has ribose sugar. Due to differential reaction of diphenylamine with deoxyribose sugar only, DNA gives a positive result (blue color) whereas RNA gives a negative result.
#6. Glucose is a hexose (6C) aldose sugar. Ribose is a pentose (5C) aldose sugar.
Diphenylamine test does not work with glucose because it does not have a “deoxy” carbon in it.