BACE - Biotechnician Assistant Credentialing DNA Extraction and Analysis Questions and Answers 1 — Questions and Answers
Question 1: During agarose gel electrophoresis, what is the primary reason that DNA fragments migrate towards the positive electrode?
- The agarose gel matrix is positively charged.
- The phosphate backbone of DNA carries a strong negative charge. (Correct answer)
- The buffer system creates a pH gradient that attracts the DNA.
- Smaller DNA fragments are naturally drawn to a positive pole.
Correct answer: The phosphate backbone of DNA carries a strong negative charge.
DNA has a net negative charge due to the phosphate groups in its sugar-phosphate backbone. When an electric current is applied to the gel, the negatively charged DNA molecules are repelled by the negative electrode (cathode) and attracted to the positive electrode (anode), causing them to migrate through the gel.
Question 2: A biotechnician is performing a plasmid DNA extraction from E. coli using a spin-column based kit. The final step involves adding an elution buffer to the silica membrane. What is the primary purpose of this step?
- To precipitate the plasmid DNA out of the solution.
- To wash away any remaining proteins and salts.
- To release the bound plasmid DNA from the silica membrane. (Correct answer)
- To digest any remaining RNA in the sample.
Correct answer: To release the bound plasmid DNA from the silica membrane.
In spin-column based DNA extraction, DNA binds to the silica membrane under high-salt conditions. The final step uses a low-salt elution buffer to decrease the affinity of the DNA for the silica, allowing the purified DNA to be released from the column and collected.
Question 3: Which of the following is a critical step for precipitating DNA from an aqueous solution after cell lysis and removal of cellular debris?
- Adding a detergent to the solution.
- Adding ice-cold ethanol or isopropanol. (Correct answer)
- Heating the solution to 95°C.
- Vortexing the sample vigorously for 5 minutes.
Correct answer: Adding ice-cold ethanol or isopropanol.
DNA is soluble in water but insoluble in alcohols like ethanol or isopropanol, especially in the presence of salt. Adding ice-cold alcohol causes the DNA to precipitate out of the aqueous solution, making it visible and allowing it to be collected by spooling or centrifugation.
Question 4: After running a DNA sample on a 1% agarose gel, a technician observes a single, very bright band located very close to the loading well. What is the most likely interpretation of this result?
- The sample contains very small DNA fragments.
- The electrophoresis was run for too long.
- The sample contains high molecular weight, undigested genomic DNA. (Correct answer)
- The incorrect buffer was used to make the gel.
Correct answer: The sample contains high molecular weight, undigested genomic DNA.
In agarose gel electrophoresis, larger molecules move more slowly through the gel matrix. A bright band remaining near the well indicates a high concentration of large DNA molecules, such as intact genomic DNA, which have not been fragmented and have struggled to migrate into the gel.
Question 5: A technician needs to isolate DNA from a tough plant tissue sample for PCR analysis. Which of the following steps is most likely required at the beginning of the extraction protocol for this sample type, but not for a bacterial cell culture?
- Addition of a lysis buffer containing detergents.
- Centrifugation to pellet the cellular material.
- Precipitation of DNA using isopropanol.
- Mechanical disruption, such as grinding in liquid nitrogen. (Correct answer)
Correct answer: Mechanical disruption, such as grinding in liquid nitrogen.
Plant cells have a rigid cell wall made of cellulose that must be broken to release the cellular contents, including the DNA. This often requires a harsh mechanical disruption method, like grinding with a mortar and pestle in liquid nitrogen, which is not necessary for bacterial cells that lack a cellulose wall.
Question 6: To assess the purity of a DNA sample extracted from mammalian cells, a spectrophotometer is used to measure the absorbance at 260 nm and 280 nm. A ratio of A260/A280 of approximately 1.8 is generally considered to indicate a pure DNA sample. What does a significantly lower ratio (e.g., 1.5) suggest?
- The sample is contaminated with RNA.
- The DNA concentration is very high.
- The sample is contaminated with protein. (Correct answer)
- The sample is contaminated with salts from the buffer.
Correct answer: The sample is contaminated with protein.
Nucleic acids (DNA and RNA) have a peak absorbance at 260 nm, while proteins have a peak absorbance at 280 nm. An A260/A280 ratio of ~1.8 is indicative of pure DNA. A ratio lower than this suggests the presence of contaminating proteins, which absorb at 280 nm and lower the ratio.
BACE - Biotechnician Assistant Credentialing DNA Extraction and Analysis Questions and Answers 1
During agarose gel electrophoresis, what is the primary reason that DNA fragments migrate towards the positive electrode?