biology : class12 Plasmid DNA Isolation : DNA Isolation, Restriction Enzymes, Vector, Ligation, Host Cell : Recombinant DNA Technology MCQ Questions and Answers
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Plasmid DNA Isolation
MCQ questions and answers.
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MCQ Questions
1. During alkaline lysis-based plasmid DNA isolation, what is the primary reason for adding sodium dodecyl sulfate (SDS) together with NaOH?
A.
SDS denatures proteins and disrupts the bacterial membrane, while NaOH denatures chromosomal and plasmid DNA
B.
SDS precipitates plasmid DNA, while NaOH hydrolyses its phosphodiester bonds
C.
SDS selectively digests chromosomal DNA, while NaOH protects plasmid DNA from nuclease activity
D.
SDS neutralizes the alkaline solution, while NaOH selectively lyses only plasmid-bearing cells
Correct Answer:
SDS denatures proteins and disrupts the bacterial membrane, while NaOH denatures chromosomal and plasmid DNA
Explanation:
In alkaline lysis, SDS disrupts the bacterial membrane and denatures proteins, whereas NaOH causes DNA denaturation. Properly controlled conditions allow small, covalently closed plasmids to renature later, while large chromosomal DNA remains prone to irreversible aggregation.
2. Why must the alkaline lysis step in plasmid DNA isolation not be prolonged excessively?
A.
Prolonged exposure causes plasmid DNA to become irreversibly denatured and may reduce its recovery in the soluble fraction
B.
Prolonged exposure converts plasmid DNA into RNA through alkaline hydrolysis
C.
Prolonged exposure prevents SDS from disrupting the bacterial membrane
D.
Prolonged exposure causes chromosomal DNA to remain completely soluble after neutralization
Correct Answer:
Prolonged exposure causes plasmid DNA to become irreversibly denatured and may reduce its recovery in the soluble fraction
Explanation:
Plasmid DNA can normally renature after controlled alkaline denaturation because its two strands remain topologically linked. Excessive alkaline treatment can damage or irreversibly denature plasmid DNA, lowering the quality and yield of the isolated preparation.
3. What is the principal function of potassium acetate in the neutralization step of alkaline plasmid DNA isolation?
A.
It neutralizes the alkaline solution and promotes precipitation of SDS, proteins, and aggregated chromosomal DNA
B.
It selectively degrades RNA while keeping SDS in solution
C.
It lyses the bacterial cell wall and denatures plasmid DNA
D.
It binds plasmid DNA irreversibly so that it sediments with cellular debris
Correct Answer:
It neutralizes the alkaline solution and promotes precipitation of SDS, proteins, and aggregated chromosomal DNA
Explanation:
Potassium acetate lowers the pH after alkaline lysis and forms insoluble potassium dodecyl sulfate with SDS. Proteins, membrane components, and large chromosomal DNA become trapped in the precipitate, while properly renatured plasmid DNA remains in the supernatant.
4. A student vigorously vortexes a bacterial suspension after adding the alkaline SDS lysis solution. What is the most likely consequence?
A.
Increased shearing of chromosomal DNA, causing it to remain in the supernatant and contaminate the plasmid preparation
B.
Selective fragmentation of plasmid DNA, causing all plasmid molecules to precipitate immediately
C.
Enhanced separation of plasmid DNA from chromosomal DNA because chromosomal DNA becomes more compact
D.
Complete removal of RNA because mechanical agitation activates RNase
Correct Answer:
Increased shearing of chromosomal DNA, causing it to remain in the supernatant and contaminate the plasmid preparation
Explanation:
After lysis, chromosomal DNA is large and fragile. Vigorous mixing mechanically shears it into smaller fragments that may not precipitate efficiently during neutralization, resulting in chromosomal DNA contamination of the plasmid-containing supernatant.
5. Why is RNase commonly included in the resuspension buffer used before alkaline lysis during plasmid DNA isolation?
A.
To degrade cellular RNA so that it does not contaminate the purified plasmid DNA
B.
To digest chromosomal DNA before it can be released from the cell
C.
To hydrolyse SDS and improve bacterial membrane disruption
D.
To prevent plasmid DNA from renaturing during neutralization
Correct Answer:
To degrade cellular RNA so that it does not contaminate the purified plasmid DNA
Explanation:
RNA is released during bacterial lysis and can remain in the soluble fraction with plasmid DNA. RNase degrades this RNA, improving the purity and interpretability of the isolated plasmid preparation without specifically digesting DNA.
6. After neutralization and centrifugation in an alkaline lysis protocol, where is the desired plasmid DNA primarily located?
A.
In the clear supernatant
B.
In the compact pellet containing cell debris and chromosomal DNA
C.
In the insoluble SDS layer at the interface
D.
In the discarded bacterial culture medium
Correct Answer:
In the clear supernatant
Explanation:
Small plasmid molecules can renature correctly after neutralization and remain soluble. In contrast, denatured proteins, membrane material, SDS complexes, and aggregated chromosomal DNA form a pellet during centrifugation.
7. A plasmid preparation shows a strong high-molecular-weight band near the well on agarose gel, along with the expected plasmid bands. Which isolation error most plausibly explains this result?
A.
Vigorous mixing after cell lysis caused chromosomal DNA shearing and carryover
B.
Insufficient RNase treatment converted RNA into high-molecular-weight DNA
C.
Excessive ethanol washing selectively concentrated chromosomal DNA
D.
Gentle resuspension of the pellet prevented plasmid DNA from entering the gel
Correct Answer:
Vigorous mixing after cell lysis caused chromosomal DNA shearing and carryover
Explanation:
A high-molecular-weight band near the well commonly indicates genomic DNA contamination. Harsh mixing shears chromosomal DNA, allowing fragments to remain in the supernatant instead of precipitating with the insoluble material.
8. Why is complete resuspension of the bacterial pellet important before adding the alkaline lysis solution?
A.
It ensures uniform exposure of all cells to the lysis reagents and reduces incomplete plasmid release
B.
It causes plasmid DNA to precipitate before chromosomal DNA is released
C.
It removes the need for neutralization because every cell is mechanically lysed
D.
It prevents RNA degradation by keeping RNase away from the cells
Correct Answer:
It ensures uniform exposure of all cells to the lysis reagents and reduces incomplete plasmid release
Explanation:
Clumps of incompletely resuspended cells may not contact the alkaline SDS solution uniformly. This leads to incomplete lysis and reduced plasmid recovery, making thorough but gentle resuspension an important isolation step.
9. During plasmid DNA isolation, why is the cleared lysate generally transferred without disturbing the pellet formed after neutralization?
A.
The pellet contains precipitated chromosomal DNA, proteins, membrane debris, and SDS complexes that would contaminate the plasmid DNA
B.
The pellet contains only plasmid DNA, whereas the supernatant contains chromosomal DNA
C.
The pellet contains RNase that would destroy plasmid DNA in the supernatant
D.
The pellet consists of residual ethanol that prevents plasmid DNA from binding to silica
Correct Answer:
The pellet contains precipitated chromosomal DNA, proteins, membrane debris, and SDS complexes that would contaminate the plasmid DNA
Explanation:
Neutralization causes large cellular contaminants and SDS-protein complexes to aggregate and sediment. Avoiding disturbance of this pellet helps retain the plasmid-enriched supernatant and prevents genomic DNA and debris from entering later purification steps.
10. Which observation most strongly indicates that the alkaline lysis and neutralization conditions preserved plasmid DNA in a usable form?
A.
The preparation contains a prominent covalently closed circular plasmid band with limited genomic DNA contamination
B.
The preparation contains only a diffuse high-molecular-weight smear near the gel well
C.
The preparation remains insoluble immediately after neutralization
D.
The preparation contains abundant RNA but no DNA bands
Correct Answer:
The preparation contains a prominent covalently closed circular plasmid band with limited genomic DNA contamination
Explanation:
Properly isolated plasmid DNA commonly appears as a compact, covalently closed circular form on agarose gel, often accompanied by smaller amounts of other topological forms. Limited genomic contamination indicates that lysis and neutralization separated plasmid DNA from chromosomal DNA effectively.
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