Cloning vectors, restriction endonucleases and DNA ligase are fundamental components of recombinant DNA technology that every B.Pharm student should master. Cloning vectors (plasmids, bacteriophage, cosmids, shuttle vectors) provide a vector backbone with origin of replication, multiple cloning site and selection markers. Restriction endonucleases (Type II enzymes) recognize specific palindromic restriction sites to create sticky or blunt ends, while DNA ligase (e.g., T4 DNA ligase) catalyzes phosphodiester bond formation during ligation using ATP or NAD+. Understanding cohesive vs. blunt-end cloning, dephosphorylation to prevent recircularization, directional cloning, transformation methods and selection strategies improves cloning efficiency and downstream expression studies. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. Which feature of a cloning vector allows it to replicate independently within a bacterial host?
- Multiple cloning site
- Origin of replication
- Selection marker
- Reporter gene
Correct Answer: Origin of replication
Q2. Type II restriction endonucleases are most useful in cloning because they:
- Require ATP to cut DNA
- Cut at random sequences
- Recognize specific DNA sequences and cut at defined positions
- Only cut RNA-DNA hybrids
Correct Answer: Recognize specific DNA sequences and cut at defined positions
Q3. A “sticky end” produced by a restriction enzyme refers to:
- An end with covalently closed hairpin structure
- A blunt, flush end with no overhang
- A single-stranded overhang that can base-pair with complementary ends
- An end that is methylated and protected
Correct Answer: A single-stranded overhang that can base-pair with complementary ends
Q4. Which enzyme is primarily responsible for joining DNA fragments during ligation?
- DNA polymerase I
- Restriction endonuclease
- DNA ligase
- RNase H
Correct Answer: DNA ligase
Q5. T4 DNA ligase requires which cofactor to catalyze ligation in most laboratory protocols?
- Mg2+ and ATP
- NAD+ only
- FAD
- No cofactor is required
Correct Answer: Mg2+ and ATP
Q6. Directional cloning is achieved by:
- Using a single restriction enzyme for both ends
- Using two different restriction enzymes that create incompatible ends
- Dephosphorylating the vector and insert
- Using blunt-end ligation only
Correct Answer: Using two different restriction enzymes that create incompatible ends
Q7. Why is alkaline phosphatase treatment used on linearized vectors before ligation?
- To increase vector copy number
- To remove 3′ hydroxyl groups
- To dephosphorylate 5′ phosphate groups and prevent self-ligation
- To add methyl groups to restriction sites
Correct Answer: To dephosphorylate 5′ phosphate groups and prevent self-ligation
Q8. Blue-white screening uses disruption of which reporter gene to identify recombinants?
- GFP
- lacZ (β-galactosidase)
- ampR
- ccdB
Correct Answer: lacZ (β-galactosidase)
Q9. Which statement about blunt-end ligation is correct?
- Blunt-end ligation is generally more efficient than cohesive-end ligation
- Blunt-end ligation does not require DNA ligase
- Blunt-end ligation can join any two blunt fragments regardless of sequence complementarity
- Blunt ends cannot be ligated at all
Correct Answer: Blunt-end ligation can join any two blunt fragments regardless of sequence complementarity
Q10. A multiple cloning site (MCS) in a plasmid contains:
- A series of unique restriction sites for easy insertion
- Only one restriction site repeated many times
- The origin of replication
- A gene encoding antibiotic resistance
Correct Answer: A series of unique restriction sites for easy insertion
Q11. Which vector type is best suited to shuttle DNA between E. coli and yeast?
- Plasmid with only bacterial origin
- Shuttle vector with both bacterial and yeast origins
- Bacteriophage λ vector
- Tn5 transposon
Correct Answer: Shuttle vector with both bacterial and yeast origins
Q12. Methylation of DNA at restriction sites can:
- Enhance cleavage by restriction enzymes
- Prevent cleavage by certain restriction endonucleases
- Convert sticky ends to blunt ends
- Be required for ligation
Correct Answer: Prevent cleavage by certain restriction endonucleases
Q13. Which antibiotic is commonly used as a selection marker in many cloning plasmids?
- Penicillin G
- Ampicillin
- Chloramphenicol acetyltransferase
- Sucrose
Correct Answer: Ampicillin
Q14. The role of the origin of replication (ori) in a plasmid affects:
- Restriction enzyme specificity
- Copy number of the plasmid in host cells
- The sequence of inserted gene
- Ability to form sticky ends
Correct Answer: Copy number of the plasmid in host cells
Q15. Which enzyme would you use to map restriction sites by cutting DNA at many random locations?
- Type II restriction endonuclease with known site
- DNase I
- T4 DNA ligase
- DNA polymerase
Correct Answer: DNase I
Q16. A common practice to increase ligation efficiency for sticky ends is to perform ligation at:
- 37°C for 1 minute
- 16°C overnight
- 95°C for 10 minutes
- Room temperature without buffer
Correct Answer: 16°C overnight
Q17. Which of the following describes a palindromic restriction site?
- A sequence that reads the same on both strands in 5’→3′ direction
- A sequence that is methylated on alternate bases
- A sequence that forms a hairpin only in RNA
- A sequence that cannot be cleaved by restriction enzymes
Correct Answer: A sequence that reads the same on both strands in 5’→3′ direction
Q18. The lacZ α-complementation used in blue-white screening relies on:
- Restoration of β-galactosidase activity by two peptide fragments
- Antibiotic inactivation
- Color change due to methylation
- Loss of plasmid origin
Correct Answer: Restoration of β-galactosidase activity by two peptide fragments
Q19. Which ligase is commonly used for joining nicks in double-stranded DNA during replication in vivo?
- T4 DNA ligase
- E. coli DNA ligase (NAD+-dependent)
- RNA ligase
- DNA gyrase
Correct Answer: E. coli DNA ligase (NAD+-dependent)
Q20. Inserting an insert with non-compatible cohesive ends into a vector will most likely result in:
- Efficient ligation without any modification
- No ligation unless ends are made compatible or blunted
- Automatic repair by host without ligase
- Higher transformation efficiency
Correct Answer: No ligation unless ends are made compatible or blunted
Q21. Colony PCR is used after transformation primarily to:
- Increase plasmid copy number
- Screen colonies for presence and size of insert rapidly
- Sequence the insert
- Introduce restriction sites
Correct Answer: Screen colonies for presence and size of insert rapidly
Q22. Which statement about high-copy-number plasmids is true?
- They typically carry eukaryotic origins of replication
- They replicate at a low number per cell
- They often yield more recombinant DNA per cell than low-copy plasmids
- They cannot carry selection markers
Correct Answer: They often yield more recombinant DNA per cell than low-copy plasmids
Q23. The enzyme that protects host DNA from its own restriction system is:
- DNA methyltransferase
- DNA polymerase
- T4 endonuclease
- RNA polymerase
Correct Answer: DNA methyltransferase
Q24. Which of the following best describes “recombinant DNA”?
- DNA isolated directly from environmental samples
- DNA formed by joining DNA fragments from different sources
- Only viral genomes cloned into plasmids
- DNA that has been methylated
Correct Answer: DNA formed by joining DNA fragments from different sources
Q25. The ccdB gene in some cloning vectors is used for:
- Providing antibiotic resistance
- Counterselection by killing cells carrying non-recombinant plasmid
- Blue-white screening
- Enhancing plasmid replication
Correct Answer: Counterselection by killing cells carrying non-recombinant plasmid
Q26. If a restriction enzyme recognizes a 6-base sequence, how often will that site occur on average in random DNA?
- Every 4 bases
- Every 64 bases
- Every 4096 bases
- Every 262144 bases
Correct Answer: Every 4096 bases
Q27. Which method increases transformation efficiency for large plasmids?
- Using smaller competent cells
- Electroporation instead of chemical transformation
- Increasing salt concentration during plating
- Heating cells to 70°C before transformation
Correct Answer: Electroporation instead of chemical transformation
Q28. A vector engineered for high-level protein expression typically contains:
- A weak promoter and no ribosome binding site
- A strong promoter, optimized ribosome binding site and selection marker
- Only an origin of replication and no selection marker
- Multiple restriction enzymes inside the gene coding region
Correct Answer: A strong promoter, optimized ribosome binding site and selection marker
Q29. Which procedure helps confirm that an insert is in the correct orientation in a plasmid?
- Digesting with a single cutter outside the MCS
- Sequencing across the insert–vector junction or using diagnostic restriction digests
- Only performing colony color screening
- Measuring plasmid copy number
Correct Answer: Sequencing across the insert–vector junction or using diagnostic restriction digests
Q30. Why are cohesive-end ligations generally preferred over blunt-end ligations in cloning?
- Cohesive-end ligations are irreversible and do not require ligase
- Cohesive ends provide base-pairing that orients fragments and increases ligation efficiency
- Cohesive-end ligations do not require competent cells for transformation
- Cohesive ends eliminate the need for selection markers
Correct Answer: Cohesive ends provide base-pairing that orients fragments and increases ligation efficiency
I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.
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