Recombinant DNA technology: restriction enzymes and vectors MCQs With Answer

Introduction: Recombinant DNA technology underpins modern molecular pharmacology, enabling the manipulation of genes for drug discovery, therapeutic protein production, and targeted gene delivery. This set of MCQs focuses on restriction enzymes and vectors—core tools used to cut, join, and propagate DNA fragments. The questions explore enzyme classification, recognition sequences, cleavage patterns (sticky vs blunt), methylation effects, enzyme specificity and star activity, and practical cloning strategies such as directional cloning, use of adaptors/linkers, and selection markers. In addition, vector types (plasmids, bacteriophages, cosmids, BACs, YACs), origins of replication, copy-number control, and expression vs shuttle vectors are covered to prepare M.Pharm students for both conceptual understanding and laboratory application.

Q1. What distinguishes Type II restriction enzymes from other types of restriction-modification systems?

• They require ATP and cleave DNA at variable distances from recognition sites
• They recognize non-palindromic sequences and methylate DNA
• They cleave within or very close to their specific recognition sequence and do not require ATP
• They only cut modified (methylated) DNA

Correct Answer: They cleave within or very close to their specific recognition sequence and do not require ATP

Q2. Which property best describes a palindromic restriction site?

• It is rich in AT content and never contains CG dinucleotides
• Sequence reads the same 5’→3′ on both complementary strands
• It is asymmetric and orientation-dependent
• It only occurs in single-stranded DNA

Correct Answer: Sequence reads the same 5’→3′ on both complementary strands

Q3. What is “sticky end” cleavage and why is it useful in cloning?

• Cleavage that results in blunt ends; useful for ligating any two DNA fragments without orientation
• Cleavage yielding single-stranded overhangs that facilitate complementary base pairing and directional ligation
• Cleavage that generates RNA fragments which are ligated to DNA vectors
• Cleavage only at methylated sites to prevent self-ligation

Correct Answer: Cleavage yielding single-stranded overhangs that facilitate complementary base pairing and directional ligation

Q4. Which enzyme is essential for covalently joining DNA fragments during recombinant DNA construction?

• DNA polymerase I
• Alkaline phosphatase
• T4 DNA ligase
• Restriction endonuclease

Correct Answer: T4 DNA ligase

Q5. What is an isoschizomer?

• A restriction enzyme that recognizes the same sequence but cuts at a different position relative to the site
• A restriction enzyme that recognizes an entirely different sequence but produces identical overhangs
• A pair of restriction enzymes from different organisms that recognize the same DNA sequence and cleave at the same position
• A mutant restriction enzyme that lacks methylation sensitivity

Correct Answer: A pair of restriction enzymes from different organisms that recognize the same DNA sequence and cleave at the same position

Q6. What is “star activity” of restriction enzymes?

• Enhanced sequence specificity at optimal buffer conditions
• Nonspecific cleavage at sites similar but not identical to the recognition sequence under suboptimal conditions
• Methylation of host DNA to protect it from cleavage
• Temperature-dependent activation that increases enzyme fidelity

Correct Answer: Nonspecific cleavage at sites similar but not identical to the recognition sequence under suboptimal conditions

Q7. Which vector type is most suitable for cloning very large DNA fragments (hundreds of kilobases)?

• Plasmid vectors
• Bacteriophage lambda vectors
• Bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs)
• Cosmids with standard multiple cloning sites

Correct Answer: Bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs)

Q8. Why is an origin of replication (ori) crucial in a plasmid vector?

• It provides a promoter for gene expression
• It allows the plasmid to be replicated independently within the host cell
• It facilitates integration into the host chromosome
• It encodes antibiotic resistance

Correct Answer: It allows the plasmid to be replicated independently within the host cell

Q9. In directional cloning, what strategy ensures insert orientation?

• Ligating blunt-ended fragments without restriction digestion
• Using two different restriction enzymes that generate incompatible ends on the vector and insert
• Using the same enzyme on both vector and insert so ends match perfectly
• Using methylation to prevent vector recircularization

Correct Answer: Using two different restriction enzymes that generate incompatible ends on the vector and insert

Q10. What is the primary role of a multiple cloning site (MCS) in a plasmid vector?

• Provide a high-expression promoter for heterologous genes
• Contain several unique restriction sites to facilitate insertion of DNA fragments
• Encode a selectable marker such as antibiotic resistance
• Act as an origin of replication for high copy number

Correct Answer: Contain several unique restriction sites to facilitate insertion of DNA fragments

Q11. How do methylases protect host DNA from restriction enzymes in bacteria?

• By acetylating lysines on histones to hide restriction sites
• By adding methyl groups to bases within recognition sequences, preventing cleavage by cognate restriction endonucleases
• By degrading restriction enzymes before they act
• By introducing nicks that block restriction enzyme binding

Correct Answer: By adding methyl groups to bases within recognition sequences, preventing cleavage by cognate restriction endonucleases

Q12. Which selection marker would you choose to ensure maintenance of a plasmid in bacterial culture?

• lacZ alpha fragment for blue/white screening only
• A gene providing auxotrophy that is not selectable in rich media
• An antibiotic resistance gene such as ampicillin or kanamycin resistance
• Origin of replication

Correct Answer: An antibiotic resistance gene such as ampicillin or kanamycin resistance

Q13. What distinguishes a shuttle vector from a standard plasmid vector?

• Shuttle vectors can replicate in multiple host species because they carry more than one origin of replication
• Shuttle vectors are only used for bacterial expression and cannot be transferred
• Shuttle vectors lack selectable markers to reduce size
• Shuttle vectors are integrated into host chromosomes permanently

Correct Answer: Shuttle vectors can replicate in multiple host species because they carry more than one origin of replication

Q14. In TA cloning, why are T-overhangs used on the vector?

• To prevent any ligation and ensure linearization
• To facilitate ligation with PCR products that commonly have 3′ A-overhangs generated by Taq polymerase
• To create blunt-end ligation conditions
• To allow directional cloning using asymmetric restriction sites

Correct Answer: To facilitate ligation with PCR products that commonly have 3′ A-overhangs generated by Taq polymerase

Q15. Which factor most influences copy number of a plasmid in a bacterial host?

• The presence of a lac operator in the multiple cloning site
• The specific origin of replication and its control elements
• The antibiotic used for selection
• The GC content of the inserted DNA fragment

Correct Answer: The specific origin of replication and its control elements

Q16. What is the function of alkaline phosphatase treatment of a linearized vector during cloning?

• To ligate the insert into vector more efficiently
• To remove 5′ phosphate groups from the vector, preventing self-ligation
• To denature the vector DNA creating single-stranded tails
• To methylate vector DNA to prevent restriction digestion

Correct Answer: To remove 5′ phosphate groups from the vector, preventing self-ligation

Q17. Which vector feature is most important for high-level protein expression in E. coli?

• Multiple cloning site with many restriction sites only
• A strong, regulatable promoter and ribosome binding site upstream of the cloning site
• High GC content to stabilize the plasmid
• Multiple origins of replication to overload replication machinery

Correct Answer: A strong, regulatable promoter and ribosome binding site upstream of the cloning site

Q18. Why might one choose a low-copy-number plasmid for cloning a toxic gene?

• To ensure very high expression of the toxic protein
• To reduce metabolic burden and lower expression level, minimizing host toxicity
• To promote rapid loss of the plasmid from the population
• To permit unlimited replication and accumulation of plasmid copies

Correct Answer: To reduce metabolic burden and lower expression level, minimizing host toxicity

Q19. What is the advantage of using a cosmid vector over a plasmid for genomic library construction?

• Cosmids can carry much larger DNA inserts (around 35–45 kb) combining lambda packaging with plasmid replication
• Cosmids have no requirement for selectable markers
• Cosmids replicate as single-stranded DNA only
• Cosmids are incapable of being packaged into phage particles

Correct Answer: Cosmids can carry much larger DNA inserts (around 35–45 kb) combining lambda packaging with plasmid replication

Q20. When mapping restriction sites on a plasmid, what technique uses partial digestion to determine relative positions of sites?

• Complete digestion followed by sequencing only
• Partial (limited) digestion with varying enzyme concentrations and gel analysis to infer order and distance
• Methylation mapping that prevents any digestion
• Using only blunt-end cutters to avoid complexity

Correct Answer: Partial (limited) digestion with varying enzyme concentrations and gel analysis to infer order and distance

Download