Cloning strategies and procedures MCQs With Answer

Introduction

This quiz set on Cloning strategies and procedures is designed specifically for M.Pharm students preparing for advanced biotechnology examinations and practical work. It covers core concepts such as vector selection, cloning methods (restriction-ligation, TA, Gibson, Golden Gate, Gateway), host strain choice, screening and confirmation techniques, expression considerations, library construction, and specialized vectors (BAC, YAC, phagemids). Questions emphasize procedural reasoning, troubleshooting, and decision-making encountered in laboratory cloning workflows. Use these MCQs to assess and deepen your conceptual understanding, reinforce practical choices, and prepare for viva and written assessments in molecular cloning and recombinant DNA technology.

Q1. Which cloning method eliminates the need for restriction enzymes and ligase by creating overlapping ends for seamless assembly?

• Restriction–ligation cloning using compatible cohesive ends
• TA cloning into T-overhang vectors
• Gibson assembly that uses exonuclease, polymerase and ligase
• Topoisomerase-mediated TOPO cloning

Correct Answer: Gibson assembly that uses exonuclease, polymerase and ligase

Q2. When performing directional cloning to ensure insert orientation, which strategy is most reliable?

• Using a single restriction enzyme to produce identical cohesive ends
• Using two different restriction enzymes producing noncompatible ends
• Using blunt-end ligation into a blunt-cut vector
• Using TA cloning into a T-vector

Correct Answer: Using two different restriction enzymes producing noncompatible ends

Q3. Which E. coli strain is most commonly used for protein expression of toxic proteins after cloning in pET vectors?

• DH5α
• JM109
• BL21(DE3) or a derivative such as BL21(DE3)pLysS
• HB101

Correct Answer: BL21(DE3) or a derivative such as BL21(DE3)pLysS

Q4. Blue-white screening exploits disruption of which gene to identify recombinant clones?

• ampR beta-lactamase gene
• lacZ alpha fragment encoding β-galactosidase complementing activity
• oriT origin of transfer
• ccdB toxin gene

Correct Answer: lacZ alpha fragment encoding β-galactosidase complementing activity

Q5. Which vector feature is most important when cloning very large genomic fragments (hundreds of kb) for stability?

• High-copy plasmid origin of replication
• Low-copy BAC (bacterial artificial chromosome) with stability elements
• pUC-based vector with strong promoter
• pGEM-T vector for TA cloning

Correct Answer: Low-copy BAC (bacterial artificial chromosome) with stability elements

Q6. TA cloning is particularly useful for PCR products generated by Taq polymerase because:

• Taq creates 5′ phosphate groups for ligation into blunt vectors
• Taq adds a single 3′ adenine overhang that complements T-overhang vectors
• Taq produces sticky 5′ ends compatible with restriction sites
• Taq inserts direct repeats that facilitate recombination cloning

Correct Answer: Taq adds a single 3′ adenine overhang that complements T-overhang vectors

Q7. Which method is best to rapidly confirm the presence and approximate size of an insert in bacterial colonies?

• Sanger sequencing of plasmid DNA from each colony
• Colony PCR using insert-specific primers
• Southern blot on colony lysates
• Restriction mapping after full plasmid purification

Correct Answer: Colony PCR using insert-specific primers

Q8. Gateway cloning relies on which biological system to mediate recombination between entry and destination clones?

• Cre-Lox recombinase system
• PhiC31 integrase system
• att site recombination by bacteriophage lambda integrase (BP and LR reactions)
• Type IIS restriction enzymes and ligases

Correct Answer: att site recombination by bacteriophage lambda integrase (BP and LR reactions)

Q9. Golden Gate cloning is advantageous for modular assembly because it uses:

• Type I restriction enzymes that cut within their recognition sequence producing blunt ends
• Type IIS restriction enzymes that cut outside recognition sites to create custom overhangs
• Topoisomerase for single-step insertion
• Site-specific recombinases for scarless integration

Correct Answer: Type IIS restriction enzymes that cut outside recognition sites to create custom overhangs

Q10. Which selection marker allows selection of eukaryotic clones stably expressing a transgene after transfection?

• ampicillin resistance
• kanamycin resistance
• neomycin resistance (G418) or hygromycin resistance genes for mammalian cells
• lacZ reporter for blue-white screening

Correct Answer: neomycin resistance (G418) or hygromycin resistance genes for mammalian cells

Q11. In subcloning a coding sequence for high-level protein expression, which promoter element is most critical for strong transcription in E. coli expression systems?

• CMV promoter
• T7 promoter recognized by T7 RNA polymerase
• Poly(A) signal
• lac promoter with no enhancements

Correct Answer: T7 promoter recognized by T7 RNA polymerase

Q12. TOPO cloning exploits which enzyme activity present on the vector ends to covalently join PCR product without ligase?

• DNA ligase I activity
• Topoisomerase I covalently bound to vector ends that ligates DNA ends
• DNA polymerase activity to fill gaps
• Restriction endonuclease activity to cut and ligate

Correct Answer: Topoisomerase I covalently bound to vector ends that ligates DNA ends

Q13. When cloning eukaryotic genes for bacterial expression, which modification is most often required to achieve efficient translation?

• Retaining introns to increase transcript complexity
• Codon optimization and removal of introns to match bacterial codon usage and avoid splicing dependence
• Adding eukaryotic polyadenylation signals
• Using only genomic DNA without modification

Correct Answer: Codon optimization and removal of introns to match bacterial codon usage and avoid splicing dependence

Q14. Which cloning approach allows creation of cDNA libraries that represent only expressed genes, excluding introns?

• Genomic library construction in BACs
• cDNA library construction using reverse-transcribed mRNA and oligo-dT or random primers
• Shotgun genomic sequencing of whole chromosomes
• Cloning PCR-amplified promoter regions

Correct Answer: cDNA library construction using reverse-transcribed mRNA and oligo-dT or random primers

Q15. Which screening method directly verifies that cloned DNA sequence is present and has expected restriction fragments?

• Phenotypic antibiotic selection only
• Restriction digestion mapping of isolated plasmid DNA followed by gel electrophoresis
• Measuring optical density of bacterial culture
• Blue-white screening without further analysis

Correct Answer: Restriction digestion mapping of isolated plasmid DNA followed by gel electrophoresis

Q16. Which factor most increases transformation efficiency when introducing plasmid DNA into chemically competent E. coli by heat shock?

• Using very large plasmids (>20 kb) only
• Using highly competent cells prepared for high transformation efficiency and optimized heat-shock conditions
• Overloading with large amounts of nonpurified genomic DNA
• Skipping recovery step after heat shock

Correct Answer: Using highly competent cells prepared for high transformation efficiency and optimized heat-shock conditions

Q17. For cloning applications that require negative selection against empty vectors, which gene-containing cassette is useful to kill cells retaining nonrecombinant plasmids?

• ampR selection gene
• lacZ alpha fragment for blue colonies
• ccdB toxic gene that kills cells unless disrupted by insert
• GFP reporter that fluoresces when empty

Correct Answer: ccdB toxic gene that kills cells unless disrupted by insert

Q18. When subcloning into an expression vector to produce a soluble fusion protein for purification, which tag is commonly used and easily purified by immobilized metal affinity chromatography (IMAC)?

• GST tag requiring glutathione resin
• His6 (polyhistidine) tag binding to Ni-NTA or Co2+ resins
• Maltose-binding protein (MBP) requiring amylose resin
• FLAG tag requiring anti-FLAG antibodies

Correct Answer: His6 (polyhistidine) tag binding to Ni-NTA or Co2+ resins

Q19. Which cloning strategy is best when assembling multiple DNA fragments in a defined order in a single reaction with minimal scars?

• Classical sequential restriction–ligation using only compatible single-site enzymes
• Gibson assembly or Golden Gate assembly designed for multi-fragment, scarless assembly
• TA cloning into serial T-vectors
• Phage λ packaging of genomic fragments

Correct Answer: Gibson assembly or Golden Gate assembly designed for multi-fragment, scarless assembly

Q20. During cloning workflow, which final step is definitive to confirm sequence accuracy and absence of mutations introduced during PCR or cloning?

• Antibiotic selection and colony morphology observation
• Sanger sequencing of the plasmid insert (or next-generation sequencing for larger constructs)
• Restriction digestion alone
• Measuring expression by SDS-PAGE without sequence confirmation

Correct Answer: Sanger sequencing of the plasmid insert (or next-generation sequencing for larger constructs)

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