Recombinant selection and screening MCQs With Answer

Recombinant selection and screening MCQs With Answer

This quiz set is designed for M.Pharm students studying Advanced Pharmaceutical Biotechnology. It focuses on recombinant selection and screening methods used to identify, isolate and validate genetically modified clones in bacteria, yeast, mammalian cells and plants. Questions emphasize conceptual understanding and practical considerations — selectable and counter-selectable markers, reporter systems (lacZ, GFP, luciferase), antibiotic and auxotrophic selection, colony PCR, hybridization, high-throughput screening (FACS, microfluidics), phage display and recombineering strategies. The items will help reinforce laboratory decision-making, troubleshoot common pitfalls (satellite colonies, false positives/negatives) and prepare for exams and research applications where robust recombinant screening is essential.

Q1. What is the primary difference between selection and screening in recombinant clone identification?

• Selection uses molecular probes to identify recombinants while screening relies on growth conditions
• Selection eliminates non-recombinants by providing a growth advantage to recombinants; screening requires assaying individual colonies for the desired trait
• Screening always uses antibiotics whereas selection always uses colorimetric substrates
• Selection is used only in eukaryotes and screening only in prokaryotes

Correct Answer: Selection eliminates non-recombinants by providing a growth advantage to recombinants; screening requires assaying individual colonies for the desired trait

Q2. What is the molecular basis of blue-white screening used with many cloning vectors?

• Recombinant plasmids produce a pigment that turns colonies blue
• Insertional inactivation of lacZ α-fragment prevents β-galactosidase α-complementation so transformants with inserts form white colonies on X-gal/IPTG plates
• Inserted DNA encodes an enzyme that converts IPTG to a colorless product
• Blue colonies indicate loss of plasmid due to recombination

Correct Answer: Insertional inactivation of lacZ α-fragment prevents β-galactosidase α-complementation so transformants with inserts form white colonies on X-gal/IPTG plates

Q3. How does the sacB gene function as a counter-selectable marker in Gram-negative bacteria?

• sacB confers resistance to sucrose, allowing selection of recombinants on sucrose plates
• sacB encodes levansucrase whose activity produces toxic products from sucrose that kill sacB-expressing cells, enabling counter-selection on sucrose
• sacB confers antibiotic resistance to ampicillin
• sacB methylates genomic DNA to prevent restriction enzyme activity

Correct Answer: sacB encodes levansucrase whose activity produces toxic products from sucrose that kill sacB-expressing cells, enabling counter-selection on sucrose

Q4. In yeast genetics, how is URA3 used for both positive selection and counter-selection?

• URA3 confers resistance to uracil-synthesis inhibitors and cannot be counter-selected
• URA3 allows growth on media lacking uracil (positive selection); presence of 5-fluoroorotic acid (5-FOA) kills URA3-positive cells (counter-selection)
• URA3 is only a reporter and does not affect growth
• URA3 encodes antibiotic resistance used in yeast selection

Correct Answer: URA3 allows growth on media lacking uracil (positive selection); presence of 5-fluoroorotic acid (5-FOA) kills URA3-positive cells (counter-selection)

Q5. Which selectable marker is most commonly used to select for stable transfectants in mammalian cells with G418?

• hygromycin phosphotransferase (Hyg)
• puromycin N-acetyltransferase (Pac)
• neomycin phosphotransferase II (nptII) conferring G418 resistance
• chloramphenicol acetyltransferase (cat)

Correct Answer: neomycin phosphotransferase II (nptII) conferring G418 resistance

Q6. What is the main advantage of colony PCR when screening bacterial transformants?

• It provides exact copy number quantification of plasmids
• It allows rapid, direct testing of colonies for presence and size of an insert without miniprepping plasmid DNA
• It replaces sequencing for final confirmation of constructs
• It can identify post-translational modifications of expressed proteins

Correct Answer: It allows rapid, direct testing of colonies for presence and size of an insert without miniprepping plasmid DNA

Q7. What causes satellite colonies when using ampicillin selection on plates?

• High plasmid copy number producing pigment
• Degradation of ampicillin by secreted β-lactamase from large colonies creates local antibiotic-free zones allowing growth of non-resistant cells
• Ampicillin directly stimulates growth of non-transformants
• Inadequate agar solidification causes colony merging

Correct Answer: Degradation of ampicillin by secreted β-lactamase from large colonies creates local antibiotic-free zones allowing growth of non-resistant cells

Q8. For very sensitive, quantitative reporter assays of promoter activity, which reporter is generally most sensitive?

• Green fluorescent protein (GFP) only
• β-galactosidase with X-gal
• Firefly luciferase luminescence assays
• Chloramphenicol acetyltransferase colorimetric assay

Correct Answer: Firefly luciferase luminescence assays

Q9. Which statement best describes phage display panning for selecting peptide ligands?

• Panning amplifies all phage equally without enrichment
• Panning involves iterative binding of a phage library to immobilized target, washing away non-binders, eluting bound phage and amplifying them to enrich high-affinity binders over multiple rounds
• Panning uses antibiotics to select high-affinity phage
• Panning is only used to select DNA sequences, not peptides

Correct Answer: Panning involves iterative binding of a phage library to immobilized target, washing away non-binders, eluting bound phage and amplifying them to enrich high-affinity binders over multiple rounds

Q10. What feature makes FACS (flow cytometry cell sorting) powerful for recombinant screening?

• It can sort cells based on colony color on agar plates
• It sorts single cells in suspension by fluorescence intensity, enabling isolation of rare fluorescently labeled recombinants at high throughput
• It selectively sequences DNA from colonies
• It only measures cell size, not fluorescence

Correct Answer: It sorts single cells in suspension by fluorescence intensity, enabling isolation of rare fluorescently labeled recombinants at high throughput

Q11. Which gene is commonly used as a negative selection marker in cloning systems because its expression is toxic to E. coli?

• lacZ
• ccdB
• gfp
• kanR (nptII)

Correct Answer: ccdB

Q12. What does colony hybridization detect when screening transformants?

• Protein expression by antibody probing of colonies directly
• Presence of specific DNA sequences in colonies by hybridizing labeled nucleic acid probes to colony DNA transferred to a membrane
• Enzymatic activity in liquid culture supernatants
• Antibiotic resistance levels in colonies

Correct Answer: Presence of specific DNA sequences in colonies by hybridizing labeled nucleic acid probes to colony DNA transferred to a membrane

Q13. Which mechanism provides plasmid stability in a host without continuous antibiotic selection?

• Using only low-copy-number origins without partitioning systems
• Plasmid addiction systems (toxin-antitoxin), or partitioning loci that ensure plasmid segregation, maintain plasmids without antibiotics
• Removing the origin of replication to prevent plasmid loss
• Repeatedly curing and retransforming cells

Correct Answer: Plasmid addiction systems (toxin-antitoxin), or partitioning loci that ensure plasmid segregation, maintain plasmids without antibiotics

Q14. How do droplet-based microfluidic systems accelerate high-throughput screening of enzyme libraries?

• By growing colonies on large agar plates only
• By encapsulating single cells or enzymes in picoliter droplets that act as isolated microreactors, enabling massively parallel activity assays and sorting based on signal
• By increasing antibiotic concentration in bulk cultures
• By using only fluorescence microscopy of plates

Correct Answer: By encapsulating single cells or enzymes in picoliter droplets that act as isolated microreactors, enabling massively parallel activity assays and sorting based on signal

Q15. Which selectable marker is commonly used to confer herbicide resistance in plant transformation?

• nptII (kanamycin resistance)
• bar gene conferring resistance to phosphinothricin (bialaphos)
• ccdB toxin gene
• lacZ reporter

Correct Answer: bar gene conferring resistance to phosphinothricin (bialaphos)

Q16. Which plate-based assay is useful to screen colonies for secreted protease activity?

• Blue-white screening with X-gal
• Hydrolysis halo assay on skim milk or casein agar where protease-producing colonies create clear zones
• Antibiotic selection on ampicillin plates
• Colony PCR for protease gene size

Correct Answer: Hydrolysis halo assay on skim milk or casein agar where protease-producing colonies create clear zones

Q17. When confirming genomic integration of a transgene, which method directly demonstrates integration site or copy number?

• Western blot of proteins only
• Southern blot analysis of genomic DNA probed for the transgene
• Blue-white screening
• Colony PCR from bacterial transformants

Correct Answer: Southern blot analysis of genomic DNA probed for the transgene

Q18. What practical step reduces false negatives in colony PCR screening?

• Picking very large amounts of colony material into the PCR reaction
• Using a small amount of colony, performing a brief heat-lysis or alkaline lysis to release DNA and using polymerases tolerant to inhibitors
• Skipping lysis and adding whole colony directly to PCR without optimization
• Only using restriction digestion to confirm inserts

Correct Answer: Using a small amount of colony, performing a brief heat-lysis or alkaline lysis to release DNA and using polymerases tolerant to inhibitors

Q19. What is a potential downside of increasing selection stringency (e.g., higher antibiotic) when isolating recombinant clones from a library?

• It has no effect on library composition and always improves diversity
• It can reduce library diversity by favoring fast-growing, high-copy or spontaneously resistant mutants and may eliminate slow but functionally desirable clones
• It guarantees isolation of the highest affinity binder in a library
• It eliminates the need for any screening after selection

Correct Answer: It can reduce library diversity by favoring fast-growing, high-copy or spontaneously resistant mutants and may eliminate slow but functionally desirable clones

Q20. In bacterial recombineering workflows using lambda Red, why are selection cassettes often flanked by FRT sites?

• FRT sites prevent recombination entirely
• FRT sites allow subsequent removal of the selectable marker using FLP recombinase to leave a clean, marker-free modification
• FRT sites encode antibiotic resistance themselves
• FRT sites increase plasmid copy number

Correct Answer: FRT sites allow subsequent removal of the selectable marker using FLP recombinase to leave a clean, marker-free modification

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