Site-directed mutagenesis MCQs With Answer

Introduction: Site-directed mutagenesis MCQs With Answer is designed for M.Pharm students to consolidate advanced concepts in protein engineering and molecular biology. This set covers fundamental principles, practical strategies, and modern techniques used to introduce defined mutations into DNA — from classical oligonucleotide-directed and PCR-based methods to contemporary genome-editing approaches such as CRISPR-based base editors. Questions emphasize primer design, choice of polymerase, selection and screening strategies, common troubleshooting steps, and applications like structure–function analysis, enzyme optimization, and therapeutic protein design. These MCQs focus on both theory and lab-practical implications to prepare students for exams and research-oriented problem solving.

Q1. What is the principal purpose of site-directed mutagenesis?

• To randomly alter the whole genome of an organism
• To introduce specific, predefined changes at precise nucleotide positions
• To increase plasmid copy number in a host cell
• To delete entire chromosomes from cells

Correct Answer: To introduce specific, predefined changes at precise nucleotide positions

Q2. In the classical oligonucleotide-directed method, what role does the synthetic primer play?

• Acts as a nonspecific primer for random amplification
• Introduces the desired nucleotide change when incorporated during DNA synthesis
• Degrades the template strand to facilitate recombination
• Serves to methylate the template DNA

Correct Answer: Introduces the desired nucleotide change when incorporated during DNA synthesis

Q3. Which enzyme is commonly used to selectively remove the parental methylated plasmid after PCR-based mutagenesis (e.g., QuikChange)?

• Exonuclease I
• DpnI
• HaeIII
• Proteinase K

Correct Answer: DpnI

Q4. Overlap extension PCR (OE-PCR) is best described as:

• A method to amplify entire genomes without primers
• A PCR technique using fragments with complementary overlaps to assemble specific mutations or chimeric constructs
• A method to selectively degrade mutated strands
• A cloning-free method to label proteins with fluorescent tags

Correct Answer: A PCR technique using fragments with complementary overlaps to assemble specific mutations or chimeric constructs

Q5. Which property of a mutagenic primer is most important to ensure the primer binds tightly despite the mismatch at the mutation site?

• Very low GC content overall
• High melting temperature (Tm) by sufficient length and GC content
• Introduction of multiple mismatches far from the 3′ end
• Use of RNA bases instead of DNA

Correct Answer: High melting temperature (Tm) by sufficient length and GC content

Q6. Which DNA polymerase characteristic is most desirable for PCR-based site-directed mutagenesis?

• High error rate to generate diversity
• High fidelity (proofreading activity) to prevent off-target mutations
• Preference for incorporating ribonucleotides
• Extremely low processivity to stop early

Correct Answer: High fidelity (proofreading activity) to prevent off-target mutations

Q7. Saturation mutagenesis at a single codon commonly uses degenerate codons like NNK. What advantage does NNK have?

• It restricts substitutions to only hydrophobic amino acids
• It encodes all 20 amino acids while minimizing stop codons
• It eliminates all cysteine residues
• It ensures only synonymous codons are produced

Correct Answer: It encodes all 20 amino acids while minimizing stop codons

Q8. In CRISPR/Cas9-mediated site-directed mutagenesis relying on homology-directed repair (HDR), what is essential to introduce a precise nucleotide change?

• Double-strand break only, without donor template
• Single-stranded or double-stranded donor DNA carrying the desired mutation and homology arms
• Continuous Cas9 expression with no repair template
• Blocking all cellular repair pathways

Correct Answer: Single-stranded or double-stranded donor DNA carrying the desired mutation and homology arms

Q9. Which technique is most appropriate for scanning the functional contribution of individual residues across a protein domain?

• Alanine scanning mutagenesis
• Random chemical mutagenesis
• Whole genome sequencing
• RNA interference

Correct Answer: Alanine scanning mutagenesis

Q10. During QuikChange-style mutagenesis, why are primers designed as complementary and overlapping and oriented back-to-back?

• To create linear fragments that cannot be ligated
• To enable amplification of the entire plasmid with the mutation incorporated on both strands
• To avoid amplification of the plasmid altogether
• To produce single-stranded DNA only

Correct Answer: To enable amplification of the entire plasmid with the mutation incorporated on both strands

Q11. Which screening strategy distinguishes mutated clones from parental plasmid when DpnI digestion is incomplete?

• Antibiotic selection alone without screening
• Colony PCR or sequencing of individual colonies
• Heat shock of plasmid DNA
• Measuring plasmid copy number

Correct Answer: Colony PCR or sequencing of individual colonies

Q12. What is the likely result of placing the intended mismatch near the 3’ terminal nucleotide of a mutagenic primer?

• Increased chance that polymerase will extend efficiently, producing the mutation
• Reduced primer extension because a 3′ mismatch can block polymerase initiation
• Guaranteed removal of parental template by exonuclease
• Creation of multiple off-target mutations elsewhere

Correct Answer: Reduced primer extension because a 3′ mismatch can block polymerase initiation

Q13. Which of these is a primary advantage of using high-fidelity polymerases like Pfu or Phusion for site-directed mutagenesis?

• They add adenine overhangs for TA cloning
• They reduce unintended background mutations introduced during PCR
• They require no primers
• They only synthesize RNA strands

Correct Answer: They reduce unintended background mutations introduced during PCR

Q14. In cassette mutagenesis, what is replaced by the synthetic DNA cassette?

• A small, defined region of the target gene to introduce multiple contiguous mutations or insertions
• The entire host chromosome
• Only the promoter far upstream of the gene
• The ribosome binding site exclusively

Correct Answer: A small, defined region of the target gene to introduce multiple contiguous mutations or insertions

Q15. What distinguishes base editors from classical CRISPR/Cas9 HDR-based editing for point mutations?

• Base editors produce double-strand breaks to increase HDR
• Base editors chemically change a single base (e.g., C→T or A→G) without double-strand breaks
• Base editors randomly mutate entire genes
• Base editors delete large genomic regions

Correct Answer: Base editors chemically change a single base (e.g., C→T or A→G) without double-strand breaks

Q16. When designing a primer for site-directed mutagenesis, which of the following helps minimize formation of primer dimers and secondary structure?

• Include long stretches of complementary bases at the 3′ ends of both primers
• Avoid self-complementarity and hairpin-forming sequences in primer design
• Maximize G runs at both ends to encourage pairing
• Use primers longer than 200 nucleotides always

Correct Answer: Avoid self-complementarity and hairpin-forming sequences in primer design

Q17. Which mutagenesis approach is typically used to create libraries for directed evolution experiments?

• Site-directed single-point correction only
• Saturation mutagenesis and error-prone PCR combined with screening or selection
• Chromosomal excision of target gene
• RNA splicing without DNA changes

Correct Answer: Saturation mutagenesis and error-prone PCR combined with screening or selection

Q18. What is a common reason to include silent (synonymous) mutations when designing a repair template for genome editing?

• To create a frameshift mutation
• To disrupt the protospacer-adjacent motif (PAM) or guide binding and prevent recleavage after repair
• To introduce premature stop codons intentionally
• To prevent translation of the gene

Correct Answer: To disrupt the protospacer-adjacent motif (PAM) or guide binding and prevent recleavage after repair

Q19. Which method allows introduction of multiple distant mutations simultaneously within a gene?

• Single short primer QuikChange only
• Multi-fragment overlap extension PCR or assembly methods using multiple mutagenic primers
• Exposure to UV light
• Restriction enzyme digestion alone

Correct Answer: Multi-fragment overlap extension PCR or assembly methods using multiple mutagenic primers

Q20. After performing site-directed mutagenesis and transformation, what is the most definitive method to confirm the intended mutation without unintended changes?

• Measuring protein activity only
• Sanger sequencing (or deep sequencing) of the plasmid insert
• Antibiotic resistance phenotype alone
• Restriction digest without sequence verification

Correct Answer: Sanger sequencing (or deep sequencing) of the plasmid insert

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