Techniques of gene manipulation MCQs With Answer

Introduction: This blog presents a focused set of multiple-choice questions on techniques of gene manipulation tailored for M.Pharm students studying Advanced Pharmaceutical Biotechnology. The questions cover foundational principles and practical applications of molecular tools used in cloning, genome editing, nucleic acid amplification, sequencing, gene delivery and expression analysis. Each MCQ emphasizes conceptual clarity, experimental design, troubleshooting and interpretation of results to reinforce both theoretical knowledge and laboratory decision-making. Ideal for exam revision and self-assessment, the set includes answers and targets competencies expected at postgraduate level, such as vector selection, CRISPR mechanisms, PCR variants, and safety considerations. Use these items to identify gaps and deepen understanding prior to exams or research work.

Q1. Which enzyme is primarily responsible for creating site-specific double-strand breaks in classical CRISPR-Cas9 genome editing?

• Cas12a (Cpf1)
• Cas9 endonuclease
• Taq polymerase
• Reverse transcriptase

Correct Answer: Cas9 endonuclease

Q2. In recombinant DNA cloning, what is the main advantage of using a shuttle vector?

• Higher transformation efficiency into prokaryotes only
• Ability to replicate and be selected in two different host species
• Automatic removal of antibiotic resistance markers
• Promotion of genomic integration without selection

Correct Answer: Ability to replicate and be selected in two different host species

Q3. Which PCR variant is most appropriate for quantifying gene expression levels in real time?

• Nested PCR
• Reverse transcription PCR without quantitation
• Quantitative real-time PCR (qRT-PCR)
• Long-range PCR

Correct Answer: Quantitative real-time PCR (qRT-PCR)

Q4. During agarose gel electrophoresis, DNA fragments are separated primarily based on which property?

• Base sequence
• Secondary structure
• Molecular size (length)
• Charge distribution independent of size

Correct Answer: Molecular size (length)

Q5. What is the purpose of using a selectable marker in a cloning vector?

• To increase plasmid copy number
• To visually track protein expression
• To allow selection of cells that have taken up the vector
• To inhibit host cell growth permanently

Correct Answer: To allow selection of cells that have taken up the vector

Q6. Which method introduces DNA directly into mammalian cells by creating transient pores in the membrane using electrical pulses?

• Lipofection
• Microinjection
• Electroporation
• Calcium phosphate transfection

Correct Answer: Electroporation

Q7. Gibson assembly differs from traditional restriction-ligation cloning because it:

• Requires compatible restriction sites flanking inserts
• Assembles multiple fragments using single-pot exonuclease, polymerase and ligase activities
• Uses only ligase to join blunt-ended fragments
• Depends on in vivo recombination in yeast

Correct Answer: Assembles multiple fragments using single-pot exonuclease, polymerase and ligase activities

Q8. Which sequencing method is based on chain termination using dideoxynucleotides?

• Illumina sequencing by synthesis
• Sanger dideoxy sequencing
• Nanopore sequencing
• Pyrosequencing

Correct Answer: Sanger dideoxy sequencing

Q9. A cDNA library differs from a genomic library in that it:

• Contains intronic and intergenic sequences
• Represents only expressed genes as complementary DNA
• Is constructed directly from genomic DNA fragments
• Includes promoter and enhancer regions upstream of genes

Correct Answer: Represents only expressed genes as complementary DNA

Q10. In RNA interference (RNAi) mediated gene silencing, which molecule is central to guiding the silencing complex to target mRNA?

• Short interfering RNA (siRNA)
• Plasmid DNA
• Single-stranded DNA oligo
• Ribosomal RNA

Correct Answer: Short interfering RNA (siRNA)

Q11. Which viral vector is commonly preferred for stable gene integration into the genome of dividing and non-dividing mammalian cells?

• Adenovirus
• Adeno-associated virus (AAV)
• Lentivirus
• Baculovirus

Correct Answer: Lentivirus

Q12. Site-directed mutagenesis is most often used to:

• Randomly fragment genomic DNA for library construction
• Introduce specific nucleotide changes to study protein function
• Quantify mRNA expression across tissues
• Increase plasmid yield during fermentation

Correct Answer: Introduce specific nucleotide changes to study protein function

Q13. Which restriction enzyme type cleaves DNA at sites distant from its recognition sequence and often requires ATP?

• Type I restriction enzymes
• Type II restriction enzymes
• Type III restriction enzymes
• Type IV restriction enzymes

Correct Answer: Type I restriction enzymes

Q14. Golden Gate assembly primarily exploits which enzyme activity to assemble DNA fragments in a defined order?

• T4 DNA ligase blunt-end ligation without overhangs
• Type IIS restriction enzymes creating unique overhangs
• Exonuclease chewing back ends for homologous overlap
• Reverse transcriptase-mediated copying

Correct Answer: Type IIS restriction enzymes creating unique overhangs

Q15. What advantage does codon optimization provide when expressing a heterologous protein in E. coli?

• Alters the amino acid sequence to improve function
• Reduces mRNA stability to prevent expression
• Matches codon usage to host tRNA abundance to improve translation efficiency
• Ensures integration into the host genome

Correct Answer: Matches codon usage to host tRNA abundance to improve translation efficiency

Q16. In Southern blotting, the primary purpose of the labeled probe is to:

• Digest DNA at specific restriction sites
• Visualize proteins separated on a gel
• Hybridize to complementary DNA sequences on the membrane for detection
• Amplify target DNA fragments by PCR

Correct Answer: Hybridize to complementary DNA sequences on the membrane for detection

Q17. Which of the following is a non-viral physical method for delivering nucleic acids into cells with high spatial precision?

• Electroporation
• Microinjection
• Lentiviral transduction
• Calcium phosphate precipitation

Correct Answer: Microinjection

Q18. When designing a CRISPR guide RNA, off-target cutting is most influenced by:

• Length of the PAM sequence only
• Sequence homology between guide RNA and non-target genomic sites
• The concentration of Cas9 protein in the buffer only
• Type of cloning vector used for guide RNA expression

Correct Answer: Sequence homology between guide RNA and non-target genomic sites

Q19. Which reporter gene is commonly used for non-invasive in vivo imaging because it emits bioluminescence?

• Green fluorescent protein (GFP)
• Beta-galactosidase
• Firefly luciferase
• Calyculin A

Correct Answer: Firefly luciferase

Q20. Which safety consideration is essential when working with recombinant lentiviral vectors in the laboratory?

• No containment is needed because lentiviruses are harmless
• Use of biosafety level containment and split-vector systems to minimize replication-competent virus formation
• Only chemical sterilization of surfaces is required; PPE is optional
• Operate without institutional biosafety oversight to speed experiments

Correct Answer: Use of biosafety level containment and split-vector systems to minimize replication-competent virus formation

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