Polymerase Chain Reaction (PCR) – principle and applications MCQs With Answer

Polymerase Chain Reaction (PCR) is an essential molecular technique in pharmaceutical sciences that amplifies specific DNA sequences through repeated thermal cycling. Understanding the PCR principle—denaturation, annealing and extension—and critical components such as primers, template DNA, dNTPs, Mg2+ and thermostable polymerases (e.g., Taq) is vital for B. Pharm students. Applications span diagnostics, pharmacogenetics, cloning, microbial detection, viral load monitoring, and quality control. Advanced formats like real-time PCR (qPCR), reverse transcription PCR (RT-PCR), multiplex PCR and digital PCR expand analytical power. Mastery of PCR optimization, troubleshooting and interpretation underpins reliable laboratory work. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the primary role of primers in PCR?

• Act as a source of nucleotides for DNA synthesis
• Provide a starting point with a free 3′-OH group for DNA polymerase
• Denature double-stranded DNA into single strands
• Control the temperature of the thermal cycler

Correct Answer: Provide a starting point with a free 3′-OH group for DNA polymerase

Q2. Which enzyme is most commonly used in conventional PCR for DNA strand synthesis?

• DNA ligase
• Reverse transcriptase
• Taq DNA polymerase
• RNA polymerase

Correct Answer: Taq DNA polymerase

Q3. During PCR, what is the typical purpose of the denaturation step?

• Allow primers to anneal to template
• Extend new DNA strands
• Separate double-stranded DNA into single strands by heating
• Activate fluorescent probes

Correct Answer: Separate double-stranded DNA into single strands by heating

Q4. Which factor most directly influences primer annealing specificity?

• Concentration of dNTPs
• Annealing temperature relative to primer melting temperature (Tm)
• Volume of PCR reaction
• Number of thermal cycles

Correct Answer: Annealing temperature relative to primer melting temperature (Tm)

Q5. What is the consequence of using too high Mg2+ concentration in a PCR?

• Increased primer specificity and decreased yield
• No effect on the reaction
• Inhibition of polymerase activity leading to no product
• Increased non-specific amplification and primer-dimer formation

Correct Answer: Increased non-specific amplification and primer-dimer formation

Q6. In RT-PCR, what is the function of reverse transcriptase?

• To synthesize RNA from an RNA template
• To amplify DNA using thermal cycling
• To convert RNA into complementary DNA (cDNA)
• To cleave RNA at specific sites

Correct Answer: To convert RNA into complementary DNA (cDNA)

Q7. Which PCR variant allows simultaneous amplification of multiple targets in one reaction?

• Nested PCR
• Multiplex PCR
• Touchdown PCR
• Inverse PCR

Correct Answer: Multiplex PCR

Q8. What parameter is commonly used in real-time PCR (qPCR) to quantify starting template?

• Gel band intensity after 40 cycles
• Cycle threshold (Ct) or quantification cycle (Cq)
• Total reaction volume
• The length of the primer

Correct Answer: Cycle threshold (Ct) or quantification cycle (Cq)

Q9. Which dye-based method in qPCR binds to any double-stranded DNA and fluoresces?

• TaqMan probe
• Molecular beacon
• SYBR Green
• FRET probe

Correct Answer: SYBR Green

Q10. What is the main advantage of high-fidelity DNA polymerases over Taq polymerase?

• Higher optimal temperature for denaturation
• Lower cost per reaction
• 3’→5′ exonuclease proofreading activity resulting in fewer errors
• The ability to synthesize RNA

Correct Answer: 3’→5′ exonuclease proofreading activity resulting in fewer errors

Q11. Which contamination control is most effective to prevent carryover PCR product contamination?

• Using higher Mg2+ concentrations
• Incorporating dUTP and using uracil-DNA glycosylase (UDG) treatment
• Reducing primer length to below 12 bases
• Running fewer cycles only

Correct Answer: Incorporating dUTP and using uracil-DNA glycosylase (UDG) treatment

Q12. In a PCR reaction, which component provides the building blocks for new DNA strands?

• Primers
• Template DNA
• dNTPs (deoxynucleotide triphosphates)
• Buffer

Correct Answer: dNTPs (deoxynucleotide triphosphates)

Q13. What is primer-dimer formation and why is it problematic?

• Annealing of primers to the template causing desired product; not problematic
• Primers forming secondary structures with template DNA increasing yield
• Primers annealing to each other and being amplified, consuming reagents and reducing target yield
• Degradation of primers by exonuclease activity

Correct Answer: Primers annealing to each other and being amplified, consuming reagents and reducing target yield

Q14. Which PCR modification reduces non-specific amplification by delaying polymerase activity until initial high-temperature step?

• Multiplex PCR
• Hot-start PCR
• Nested PCR
• Reverse transcription PCR

Correct Answer: Hot-start PCR

Q15. For a 1 kb target, approximately how long should the extension time be per cycle when using Taq polymerase (∼1 kb/min)?

• 10 seconds
• 30 seconds
• 1 minute
• 10 minutes

Correct Answer: 1 minute

Q16. Which technique uses a nested set of primers to increase specificity and sensitivity?

• Touchdown PCR
• Nested PCR
• RT-PCR
• Digital PCR

Correct Answer: Nested PCR

Q17. What does a melting curve analysis after qPCR primarily assess?

• The concentration of Mg2+ in the reaction
• The size of the PCR product via gel visualization
• The specificity of amplified products by their melting temperatures
• The initial template concentration directly

Correct Answer: The specificity of amplified products by their melting temperatures

Q18. Which real-time PCR probe type uses fluorescence resonance energy transfer (FRET) between two dyes?

• SYBR Green
• TaqMan probe
• Scorpion probe
• Molecular beacon

Correct Answer: TaqMan probe

Q19. Which application of PCR is directly relevant to personalized medicine and drug therapy selection?

• Cloning of plasmids
• Pharmacogenetic genotyping to detect SNPs affecting drug metabolism
• Gel electrophoresis size markers
• Routine staining techniques

Correct Answer: Pharmacogenetic genotyping to detect SNPs affecting drug metabolism

Q20. What is digital PCR primarily used for compared to conventional qPCR?

• Qualitative detection only
• Absolute quantification of target molecules without standard curves
• Faster thermal cycling at lower temperatures
• Amplifying longer fragments (>10 kb) efficiently

Correct Answer: Absolute quantification of target molecules without standard curves

Q21. Which factor decreases PCR efficiency when template contains high GC content?

• Lower annealing temperature improves denaturation of GC-rich regions
• Strong secondary structures and higher melting temperature impede denaturation and polymerase progress
• GC-rich templates always increase yield
• GC content has no effect on PCR

Correct Answer: Strong secondary structures and higher melting temperature impede denaturation and polymerase progress

Q22. Which step in PCR determines the length of the amplified product?

• Denaturation step temperature
• Positioning of forward and reverse primers on the template
• Concentration of Mg2+
• Type of fluorescent dye used

Correct Answer: Positioning of forward and reverse primers on the template

Q23. In PCR primer design, which feature should be avoided to reduce secondary structure formation?

• Balanced GC content (40–60%)
• Repetitive sequences and long runs of a single nucleotide
• Similar melting temperatures for forward and reverse primers
• 3′ end ending in G or C for stronger binding

Correct Answer: Repetitive sequences and long runs of a single nucleotide

Q24. Which control indicates whether PCR reagents are contaminated with target DNA?

• Positive control containing known template
• No-template control (NTC) lacking template DNA
• Blank electrophoresis lane

Correct Answer: No-template control (NTC) lacking template DNA

Q25. Which application of PCR is commonly used in forensic science?

• Amplification of mitochondrial DNA and STR loci for human identification
• Measuring blood glucose levels
• Determining drug purity by HPLC
• Synthesis of large proteins

Correct Answer: Amplification of mitochondrial DNA and STR loci for human identification

Q26. What is the role of buffer in a PCR reaction?

• Provide proper pH and ionic strength for optimal enzyme activity
• Serve as the template for amplification
• Denature DNA strands
• Act as a fluorescent probe

Correct Answer: Provide proper pH and ionic strength for optimal enzyme activity

Q27. Touchdown PCR is primarily used to:

• Decrease reaction volume for cost savings
• Gradually increase annealing temperature to promote non-specific binding
• Start with a higher annealing temperature and decrease it to improve specificity
• Amplify RNA directly without reverse transcription

Correct Answer: Start with a higher annealing temperature and decrease it to improve specificity

Q28. Which statement about Taq polymerase is correct?

• It has strong 3’→5′ proofreading exonuclease activity
• It is inactivated at 95°C
• It is thermostable and functions well at high temperatures used during extension
• It synthesizes RNA primers during PCR

Correct Answer: It is thermostable and functions well at high temperatures used during extension

Q29. In qPCR using hydrolysis probes (TaqMan), what causes the increase in fluorescence during amplification?

• Intercalation of dye into double-stranded DNA
• Cleavage of the probe by the 5’→3′ exonuclease activity of Taq polymerase separating fluorophore from quencher
• Formation of primer-dimers
• Decrease in magnesium concentration

Correct Answer: Cleavage of the probe by the 5’→3′ exonuclease activity of Taq polymerase separating fluorophore from quencher

Q30. Which consideration is essential when interpreting PCR results in clinical diagnostics?

• Only the number of cycles matters, independent of contamination or controls
• Presence of amplification always indicates live infectious organism
• Appropriate controls, sample quality, inhibition, and clinical context must be evaluated alongside PCR results
• PCR cannot detect low copy number targets reliably

Correct Answer: Appropriate controls, sample quality, inhibition, and clinical context must be evaluated alongside PCR results

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