Polymerase chain reaction techniques MCQs With Answer

Polymerase chain reaction techniques MCQs With Answer

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Introduction

This quiz collection on Polymerase Chain Reaction (PCR) techniques is designed for M.Pharm students studying Advanced Pharmaceutical Biotechnology. It covers core principles, enzyme and reagent roles, advanced variants (qPCR, RT-PCR, digital PCR), primer design, optimization strategies, troubleshooting, and applications in pharmaceutical research such as genotyping, quality control and mutation detection. Each question targets conceptual understanding and practical problem-solving to strengthen laboratory decision-making and exam readiness. Use these MCQs to test knowledge, identify weak areas and prepare for both theoretical and practical assessments in molecular methods relevant to drug development, pharmacogenomics and biopharmaceutical quality assurance.

Q1. Which component of a standard PCR mix is primarily responsible for synthesizing the new DNA strand during thermal cycling?

  • Taq DNA polymerase
  • DNA ligase
  • Reverse transcriptase
  • Exonuclease I

Correct Answer: Taq DNA polymerase

Q2. In quantitative real-time PCR (qPCR) using SYBR Green, which factor most directly allows quantification of initial template amount?

  • Threshold cycle (Ct) value
  • Final reaction volume
  • Primer GC clamp
  • Annealing temperature

Correct Answer: Threshold cycle (Ct) value

Q3. Which PCR variant is most appropriate when first converting RNA into complementary DNA prior to amplification?

  • Reverse transcription PCR (RT-PCR)
  • Nested PCR
  • Hot-start PCR
  • Touchdown PCR

Correct Answer: Reverse transcription PCR (RT-PCR)

Q4. When designing primers, which of the following characteristics is least desirable because it can promote primer-dimer formation?

  • Complementarity at 3′ ends between primers
  • Balanced GC content between 40–60%
  • Absence of long runs of a single nucleotide
  • Minimal internal secondary structure

Correct Answer: Complementarity at 3′ ends between primers

Q5. Hot-start PCR improves specificity primarily by which mechanism?

  • Preventing polymerase activity until initial denaturation is complete
  • Increasing primer concentration dramatically
  • Using higher magnesium concentrations throughout
  • Extending annealing time at low temperatures

Correct Answer: Preventing polymerase activity until initial denaturation is complete

Q6. Which parameter is directly adjusted to change the stringency of primer annealing during PCR?

  • Annealing temperature
  • Extension time
  • Initial denaturation time
  • dNTP concentration

Correct Answer: Annealing temperature

Q7. In probe-based qPCR (TaqMan), how is fluorescence generated?

  • Probe cleavage by Taq polymerase 5’→3′ exonuclease activity separates reporter and quencher
  • Intercalation of dye into double-stranded DNA
  • Hybridization of two separate fluorescent primers
  • Emission by a fluorescent dNTP incorporated into the strand

Correct Answer: Probe cleavage by Taq polymerase 5’→3′ exonuclease activity separates reporter and quencher

Q8. Which PCR modification is best suited for detecting rare mutations present at very low frequencies within a high background of wild-type DNA?

  • Digital PCR
  • Standard end-point PCR
  • Multiplex PCR with many primer pairs
  • Touchdown PCR

Correct Answer: Digital PCR

Q9. What is the main advantage of nested PCR compared with conventional single-round PCR?

  • Increased specificity by using two successive primer pairs
  • Shorter overall run time due to fewer cycles
  • Quantitative measurement of initial template without standards
  • Avoidance of contamination entirely

Correct Answer: Increased specificity by using two successive primer pairs

Q10. Which reagent concentration is most commonly optimized to balance enzyme function and primer-template interactions in PCR?

  • Magnesium ion concentration
  • PEG concentration
  • SDS concentration
  • EDTA concentration

Correct Answer: Magnesium ion concentration

Q11. When interpreting qPCR melt curves generated with SYBR Green, what indicates the presence of a single specific amplicon?

  • A single sharp melting peak at the expected Tm
  • Multiple broad peaks across a range of temperatures
  • A flat baseline with no peaks
  • A shift of peak to lower temperature after each cycle

Correct Answer: A single sharp melting peak at the expected Tm

Q12. Which of the following is a common PCR inhibitor in clinical samples that can reduce amplification efficiency?

  • Hemoglobin
  • Water
  • NaCl at low physiological concentration
  • EDTA-free buffer

Correct Answer: Hemoglobin

Q13. According to MIQE guidelines, which element is essential to report for quantitative PCR experiments to ensure reproducibility?

  • Primer sequences and amplicon size
  • Color of reaction tubes
  • Operator’s initials
  • Brand of laboratory coat used

Correct Answer: Primer sequences and amplicon size

Q14. What does PCR efficiency of 100% indicate about an exponential amplification reaction?

  • Product amount doubles with each cycle during exponential phase
  • The reaction produces no nonspecific products
  • All template molecules are single-stranded initially
  • The Ct value is constant regardless of template amount

Correct Answer: Product amount doubles with each cycle during exponential phase

Q15. Touchdown PCR improves specificity by which approach?

  • Starting with higher annealing temperatures and gradually decreasing them
  • Decreasing extension time progressively each cycle
  • Using progressively lower primer concentrations
  • Increasing the magnesium concentration each cycle

Correct Answer: Starting with higher annealing temperatures and gradually decreasing them

Q16. In reverse transcription quantitative PCR (RT-qPCR), which control distinguishes genomic DNA contamination from true cDNA amplification?

  • No-RT control (reaction without reverse transcriptase)
  • No-template water control
  • Positive amplification control using plasmid
  • Internal amplification control with synthetic RNA spike-in

Correct Answer: No-RT control (reaction without reverse transcriptase)

Q17. Which method is commonly used to avoid carryover contamination in PCR when opening amplified product tubes?

  • Uracil-DNA glycosylase (UNG) system with dUTP substitution
  • Using standard dTTP exclusively
  • Reducing cycle number to below 10
  • Adding RNase A to the PCR mix

Correct Answer: Uracil-DNA glycosylase (UNG) system with dUTP substitution

Q18. Which calculated parameter from a qPCR standard curve is used to determine assay sensitivity and dynamic range?

  • Slope and linearity (R2) of the standard curve
  • Color of the standard solution
  • Initial denaturation temperature only
  • Primer secondary structure prediction

Correct Answer: Slope and linearity (R2) of the standard curve

Q19. Multiplex PCR allows simultaneous amplification of multiple targets. What is the primary challenge when designing a multiplex assay?

  • Avoiding primer–primer interactions and balancing primer efficiencies
  • Eliminating template DNA from the reaction
  • Ensuring only one cycle is performed
  • Preventing polymerase from binding to primers

Correct Answer: Avoiding primer–primer interactions and balancing primer efficiencies

Q20. Digital PCR quantifies target molecules by partitioning the sample. Which statistical model underlies absolute quantification in digital PCR?

  • Poisson distribution
  • Normal distribution
  • Binomial distribution without correction
  • Uniform distribution

Correct Answer: Poisson distribution

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