PCR Applications: Principles, instrumentation and use in gene/regulatory studies MCQs With Answer

Introduction: This quiz collection focuses on PCR applications, covering core principles, instrumentation and specific uses in gene and regulatory studies relevant to the M.Pharm curriculum. Questions are designed to test and deepen understanding of thermal cycling steps, enzyme selection, primer design, reaction optimization, real‑time quantification approaches, advanced variants such as digital, multiplex and nested PCR, and practical uses like ChIP‑PCR and mutation detection. Emphasis is placed on interpretation of qPCR data, contamination control, and selection of appropriate polymerases for high fidelity or long templates. Use these MCQs to reinforce conceptual knowledge and to prepare for advanced laboratory design and critical assessment of PCR‑based regulatory gene studies.

Q1. What are the three fundamental temperature-dependent steps of conventional PCR?

• Denaturation, annealing and extension
• Translation, transcription and replication
• Activation, ligation and termination
• Melting, hybridization and polymerization

Correct Answer: Denaturation, annealing and extension

Q2. Which statement best describes the role of Mg2+ in a PCR reaction?

• Cofactor for DNA polymerase and affects primer-template binding and fidelity
• Acts as a chelating agent to remove inhibitors from the sample
• Serves as the fluorescent reporter in real-time detection
• Denatures double-stranded DNA at high temperature

Correct Answer: Cofactor for DNA polymerase and affects primer-template binding and fidelity

Q3. Which characteristic correctly describes Taq DNA polymerase commonly used in PCR?

• Thermostable DNA polymerase from Thermus aquaticus lacking 3’→5′ exonuclease activity
• High-fidelity polymerase from Pyrococcus with intrinsic proofreading
• Reverse transcriptase that synthesizes DNA from RNA templates
• Endonuclease that digests single-stranded DNA during PCR

Correct Answer: Thermostable DNA polymerase from Thermus aquaticus lacking 3’→5′ exonuclease activity

Q4. What is the primary advantage of hot‑start PCR?

• Reduce non-specific amplification by preventing polymerase activity until initial denaturation
• Allow continuous isothermal amplification at a single temperature
• Enable direct sequencing of PCR products without purification
• Increase the melting temperature (Tm) of primers

Correct Answer: Reduce non-specific amplification by preventing polymerase activity until initial denaturation

Q5. Which real-time PCR chemistry reports signal by binding to any double‑stranded DNA product?

• SYBR Green I intercalating dye
• TaqMan hydrolysis probe
• Molecular beacon probe
• Scorpion probe

Correct Answer: SYBR Green I intercalating dye

Q6. In quantitative PCR, what does the Ct (threshold cycle) value represent?

• The cycle number at which fluorescence exceeds the threshold; inversely proportional to initial template amount
• The absolute number of target copies present in the reaction
• The final plateau fluorescence intensity after all cycles
• The melting temperature of the amplified product

Correct Answer: The cycle number at which fluorescence exceeds the threshold; inversely proportional to initial template amount

Q7. What is a main advantage of digital droplet PCR (ddPCR) over conventional qPCR?

• Absolute quantification of target molecules without the need for a standard curve
• Faster thermal cycling due to continuous flow
• Lower sensitivity to inhibitors but only qualitative results
• Generates longer amplicons (>10 kb) efficiently

Correct Answer: Absolute quantification of target molecules without the need for a standard curve

Q8. Which primer type is most selective for synthesizing cDNA from polyadenylated mRNA?

• Oligo(dT) primers anneal to poly(A) tails and preferentially prime mRNA
• Random hexamers prime uniformly across all RNA species
• Gene-specific primers only prime rRNA and tRNA
• Ribosomal RNA primers increase mRNA yield selectively

Correct Answer: Oligo(dT) primers anneal to poly(A) tails and preferentially prime mRNA

Q9. What is essential when designing a multiplex PCR assay?

• Careful primer design and non‑overlapping amplicon sizes or differently labeled probes
• Using the same primer sequences for all targets to simplify amplification
• Removing Mg2+ to avoid cross-reactivity among targets
• Limiting reaction volume to less than 10 µL to prevent competition

Correct Answer: Careful primer design and non‑overlapping amplicon sizes or differently labeled probes

Q10. What is the main purpose of nested PCR?

• Increase sensitivity and specificity by using internal primers in a second amplification
• Allow amplification of circular DNA without denaturation
• Simultaneously amplify RNA and DNA targets in one reaction
• Reduce the need for primer design by using universal primers

Correct Answer: Increase sensitivity and specificity by using internal primers in a second amplification

Q11. How is melting curve analysis used after real‑time PCR?

• Assess specificity of amplified products by measuring melting temperature profile
• Quantify absolute copy number using peak height calibration
• Control enzyme activity by determining denaturation temperature
• Calculate primer Tm for future reactions

Correct Answer: Assess specificity of amplified products by measuring melting temperature profile

Q12. What effect can excessively high dNTP concentrations have in PCR?

• Reduce fidelity and chelate Mg2+ leading to decreased polymerase accuracy
• Prevent primer annealing by increasing melting temperature dramatically
• Enhance proofreading activity of Taq polymerase
• Stabilize secondary structures and improve specificity

Correct Answer: Reduce fidelity and chelate Mg2+ leading to decreased polymerase accuracy

Q13. Which DNA polymerase is commonly chosen when high fidelity (proofreading) is required?

• Pfu DNA polymerase with 3’→5′ exonuclease proofreading activity
• Taq polymerase lacking exonuclease activity
• Reverse transcriptase derived polymerase
• Endonuclease I that cleaves mispaired bases

Correct Answer: Pfu DNA polymerase with 3’→5′ exonuclease proofreading activity

Q14. What is the primary application of ChIP‑PCR in regulatory gene studies?

• Chromatin immunoprecipitation followed by PCR to detect protein‑bound DNA regions
• Quantitative measurement of mRNA expression using chromatin markers
• Amplification of entire chromosomes to study copy number variations
• High‑throughput sequencing library preparation without enrichment

Correct Answer: Chromatin immunoprecipitation followed by PCR to detect protein‑bound DNA regions

Q15. What do the MIQE guidelines promote for qPCR experiments?

• Minimum Information for Publication of Quantitative Real‑Time PCR Experiments to improve transparency and reproducibility
• Mandatory use of SYBR Green chemistry for all published qPCR work
• Standardized primer sequences for common housekeeping genes only
• Elimination of no‑template controls to streamline workflows

Correct Answer: Minimum Information for Publication of Quantitative Real‑Time PCR Experiments to improve transparency and reproducibility

Q16. Which factors most strongly influence primer melting temperature (Tm)?

• Primer length, GC content and salt concentration in the buffer
• Polymerase type and dNTP concentration
• Template secondary structure only
• Number of PCR cycles and annealing time

Correct Answer: Primer length, GC content and salt concentration in the buffer

Q17. Which method is widely used to prevent PCR carryover contamination?

• dUTP/uracil‑N‑glycosylase (UNG) system to degrade carryover PCR products
• Using lower annealing temperatures to reduce non-specific priming
• Adding RNase to all reactions before amplification
• Substituting Mg2+ with Ca2+ in the reaction buffer

Correct Answer: dUTP/uracil‑N‑glycosylase (UNG) system to degrade carryover PCR products

Q18. How do primer‑dimers affect PCR and how can they be minimized?

• They consume reagents and reduce target yield; minimize by avoiding complementary 3′ sequences in primers
• They increase product length and improve fidelity; minimize by increasing Mg2+
• They enhance fluorescence signal in qPCR and are desirable for detection
• They convert single‑stranded templates to double‑stranded DNA and improve efficiency

Correct Answer: They consume reagents and reduce target yield; minimize by avoiding complementary 3′ sequences in primers

Q19. For amplification of long targets (>5 kb), which strategy is recommended?

• Use high‑fidelity, highly processive polymerases and optimized buffers (e.g., Phusion, LongAmp)
• Reduce extension time per cycle to prevent over‑extension
• Use only Taq polymerase at very high Mg2+ concentration
• Perform nested PCR with short amplicons to infer long target presence

Correct Answer: Use high‑fidelity, highly processive polymerases and optimized buffers (e.g., Phusion, LongAmp)

Q20. What distinguishes absolute from relative quantification in qPCR?

• Absolute quantification uses a standard curve with known template amounts; relative quantification uses an internal reference gene and ΔΔCt
• Absolute quantification is always less accurate than relative methods
• Relative quantification requires external standards with known copy numbers
• Absolute quantification cannot be used for rare targets

Correct Answer: Absolute quantification uses a standard curve with known template amounts; relative quantification uses an internal reference gene and ΔΔCt

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