Real-time PCR, RT-PCR and gene sequencing applications MCQs With Answer

Introduction:

This collection of MCQs on Real-time PCR, RT-PCR and gene sequencing applications is tailored for M.Pharm students preparing for advanced assessments in Cellular and Molecular Pharmacology. The questions cover core principles, experimental design, assay chemistries, controls, data analysis strategies such as absolute and relative quantification, and key sequencing concepts including library preparation, platform differences, quality metrics and clinical applications like pharmacogenomics and mutation detection. Each item probes practical decisions and interpretation skills you will need in the lab and in literature evaluation. Use these questions to test conceptual understanding, troubleshoot experiments, and strengthen readiness for research and exams.

Q1. Which statement best distinguishes real-time PCR (qPCR) from conventional end-point PCR?

• qPCR provides qualitative detection only at the end of amplification
• qPCR quantifies DNA during exponential amplification by measuring fluorescence each cycle
• Conventional PCR measures fluorescence in real time while qPCR measures product on a gel
• qPCR uses RNA as template while conventional PCR uses DNA

Correct Answer: qPCR quantifies DNA during exponential amplification by measuring fluorescence each cycle

Q2. In SYBR Green–based qPCR, which factor most directly causes a false increase in fluorescence signal?

• Probe degradation
• Formation of primer-dimers or non-specific products
• High template GC content only
• Using a hydrolysis probe instead of intercalating dye

Correct Answer: Formation of primer-dimers or non-specific products

Q3. The ΔΔCt method for relative quantification assumes which key condition about the target and reference amplifications?

• Both assays have identical initial template copy numbers
• Amplification efficiencies of target and reference are approximately equal and near 100%
• Reference gene expression changes proportionally with treatment
• PCR product sizes must be identical for target and reference

Correct Answer: Amplification efficiencies of target and reference are approximately equal and near 100%

Q4. Which control specifically tests for genomic DNA contamination in an RT-PCR (reverse transcription PCR) workflow?

• No-template control (NTC) containing water instead of RNA
• No-RT control where reverse transcriptase is omitted during cDNA synthesis
• Positive control with known RNA target
• Internal reference gene control such as GAPDH

Correct Answer: No-RT control where reverse transcriptase is omitted during cDNA synthesis

Q5. Which probe chemistry enables multiplex qPCR with distinct fluorescent reporters and high specificity due to probe cleavage?

• SYBR Green intercalating dye
• TaqMan hydrolysis probes
• Molecular beacons without fluorescence quenchers
• Ethidium bromide staining

Correct Answer: TaqMan hydrolysis probes

Q6. When preparing RNA for RT-qPCR, the RNA Integrity Number (RIN) is important because:

• A low RIN increases PCR efficiency
• RNA fragmentation may bias quantification of transcripts, affecting reproducibility and interpretation
• RIN determines the GC content of transcripts
• RIN measures only DNA contamination levels

Correct Answer: RNA fragmentation may bias quantification of transcripts, affecting reproducibility and interpretation

Q7. Which sequencing approach is most appropriate for detecting rare somatic mutations across a focused set of pharmacogenes at high sensitivity?

• Whole-genome sequencing at low coverage
• Targeted amplicon sequencing with deep coverage and unique molecular identifiers (UMIs)
• RNA-Seq without enrichment
• Whole-exome sequencing at standard coverage

Correct Answer: Targeted amplicon sequencing with deep coverage and unique molecular identifiers (UMIs)

Q8. In qPCR data analysis, melting curve analysis is primarily used to:

• Determine absolute copy number using a standard curve
• Assess product specificity by identifying distinct melting temperatures of amplicons
• Quantify RNA integrity before reverse transcription
• Label probes with fluorescent dyes

Correct Answer: Assess product specificity by identifying distinct melting temperatures of amplicons

Q9. A TaqMan probe is labeled at the 5′ end with a fluorophore and at the 3′ end with a quencher. How does fluorescence increase during PCR?

• Probe is incorporated intact into product and fluorescence increases due to cumulative probe concentration
• Polymerase-mediated probe cleavage separates fluorophore from quencher during extension, releasing fluorescence
• Quencher emits fluorescence when bound to polymerase
• Fluorescence increases only because of product melting at the end of PCR

Correct Answer: Polymerase-mediated probe cleavage separates fluorophore from quencher during extension, releasing fluorescence

Q10. Which parameter is most critical when designing qPCR primers to prevent amplification bias and increase efficiency?

• Primers should have highly variable melting temperatures differing by >10°C
• Primers should avoid secondary structures and have similar melting temperatures within 1–3°C and minimal dimerization potential
• Primers must always be longer than 40 bases
• Primers should always include restriction sites for cloning

Correct Answer: Primers should avoid secondary structures and have similar melting temperatures within 1–3°C and minimal dimerization potential

Q11. In absolute quantification by qPCR using a standard curve, which source of error most directly affects accurate copy number estimation?

• Using a single reference gene for normalization
• Inaccurate concentration or degradation of the standard material used to generate the curve
• Not performing a melting curve
• Using SYBR Green instead of probe chemistry

Correct Answer: Inaccurate concentration or degradation of the standard material used to generate the curve

Q12. Which sequencing quality metric is described by Phred score 30 (Q30)?

• Probability of an incorrect base call is 1 in 10
• Probability of an incorrect base call is 1 in 1000, corresponding to 99.9% accuracy
• Average read length of 30 bases
• Coverage depth of 30× across the genome

Correct Answer: Probability of an incorrect base call is 1 in 1000, corresponding to 99.9% accuracy

Q13. For measuring viral load in patient samples, which qPCR feature is most important for reliable clinical monitoring?

• Use of relative quantification without standards
• Use of a validated, quantitative standard curve and an internal extraction control to account for sample-to-sample variation
• Exclusive reliance on melting curve peaks
• Use of high cycle numbers (>50) to detect late amplification only

Correct Answer: Use of a validated, quantitative standard curve and an internal extraction control to account for sample-to-sample variation

Q14. Which statement about RNA-Seq versus RT-qPCR for gene expression analysis is most accurate?

• RT-qPCR provides unbiased transcript discovery across the whole transcriptome
• RNA-Seq gives genome-wide expression profiles and novel isoform detection, while RT-qPCR offers higher sensitivity and precision for targeted genes
• RNA-Seq cannot detect splice variants
• RT-qPCR is always more cost-effective for large-scale transcriptome profiling

Correct Answer: RNA-Seq gives genome-wide expression profiles and novel isoform detection, while RT-qPCR offers higher sensitivity and precision for targeted genes

Q15. Unique molecular identifiers (UMIs) are used in sequencing library preparation to:

• Increase fragment GC content
• Differentiate original molecules from PCR duplicates to improve quantification accuracy
• Replace adapters during sequencing runs
• Eliminate need for sequencing quality control

Correct Answer: Differentiate original molecules from PCR duplicates to improve quantification accuracy

Q16. Which of the following is a common cause of inaccurate ΔCt values in relative qPCR experiments?

• Using validated primers with no secondary structures
• Variability in cDNA synthesis efficiency between samples when reverse transcription is inconsistent
• Running all samples on the same plate with identical reagents
• Using an internal control gene with stable expression across conditions

Correct Answer: Variability in cDNA synthesis efficiency between samples when reverse transcription is inconsistent

Q17. In targeted sequencing panels for pharmacogenomics, “coverage depth” primarily impacts which outcome?

• The ability to detect low-frequency variants with statistical confidence
• The physical length of the sequencer run
• The GC bias of the sequencer
• Primer melting temperature

Correct Answer: The ability to detect low-frequency variants with statistical confidence

Q18. Which library quantification method is preferred before sequencing to ensure accurate cluster density on Illumina platforms?

• Nanodrop absorbance measurement alone
• qPCR-based library quantification targeting adapter sequences
• Visual estimation of concentration on a gel
• Measuring pH of the library solution

Correct Answer: qPCR-based library quantification targeting adapter sequences

Q19. When interpreting qPCR amplification plots, what does an earlier Ct (lower cycle threshold) indicate, all else being equal?

• Lower initial template amount
• Higher initial template amount
• Poor PCR efficiency
• Complete absence of template

Correct Answer: Higher initial template amount

Q20. Which bioinformatic approach is most appropriate for identifying novel splice variants from RNA-Seq data in pharmacology research?

• Align reads to the reference genome with a splice-aware aligner and assemble transcripts to detect novel junctions
• Use only de novo assembly without reference alignment for all samples regardless of annotation quality
• Count reads mapping only to exonic regions ignoring junction reads
• Perform qPCR instead of any computational analysis

Correct Answer: Align reads to the reference genome with a splice-aware aligner and assemble transcripts to detect novel junctions

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