Application of PCR and genomic tools in plant genome analysis MCQs With Answer

Introduction

Understanding polymerase chain reaction (PCR) and genomic tools is essential for M.Pharm students specializing in medicinal plant biotechnology. This quiz-focused blog explores practical applications of conventional PCR, quantitative and real-time PCR, next-generation sequencing (NGS), molecular markers, and advanced genotyping techniques in plant genome analysis. Questions emphasize experimental design, troubleshooting, interpretation of molecular data, and selecting appropriate tools for gene discovery, authentication of medicinal species, marker-assisted selection, and epigenetic studies. The MCQs are crafted to deepen conceptual understanding and link laboratory methods with real-world applications in phytopharmaceutical research, quality control, and breeding of medicinal plants.

Q1. Which PCR variant is most appropriate for quantifying gene expression levels of a biosynthetic enzyme in different plant tissues?

• Conventional end-point PCR analyzed by gel electrophoresis
• Northern blot hybridization
• Quantitative real-time PCR (qRT-PCR) with internal reference genes
• Random amplified polymorphic DNA (RAPD) PCR

Correct Answer: Quantitative real-time PCR (qRT-PCR) with internal reference genes

Q2. In amplification of genomic DNA from medicinal plant leaves, which of the following plant-derived compounds is most likely to inhibit Taq polymerase and give false negatives?

• Sodium chloride
• Polyphenols and polysaccharides co-extracted with DNA
• Tris-EDTA buffer
• MgCl2 at optimized concentration

Correct Answer: Polyphenols and polysaccharides co-extracted with DNA

Q3. Which molecular marker system provides co-dominant markers and is highly reproducible for fingerprinting medicinal plant cultivars?

• Random amplified polymorphic DNA (RAPD)
• Simple sequence repeats (SSRs or microsatellites)
• Arbitrarily primed PCR (AP-PCR)
• AFLP without selective primers

Correct Answer: Simple sequence repeats (SSRs or microsatellites)

Q4. For detecting single nucleotide polymorphisms (SNPs) across many samples in a candidate gene associated with alkaloid content, which approach is most cost-effective and high-throughput?

• Sanger sequencing of each PCR product individually
• Restriction fragment length polymorphism (RFLP) only
• Targeted amplicon sequencing using next-generation sequencing (NGS)
• Isoelectric focusing of proteins

Correct Answer: Targeted amplicon sequencing using next-generation sequencing (NGS)

Q5. Which PCR modification allows simultaneous amplification of multiple loci in a single reaction to genotype several markers for marker-assisted selection?

• Nested PCR
• Touchdown PCR
• Multiplex PCR with carefully designed primer sets
• Inverse PCR

Correct Answer: Multiplex PCR with carefully designed primer sets

Q6. High-resolution melting (HRM) analysis following PCR is primarily used to:

• Visualize large structural variations on agarose gels
• Detect small sequence differences like SNPs or methylation-associated changes without sequencing
• Quantify absolute copy number without standards
• Enrich for GC-rich regions prior to sequencing

Correct Answer: Detect small sequence differences like SNPs or methylation-associated changes without sequencing

Q7. Which genomic tool is most appropriate for de novo assembly of a large and highly repetitive medicinal plant genome?

• Short-read Illumina sequencing only
• Long-read sequencing technologies (PacBio or Oxford Nanopore) combined with short reads for polishing
• Sanger sequencing of random clones
• qPCR-based genome size estimation

Correct Answer: Long-read sequencing technologies (PacBio or Oxford Nanopore) combined with short reads for polishing

Q8. In DNA barcoding to authenticate medicinal plant species, which chloroplast locus is commonly used because of its balance between universality and discriminatory power?

• ITS2 nuclear ribosomal spacer only
• rbcL and matK combined as standard plant barcodes
• 18S rRNA gene
• COI mitochondrial gene used in animals

Correct Answer: rbcL and matK combined as standard plant barcodes

Q9. During qPCR assay design for a plant gene, which factor is most critical to ensure accurate relative quantification across tissues?

• Using water as no-template control (NTC)
• Selection and validation of stable internal reference (housekeeping) genes
• Running PCR for at least 45 cycles regardless of primer efficiency
• Using very long amplicons (>1000 bp)

Correct Answer: Selection and validation of stable internal reference (housekeeping) genes

Q10. Digital droplet PCR (ddPCR) offers which main advantage over conventional qPCR for detecting low-frequency alleles in heterogeneous plant tissue?

• Lower precision in quantitation
• Absolute quantification without reference standards and higher sensitivity for rare variants
• Ability to sequence the amplicon in the same reaction
• Requires no DNA extraction

Correct Answer: Absolute quantification without reference standards and higher sensitivity for rare variants

Q11. Which technique integrates PCR-based genotyping with forward genetics to identify induced point mutations in target genes of mutagenized plant populations?

• TILLING (Targeting Induced Local Lesions IN Genomes)
• RAPD profiling
• Comparative genomic hybridization (CGH)
• ChIP-Seq

Correct Answer: TILLING (Targeting Induced Local Lesions IN Genomes)

Q12. When preparing genomic DNA libraries for whole-genome bisulfite sequencing to study cytosine methylation in a medicinal plant, which step is crucial?

• Avoiding fragmentation to maintain ultra-long DNA
• Complete conversion of unmethylated cytosines to uracil by bisulfite treatment with controls for conversion efficiency
• Enriching for GC-poor regions only
• Using RNA as input material

Correct Answer: Complete conversion of unmethylated cytosines to uracil by bisulfite treatment with controls for conversion efficiency

Q13. Which approach is best for discovering genes associated with a complex trait like secondary metabolite concentration across diverse plant accessions?

• Genome-wide association study (GWAS) using high-density SNP genotyping
• Single-locus SSR genotyping in one cultivar
• RAPD profiling of a single individual
• Protein electrophoresis only

Correct Answer: Genome-wide association study (GWAS) using high-density SNP genotyping

Q14. For validating CRISPR/Cas9 edits in a medicinal plant gene, which PCR-based method allows quick screening of small insertions/deletions (indels)?

• Gel-free mass spectrometry
• High-resolution melting (HRM) analysis or T7 endonuclease I mismatch assay followed by sequencing
• Southern blotting
• RAPD-PCR

Correct Answer: High-resolution melting (HRM) analysis or T7 endonuclease I mismatch assay followed by sequencing

Q15. Which primer design strategy reduces formation of primer-dimers and non-specific products in multiplex PCR for plant marker analysis?

• Design primers with overlapping complementary 3′ ends
• Ensure primers have similar annealing temperatures and minimal mutual complementarity
• Use very low annealing temperatures for all primer pairs
• Design very long primers (>40 nt) with high GC clamps at 5′ ends

Correct Answer: Ensure primers have similar annealing temperatures and minimal mutual complementarity

Q16. When performing metabarcoding to profile endophytic fungal communities in medicinal plant roots, which sequencing strategy and marker are typically used?

• Shotgun whole-genome sequencing without amplification
• Amplicon sequencing of fungal ITS regions using NGS platforms
• Sanger sequencing of bacterial 16S rRNA
• Microscopy-based spore counting

Correct Answer: Amplicon sequencing of fungal ITS regions using NGS platforms

Q17. In allele-specific PCR to detect a known SNP affecting alkaloid biosynthesis, what is the key design principle for the discriminatory primer?

• Place the SNP at the 5′ end of the primer and include several mismatches internally
• Place the SNP at the 3′ terminal base of the allele-specific primer with deliberate destabilizing mismatches near the 3′ end
• Use extremely long primers to ensure binding despite mismatches
• Design primers in intronic regions only

Correct Answer: Place the SNP at the 3′ terminal base of the allele-specific primer with deliberate destabilizing mismatches near the 3′ end

Q18. Which statement best describes the use of chloroplast genome sequencing in medicinal plant studies?

• Chloroplast genomes are highly variable within individuals and unsuitable for phylogeny
• Complete chloroplast genome sequences provide robust markers for species identification, phylogenetics, and barcoding of plants
• Chloroplast sequencing cannot differentiate closely related species under any circumstances
• Chloroplast genomes are identical across all plant species

Correct Answer: Complete chloroplast genome sequences provide robust markers for species identification, phylogenetics, and barcoding of plants

Q19. For detecting copy number variation (CNV) of a gene encoding a key enzyme in secondary metabolism, which PCR-based method provides relative quantification across samples?

• Conventional PCR with endpoint gel band intensity comparison
• Quantitative PCR (qPCR) using a single-copy reference gene for normalization
• RAPD analysis
• In situ hybridization only

Correct Answer: Quantitative PCR (qPCR) using a single-copy reference gene for normalization

Q20. When designing an experiment to identify methylation changes associated with elicitor-induced metabolite production, which combined approach is most informative?

• RNA-Seq alone without methylation data
• Whole-genome bisulfite sequencing (WGBS) combined with RNA-Seq to correlate methylation changes with gene expression
• Protein blotting alone
• Microscopy of chromatin without sequencing

Correct Answer: Whole-genome bisulfite sequencing (WGBS) combined with RNA-Seq to correlate methylation changes with gene expression

Download