DNA fingerprinting for authentication of herbal materials MCQs With Answer

Introduction: DNA fingerprinting has become an essential tool in the authentication of herbal materials, addressing issues such as species substitution, adulteration and misidentification that compromise safety and efficacy. For M.Pharm students in Advanced Pharmacognosy-II, understanding molecular marker systems, barcoding loci, sequencing strategies and practical challenges (degraded DNA, complex mixtures, reference database quality) is crucial. This set of MCQs focuses on marker selection, laboratory workflows, data interpretation, regulatory implications and advanced techniques like metabarcoding and next-generation sequencing. The questions are designed to deepen conceptual knowledge and practical problem-solving skills needed to apply DNA-based authentication in herbal quality control and research.

Q1. What does DNA fingerprinting for herbal authentication primarily produce?

• A chemical profile based on active constituents
• A microscopic image of plant tissues
• A unique genetic profile based on polymorphic DNA markers
• A chromatographic fingerprint of impurities

Correct Answer: A unique genetic profile based on polymorphic DNA markers

Q2. Which molecular marker is known for high polymorphism, co-dominance and reproducibility in plant authentication?

• RAPD (Random Amplified Polymorphic DNA)
• SSR (Simple Sequence Repeat / microsatellite)
• AFLP (Amplified Fragment Length Polymorphism)
• RFLP (Restriction Fragment Length Polymorphism)

Correct Answer: SSR (Simple Sequence Repeat / microsatellite)

Q3. Which pair of loci is recommended as the standard two-locus barcode for land plants?

• COI and 16S
• ITS and 18S
• rbcL and matK
• trnH-psbA and COI

Correct Answer: rbcL and matK

Q4. For highly processed herbal products with fragmented DNA, which approach is most suitable for species identification?

• Full-length matK sequencing (~1500 bp)
• Microsatellite genotyping requiring long DNA
• Mini-barcodes targeting short amplicons (100–300 bp)
• RAPD using long extension times

Correct Answer: Mini-barcodes targeting short amplicons (100–300 bp)

Q5. Which method is best for simultaneous detection of multiple species in a complex herbal mixture?

• Sanger sequencing of a single PCR product
• DNA metabarcoding using high-throughput sequencing
• Single-locus SSR profiling by capillary electrophoresis
• Chromatographic fingerprinting

Correct Answer: DNA metabarcoding using high-throughput sequencing

Q6. Which marker type consists of tandemly repeated short motifs and is useful for cultivar-level discrimination?

• SNP (Single Nucleotide Polymorphism)
• SSR (Simple Sequence Repeat)
• cpDNA large inversions
• rRNA gene copy number variation

Correct Answer: SSR (Simple Sequence Repeat)

Q7. Which fingerprinting technique uses short arbitrary primers to amplify random genomic segments and is sensitive to PCR conditions?

• AFLP (Amplified Fragment Length Polymorphism)
• SSR genotyping
• RAPD (Random Amplified Polymorphic DNA)
• SNP genotyping by TaqMan assays

Correct Answer: RAPD (Random Amplified Polymorphic DNA)

Q8. What is the basic principle behind AFLP analysis?

• Direct sequencing of mitochondrial COI gene
• Restriction digestion of genomic DNA followed by selective PCR amplification of fragments
• Allele-specific hybridization on microarrays
• Cloning of full-length genes and restriction mapping

Correct Answer: Restriction digestion of genomic DNA followed by selective PCR amplification of fragments

Q9. Why is the nuclear ribosomal ITS region commonly used for plant species discrimination?

• It is a chloroplast gene with very slow evolution
• It shows high interspecific variation and has conserved priming sites
• It codes for an active metabolite used in herbal potency assays
• ITS sequences are identical across all plants

Correct Answer: It shows high interspecific variation and has conserved priming sites

Q10. Which curated database is specifically designed to support standardized DNA barcode records for species identification?

• UniProtKB
• PubChem
• BOLD (Barcode of Life Data Systems)
• EMBL-EBI small molecule database

Correct Answer: BOLD (Barcode of Life Data Systems)

Q11. For absolute and highly precise quantification of a particular species’ DNA in a mixed herbal sample, which method is most appropriate?

• Semi-quantitative agarose gel band intensity
• Conventional end-point PCR
• Digital PCR (dPCR) for absolute quantification
• RFLP followed by densitometry

Correct Answer: Digital PCR (dPCR) for absolute quantification

Q12. What is a key limitation of DNA-based authentication when assessing herbal product quality?

• DNA methods provide direct information about active chemical concentrations
• DNA cannot distinguish between closely related species at any level
• DNA fingerprinting does not inform on phytochemical potency or presence of specific bioactive compounds
• DNA analysis is always faster and cheaper than chemical assays

Correct Answer: DNA fingerprinting does not inform on phytochemical potency or presence of specific bioactive compounds

Q13. What length range is typically targeted for a mini-barcode used on degraded herbal DNA?

• 1000–1500 base pairs
• 500–800 base pairs
• 100–300 base pairs
• 10–50 base pairs

Correct Answer: 100–300 base pairs

Q14. Which extraction approach is commonly modified to remove polyphenols and polysaccharides from dried herbal powders?

• Simple water extraction with heat
• CTAB extraction with PVPP and high salt/β-mercaptoethanol modifications
• Organic solvent extraction using hexane
• Direct PCR without any purification

Correct Answer: CTAB extraction with PVPP and high salt/β-mercaptoethanol modifications

Q15. Which standard plant barcode gene codes for the large subunit of ribulose-1,5-bisphosphate carboxylase and is located in the chloroplast genome?

• ITS (Internal Transcribed Spacer)
• rbcL
• 18S rRNA
• Cytb (cytochrome b)

Correct Answer: rbcL

Q16. SNP markers are particularly useful in herbal authentication for which purpose?

• Broad intergeneric phylogeny across plant families
• High-resolution discrimination at the cultivar or population level
• Detecting long indels >1 kb in chloroplast DNA
• Visual microscopic differentiation of plant tissues

Correct Answer: High-resolution discrimination at the cultivar or population level

Q17. The term “barcoding gap” in DNA barcoding refers to what concept?

• The sequencing read length limit of a given platform
• The difference where average interspecific genetic divergence exceeds intraspecific divergence
• The PCR amplification failure zone due to inhibitors
• The chromatographic separation distance in HPLC

Correct Answer: The difference where average interspecific genetic divergence exceeds intraspecific divergence

Q18. Which practice is essential to control contamination in a DNA authentication workflow?

• Running extraction and PCR without negative controls to save reagents
• Including extraction and PCR negative controls and using physically separated pre- and post-PCR areas
• Using only disposable tips but no blanks
• Pooling samples before extraction to dilute contaminants

Correct Answer: Including extraction and PCR negative controls and using physically separated pre- and post-PCR areas

Q19. From a regulatory perspective which requirement strengthens the legal acceptance of DNA authentication results for herbal products?

• Using unpublished internal sequences without vouchers
• Relying solely on vendor-provided identity claims
• Submission of authenticated voucher specimens and validated reference sequences to public databases
• Only reporting qualitative presence/absence without method validation

Correct Answer: Submission of authenticated voucher specimens and validated reference sequences to public databases

Q20. Which advantage of next-generation sequencing (NGS) is most relevant for authentication of multi-ingredient herbal formulations?

• Ability to produce single long reads only useful for genomes
• High cost per base compared to Sanger sequencing
• Capacity to sequence mixed-template PCR amplicons and generate thousands to millions of reads for species detection
• NGS eliminates the need for DNA extraction

Correct Answer: Capacity to sequence mixed-template PCR amplicons and generate thousands to millions of reads for species detection

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