Biotechnology and its Applications Set-3

Q1. A circular plasmid of length $6000\ \text{bp}$ has unique EcoRI and HindIII restriction sites at positions $1000\ \text{bp}$ and $2500\ \text{bp}$ respectively (positions measured clockwise from a reference point). The plasmid is double-digested with EcoRI and HindIII and run on an agarose gel. Which set of fragment sizes is expected?

Q2. A eukaryotic secretory protein of size $\sim 20\ \text{kDa}$ contains three disulfide bonds and two $N$-linked glycosylation sites. Its coding sequence (without any signal peptide) is cloned into a pET vector and expressed in E. coli BL21; most recombinant protein accumulates as insoluble inclusion bodies and is inactive. Which strategy is most likely to produce correctly folded, biologically active protein with native disulfide bonds and proper glycosylation?

Q3. A therapeutic human serum glycoprotein (~$50\ \text{kDa}$) requires human‑like complex $N$-linked glycosylation to avoid immunogenic responses in patients. Which expression system will most reliably provide human-like complex N-glycans and is commonly used for large‑scale biopharmaceutical production?

Q4. Assertion (A): Integrative retroviral vectors are unsuitable for gene therapy aimed at permanent correction in terminally differentiated non‑dividing cells.
Reason (R): Gammaretroviral vectors require cell division for integration into the host genome, whereas lentiviral vectors (a subclass of retroviruses) can transduce non‑dividing cells and integrate stably.

Q5. To detect a point mutation where the wild‑type allele has base $G$ and the mutant allele has base $A$ at a specific locus, an allele‑specific PCR is planned with the forward primer's 3′ terminal base overlapping the SNP. Which primer design and experimental strategy will most effectively amplify only the mutant allele while minimizing amplification from the wild‑type allele?

Q6. A single double‑stranded DNA molecule is subjected to PCR for 25 cycles. Assuming ideal amplification (each cycle doubles the number of DNA molecules), how many double‑stranded DNA molecules will be present after 25 cycles? Use the relation $N=2^{n}$ where $n$ is the number of cycles.

Q7. A lacZ-based vector is ligated with a mammalian cDNA insert and transformed into E. coli. After plating on X‑gal/IPTG plates there are 240 blue colonies and 760 white colonies. Assume every white colony contains an insert (no background), insert orientation relative to the bacterial promoter is random (50% chance of the correct orientation), and only one of three reading frames (1/3 chance) yields a functional protein when cloned without added bases. Using $N=760\times\frac{1}{2}\times\frac{1}{3}$, how many colonies are expected to express the functional protein?

Q8. In viral transduction experiments the multiplicity of infection (MOI) is $m$. The probability that a cell receives zero viral particles is $P(0)=e^{-m}$. What minimum MOI is required so that at least 95% of cells receive at least one viral particle (i.e., probability of infection $\ge 0.95$)?

Q9. Assertion (A): To obtain active human insulin in E. coli, the insulin coding sequence must be cloned as $cDNA$ rather than genomic DNA because prokaryotes cannot splice introns.
Reason (R): Using $cDNA$ guarantees correct protein folding because introns, if present, would be translated into amino acids that cause misfolding.

Q10. A secreted mammalian enzyme requires N‑linked glycosylation and multiple disulfide bonds for activity. Which expression strategy is most likely to produce correctly folded, glycosylated, active enzyme at scale?