Biotechnology and its Applications Set-1

Q1. A restriction enzyme recognises a 6‑base palindromic sequence. Assuming equal base frequencies and random distribution of bases, estimate the expected number of fragments produced when this enzyme completely digests linear lambda phage DNA of length 48.5 kb. (Average spacing between recognition sites ≈ $4^6 = 4096$ bp.)

Q2. In a real‑time PCR (qPCR) experiment Sample A has Ct = 18 and Sample B has Ct = 21. If the amplification efficiency per cycle is 90% (amplification factor per cycle = $1.90$), by what approximate factor was the starting template in Sample A greater than in Sample B? (Use relative quantity $\propto(1+E)^{\Delta \text{Ct}}$.)

Q3. Competent E. coli cells have a measured transformation efficiency of $2\times10^{8}$ transformants per µg plasmid DNA. A researcher uses 20 ng of plasmid DNA in one transformation, recovers cells in 1 mL total volume and plates 100 µL on selective medium. Approximately how many antibiotic‑resistant colonies should be expected on that plate?

Q4. Assertion (A): Ti‑plasmid mediated Agrobacterium transformation of plant cells always results in integration of a single copy of T‑DNA at a single chromosomal locus in the host genome.
Reason (R): Transfer of T‑DNA to plant cells involves formation and transfer of a single‑stranded "T‑strand" by Vir proteins, which is then converted/integrated into the plant genome via host repair mechanisms.

Q5. A plasmid vector is cut with BamHI at the left cloning site and BglII at the right cloning site; an insert is cut with BamHI at its left end and BglII at its right end. BamHI and BglII produce compatible cohesive overhangs ($5'\text{-}GATC\text{-}3'$) but have different recognition sequences. Which statement best describes the likely cloning outcome?

Q6. A PCR reaction is set up with $50$ copies of a target DNA. Assuming ideal conditions with exact doubling of product each cycle, how many copies of the target will be present after $20$ cycles?

Q7. A circular plasmid of size $8\ \text{kb}$ is completely digested by restriction enzymes producing these fragment patterns on agarose gel: Enzyme A → $3\ \text{kb}$ and $5\ \text{kb}$; Enzyme B → $2\ \text{kb}$ and $6\ \text{kb}$; Double digest A + B → $5\ \text{kb}$, $2\ \text{kb}$ and $1\ \text{kb}$. Which statement best describes the arrangement of restriction sites?

Q8. You need to produce a functional human glycoprotein that (i) contains introns in its genomic sequence and (ii) requires authentic human N-linked glycosylation for activity. Which host system would most reliably yield a correctly folded and glycosylated functional protein with minimal additional engineering?

Q9. A researcher attempts CRISPR–Cas9 mediated precise knock‑in of a $1.5\ \text{kb}$ reporter into a human gene but supplies only a $100$‑nt single‑stranded oligonucleotide (ssODN) as donor. After transfection, most edits are NHEJ-induced indels rather than the intended precise insertion. Which modification would most increase the frequency of precise homology‑directed repair (HDR) knock‑ins?

Q10. Consider these statements about chloroplast (plastid) transformation in angiosperms: (i) plastid genomes are present in many copies per cell, often allowing very high levels of transgene expression; (ii) plastid transgenes are less likely to spread via pollen in most angiosperms because plastids are predominantly maternally inherited; (iii) chloroplasts perform eukaryotic N‑linked glycosylation identical to the endoplasmic reticulum, so chloroplast‑expressed proteins will be glycosylated just like secreted proteins. Which option correctly identifies true and false statements?