This chapter is highly important for both board and competitive exams because it links core biology with real-world applications of microorganisms and genetic engineering—covering tools like recombinant DNA, vectors, PCR, transformation, and CRISPR. Exam questions commonly test conceptual understanding (mechanisms and purpose) as well as quantitative reasoning (e.g., PCR, restriction site spacing, transformation efficiency), making a clear grasp of biotech workflows essential.
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Marking
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 ≈ bp.)
11 fragments
12 fragments
13 fragments
24 fragments
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 = ), by what approximate factor was the starting template in Sample A greater than in Sample B? (Use relative quantity .)
8‑fold
‑fold
3‑fold
1.9‑fold
Q3. Competent E. coli cells have a measured transformation efficiency of 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.
Both A and R are true, and R explains A.
Both A and R are true, but R does not explain A.
A is true, R is false.
A is false, R is true.
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 () but have different recognition sequences. Which statement best describes the likely cloning outcome?
Compatible ends enforce a single insert orientation and both original restriction sites are restored after ligation, allowing directional cloning by re‑digestion.
Compatible ends prevent ligation between vector and insert; only blunt‑end ligation would allow cloning.
Because the cohesive overhangs are identical the insert can ligate in either orientation (cloning is not strictly directional); ligation between BamHI and BglII ends yields hybrid junctions that are not recognised by either enzyme.
Ligation between BamHI and BglII ends will recreate a site recognised by one of the enzymes, enabling orientation selection by re‑digestion.
Q6. A PCR reaction is set up with 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 cycles?
copies
copies
copies
copies
Q7. A circular plasmid of size is completely digested by restriction enzymes producing these fragment patterns on agarose gel: Enzyme A → and ; Enzyme B → and ; Double digest A + B → , and . Which statement best describes the arrangement of restriction sites?
A has two recognition sites (cutting the plasmid into and fragments). B also has two recognition sites, with one B site coinciding with one A site; the other B site lies within the A fragment, splitting it into and in the double digest.
A has two recognition sites (producing and fragments), but B has two sites such that the extra (non‑overlapping) B site lies within the A fragment; hence the fragment would be split in the double digest.
Neither enzyme shares a restriction site (A and B cut at four distinct positions), so the double digest should produce four fragments rather than three.
A and B sites are adjacent in the plasmid so that the double digest yields only two fragments because one cut position is effectively redundant.
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?
Escherichia coli expression system
Saccharomyces cerevisiae (baker's yeast)
Baculovirus–insect cell expression system
Mammalian cell culture (e.g., CHO or HEK293 cells)
Q9. A researcher attempts CRISPR–Cas9 mediated precise knock‑in of a reporter into a human gene but supplies only a ‑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?
Treat cells with an NHEJ inhibitor (e.g., SCR7) while continuing to use the ‑nt ssODN donor
Synchronize cells in S/G2 phase and provide a double‑stranded DNA donor with long homology arms (e.g., each) flanking the insert
Switch to Cas9 nickase with the same ‑nt ssODN donor to reduce double‑strand break formation
Increase the concentration of Cas9–gRNA complex while leaving donor design and cell cycle status unchanged
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?
(i) and (ii) are true; (iii) is false.
Only (i) is true; (ii) and (iii) are false.
(ii) and (iii) are true; (i) is false.
All three statements (i), (ii) and (iii) are true.