Biotechnology and its Applications Set-5

Q1. In PCR each cycle ideally doubles the number of DNA copies following $N = N_0\times 2^n$. If $N_0 = 250$ and $n = 3$, what is the expected number of DNA molecules produced (assuming 100% efficiency)?

Q2. In a cloning experiment a plasmid vector linearized with EcoRI undergoes ligation where 40% of ligation events are vector recircularization (no insert) and 60% are vector–insert ligations. Because both ends are generated by the same enzyme, insert ligation can occur in either orientation with equal probability. If transformation yields 1000 colonies, approximately how many colonies would contain the insert in the correct orientation?

Q3. A genomic locus is flanked by HindIII sites at 1 kb and 7 kb producing a 6 kb HindIII fragment that hybridizes with a probe located within this region in the wild-type allele. A mutant allele contains a 2 kb insertion between those HindIII sites (so the corresponding HindIII fragment becomes 8 kb). After HindIII digestion and Southern blot with the same probe, which band pattern would you expect for an individual heterozygous for the insertion?

Q4. Statement (S): Eukaryotic proteins expressed in Escherichia coli frequently form insoluble inclusion bodies that can be purified and subsequently solubilized and refolded in vitro to recover biologically active protein.
Reason (R): Inclusion body formation in E. coli is mainly due to hyperglycosylation of eukaryotic polypeptides by bacterial enzymes; removal of those glycans during refolding restores native folding and activity.
Which of the following is correct?

Q5. A monogenic autosomal recessive liver disorder requires gene replacement therapy to restore enzyme activity in hepatocytes while minimizing the risk of insertional mutagenesis and immune responses. Which vector/strategy best balances long-term hepatocyte expression with safety?

Q6. In a PCR experiment each template molecule produces $1+e$ copies per cycle (where $e$ is polymerase efficiency as a fraction of the maximal additional strands). Starting with $10$ template molecules and $e=0.90$, after $20$ cycles the expected number of DNA molecules is approximately $N=10(1.9)^{20}$. Which value is closest to $N$?

Q7. In a ligation setup you have a plasmid vector of length $4000\ \text{bp}$ and an insert of length $1000\ \text{bp}$. You use $50\ \text{ng}$ of vector and desire an insert:vector molar ratio of $3:1$. Using the relation $m_{\text{insert}}=m_{\text{vector}}\times\text{ratio}\times\frac{\text{bp}_{\text{insert}}}{\text{bp}_{\text{vector}}}$, the mass of insert required is closest to:

Q8. In STR profiling, the probability of a heterozygous genotype $a/b$ at a locus is $2p_a p_b$. For locus A the alleles 10 and 12 have frequencies $p_{10}=0.20$ and $p_{12}=0.10$; for locus B the alleles 7 and 8 have frequencies $p_{7}=0.15$ and $p_{8}=0.25$. If an individual is 10/12 at A and 7/8 at B, what is the probability that a random unrelated person matches this genotype at both loci (assume independence between loci)?

Q9. A researcher compares expression of gene X by qPCR. Ct values are: control — $C_{t,X}=25$, $C_{t,ref}=18$; treated — $C_{t,X}=22$, $C_{t,ref}=16$. Using $\Delta C_t=C_{t,X}-C_{t,ref}$ and fold change $=2^{-\Delta\Delta C_t}$, what is the fold change of X in treated relative to control?

Q10. A transgene that contains no EcoRI recognition sites was introduced into plants. Genomic DNA from two independent transformants P and Q was digested with EcoRI and hybridized with a probe specific to the transgene. P shows a single hybridizing band; Q shows two hybridizing bands of different sizes. Which inference is best supported by these Southern blot results?

Q11. In a PCR reaction starting with $N_0=100$ template molecules, each cycle amplifies with an efficiency of $90\%$ (i.e., $E=0.9$). Using the relation $N=N_0(1+E)^n$, estimate the number of DNA molecules after $n=30$ cycles (nearest power-of-ten estimate).

Q12. A circular plasmid of total length $5000\ \text{bp}$ contains a $1200\ \text{bp}$ insert occupying positions $1000$–$2200$. Primer PF anneals at position $900$ and extends in the $5'\to3'$ direction (toward increasing position numbers). Primer PR anneals at position $2300$ and is oriented $5'\to3'$ toward decreasing position numbers (i.e., a reverse primer). Colony PCR using PF and PR yields a single product. What is the expected size of the PCR product?

Q13. The relationship between fragment size (in bp) and migration distance $d$ (in cm) in agarose gel can be approximated as $\log_{10}(\text{size})=a\,d+b$. A DNA ladder shows a $1000\ \text{bp}$ band at $d=2.0\ \text{cm}$ and a $100\ \text{bp}$ band at $d=8.0\ \text{cm}$. An unknown band migrates to $d=4.0\ \text{cm}$. Using the two ladder points to determine the linear relation, estimate the size of the unknown fragment (nearest whole bp).

Q14. A T-DNA construct (length $6\ \text{kb}$) used for plant transformation contains a single EcoRI site located $2\ \text{kb}$ from its left border. A probe internal to the T-DNA hybridizes within the transformant's genomic DNA. Genomic DNA from the transformant was digested separately with BamHI (which has no recognition sites within the T-DNA) and with EcoRI. Southern blotting with the internal probe gave: BamHI digest — a single hybridizing band of $12\ \text{kb}$; EcoRI digest — two hybridizing bands of $3\ \text{kb}$ and $9\ \text{kb}$. Which configuration of the T-DNA copies in the plant genome best explains these results?

Q15. Gene X has a processed (intronless) pseudogene in the genome whose sequence is identical to the spliced mRNA of gene X. You extract total RNA from tissue and want to detect expression of gene X by RT–PCR without false positives from genomic DNA (including the processed pseudogene). Which strategy most reliably ensures the amplified product originates from RNA-derived cDNA only?