Q.45 Identify the CORRECT statements
P. 5′ and 3′ ends of the transcripts can be mapped by utilizing polymerase chain reaction
Q. S1 nuclease can cleave the DNA strand of a DNA–RNA hybrid
R. T4 polynucleotide kinase is used for labeling 3′ end of DNA
S. Baculovirus (Autographa californica) can be used as an insect expression vector
(A) P and Q only
(B) R and S only
(C) P and S only
(D) Q and R only
Correct Answer: (D) Q and S only
S1 nuclease specifically cleaves the single-stranded DNA strand in DNA-RNA hybrids while sparing RNA (Q true), and baculovirus Autographa californica serves as a standard insect expression vector via polyhedrin/p10 promoter replacement (S true). P false (5’/3′ mapping uses RACE/3’RACE, S1 protection, primer extension—not PCR alone); R false (T4 PNK labels 5′ ends with γ-[³²P]ATP).
Option Analysis
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(A) P and Q only: Incorrect. PCR amplifies known regions; transcript ends require ligation (5’/3′ RACE) or protection assays.
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(B) R and S only: Incorrect. T4 PNK transfers 5′-phosphate from ATP to 5′-OH (forward reaction); 3′ labeling uses terminal transferase.
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(C) P and S only: Incorrect. P false; direct PCR doesn’t map unknown ends without prior adapter ligation.
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(D) Q and S only: Correct. S1 nuclease hydrolyzes ssDNA in hybrids (RNA mapping); AcMNPV infects Sf9/High Five cells for eukaryotic protein production.
S1 nuclease DNA-RNA hybrid cleavage and baculovirus Autographa californica insect expression vector define Q.45 correct statements, testing molecular biology techniques essential for biochemical engineering recombinant protein production.
Technique Validation
S1 Nuclease Mapping (Q): Hybridize end-labeled ssDNA probe to target RNA → S1 digests unprotected ssDNA → protected RNA-DNA fragment size indicates 5’/3′ ends (e.g., 200 nt protection = TSS 200 nt from label).
Protected dsRNA-DNA: resistant
ssDNA overhangs: cleaved to 5'-P oligos
pH 4.6, 25°C, 30 min optimal
Baculovirus Expression (S): AcMNPV genome (134 kbp) → transfer plasmid replaces polyhedrin gene → homologous recombination in Sf9 cells → 10⁸ pfu/mL titers → post-translational modifications superior to E. coli.
False Statements:
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P: 5′ RACE (TAP dephosphorylate → ligate adapter → RT-PCR); 3′ RACE (oligo-dT).
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R: T4 PNK: 5′-OH + [γ-³²P]ATP → 5′-³²P; KpnI leaves 3′-OH for TdT labeling.
Applications Table
| Technique | Purpose | Key Reagent |
|---|---|---|
| S1 Mapping | Transcript ends | S1 nuclease |
| Baculovirus | Insect expression | AcMNPV polyhedrin |
| 5′ RACE | 5′ UTR mapping | TAP + T4 RNA ligase |
| End Labeling | Probes | T4 PNK (5′) / TdT (3′) |
GATE Biotechnology Context
Q.45 integrates Q.35-44: S1 mapping → μ_max timing (Q.44) → Pol III replication (Q.43) → synchronous expression (Q.42) → IPTG vectors (Q.41) → pathway analysis. Critical for Sf9 vs BL21 yield comparisons and glycosylation engineering in bioprocesses.
Exam Tip: S1 spares RNA in hybrids; baculovirus = insect standard (vs mammalian AAV/SV40).
