Q.1 Which radio isotope is used to label the 5′ end of DNA or RNA?
- (1) 32S
- (2) 32P
- (3) 14C
- (4) 12C
The correct answer is option (2): 32P.
32P (phosphorus-32) is the standard radioisotope for 5′ end-labeling of DNA/RNA in molecular biology via T4 polynucleotide kinase (PNK) transfer from [γ-32P]ATP.
Option Explanations
(1) 32S – Incorrect
Sulfur-32 doesn’t exist as a significant radioisotope (S-35 is used for protein labeling via methionine/cysteine). No role in nucleic acid end-labeling.
(2) 32P – Correct
32P (β-emitter, 14.3 day half-life) labels 5′-OH of DNA/RNA. PNK catalyzes: R-OH + [γ-32P]ATP → R-[32P] + ADP. Gold standard for sequencing, footprinting, primer extension.
(3) 14C – Incorrect
Carbon-14 (long half-life, weak β-emitter) used for metabolic tracing (e.g., 14C-thymidine for DNA synthesis), not high-resolution end-labeling.
(4) 12C – Incorrect
Carbon-12 is stable, non-radioactive isotope. Cannot serve as label.
Why 32P for 5′ Ends?
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Chemical match: Phosphate backbone contains phosphorus
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High specific activity: ~9000 Ci/mmol
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Suitable β energy: 1.71 MeV penetrates gels/screens
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Enzymatic specificity: T4 PNK transfers γ-phosphate exclusively to 5′-OH
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Historical standard: Maxam-Gilbert sequencing used 32P-end-labeled DNA
Protocol: Dephosphorylate (CIP) → 5′-OH → PNK + [γ-32P]ATP → purify (G-50/denaturing PAGE)
Nucleic Acid End-Labeling Fundamentals
32P radioisotope 5′ end DNA RNA labeling is foundational molecular biology technique tested in GATE Life Sciences. T4 polynucleotide kinase transfers 32P from [γ-32P]ATP to 5′-OH groups for sequencing, footprinting, mapping.
32P Labeling Mechanism
T4 PNK catalyzes nucleotidyl transfer:
DNA/RNA-5′-OH + [γ-32P]ATP → DNA/RNA-5′-[32P] + ADPAdvantages:
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High resolution (single nucleotide)
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Quantitative (scintillation counting)
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Compatible with polyacrylamide gels/X-ray film/phosphorimaging
Common Applications
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Maxam-Gilbert sequencing (historical)
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DNaseI footprinting (protein-DNA interactions)
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Primer extension (transcription start sites)
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Ligation mapping (restriction sites)
Alternative Isotopes Comparison
Isotope Use Case Why Not 5′ End? 32P 5′/3′ nucleic acid ends GOLD STANDARD 35S Protein (Met/Cys), sequencing Sulfur absent in backbone 3H Metabolic labeling Low energy, poor resolution 14C Biosynthetic studies Long half-life, weak signal Exam Tip: Remember “P for Phosphate, 32P for 5′ Prime!”
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